Beta-glucan particles for trained immunization

Abstract

An effective amount of Saccharomyces cerevisiae-derived (1-> 3)-beta-D-glucan particles for use in a method of programming bone marrow hematopoietic stem and progenitor cells (HSPC) to promote pluripotent progenitor cell (MPP) expansion to facilitate bone marrow cell production in a subject.

Description

Field of the Invention

[0001] The present invention relates to intact β-glucan particles for inducing trained immunity in a subject. More specifically, the present invention relates to the use of intact β-glucan particles for programming bone marrow hematopoietic stem and progenitor cells (HSPCs) to provide multipotent progenitor cell (MPP) expansion to facilitate myelopoiesis. Background of the Invention

[0003] In vertebrates, the function of the immune system relies on two main pillars to fight infections and pathogens: the innate immune system and the adaptive immune system. The innate immune system is the first line of defense against pathogens, while the adaptive immune system establishes a coordinated and specific response. It has been known for many years that the adaptive immune system displays characteristics of immunological memory. For example, it is the adaptive immune system that is primarily stimulated during vaccination.

[0004] Until 2011, it was believed that patrolling immune cells, such as monocytes and macrophages, that belong to the innate immune system lacked immunological memory. However, since then, several studies have shown that innate immune cells that have been exposed to pathogens and danger signals can display features of immunological memory upon subsequent reinfection. This phenomenon is known as "trained immunity." Because patrolling innate immune cells provide our body's first immune response against invading organisms, targeting innate immune cells and "priming" them can greatly enhance the body's ability to improve immune function and fight infection.

[0005] Exposure to microbial stimuli has been shown to alter cells metabolically and epigenetically. Innate immune genes (inflammatory cytokines, chemokines) become epigenetically sensitized at the chromatin level, leading to altered (often enhanced) responses with accelerated kinetics upon maturation and re-challenge (Netea, MG et al., Defining trained immunity and its role in health and disease. Nat Rev Immuno, 2020).

[0006] It has been previously shown that trained immunity can be successfully induced by prior exposure to β-glucans from the cell wall of pathogenic yeasts, including Candida albicans, and is mechanistically driven in blood-borne monocytes through long-term epigenetic and metabolic reprogramming. Recent findings confirm that trained immunity operates at the whole organism level, targeting the bone marrow hematopoietic stem and progenitor cell (HSPC) compartment, thereby providing sustained protection against a variety of pathogens.

[0007] Much of this work has been demonstrated with fungal β-glucans derived from Candida albicans (Quintin, J. et al., Candida albicans infection affords protection against reinfection via functional reprogramming of monocytes. Cell Host Microbe, 12 223-232, 2012; Cheng, SC et al., mTOR and HI-1alpha-mediated aerobic glycolysis as metabolic basis for trained immunity. Science 345, (2014); Garcia-Valtanen, P. et al., Evaluation of trained immunity by beta-1,3(d)-glucan on murine monocytes invitro and duration of response in vivo. Immunol Cell Biol 95, 601-610, (2017)). Recent work showing in vivo training through bone marrow HSPC reprogramming and myelopoiesis was demonstrated following intraperitoneal (IP) injection of macrofungi (mushroom) Trametes versicolor or Candida albicans β-glucan (Moorlag, S. et al., beta-Glucan Induces Protective Trained Immunity against Mycobacterium tuberculosis Infection: A Key Role for IL-1. Cell Rep 31, (2020); Mitroulis et al., Modulation of Myelopoiesis Progenitors Is an Integral Component of Trained Immunity. Cell 172, 147-161 (2018)).

[0008] The anti-tuberculosis vaccine Bacillus Calmette-Guérin (BCG) has also been shown to induce innate training, and mice injected intravenously or subcutaneously with BCG showed lineage 阴性 c-Kit 阳性 Sca-1 阳性(LKS+) cell number and proportion expansion, as well as the expansion of myeloid-biased progenitors (named MPP3) at the expense of lymphoid-biased progenitors (i.e., MPP4) (Kaufmann, E. et al., BCG Educates Hematopoietic Stem Cells to Generate Protective Innate Immunity against Tuberculosis. Cell 172, 176-190 (2018) (Kaufmann et al., 2018). Similarly, intraperitoneal injection of β-glucan peptides from Trametes versicolor was shown to induce a significant increase in hematopoiesis of LKS+ cells and MPP3 (Mitroulis et al., 2018).

[0009] β-Glucans are a heterogeneous family of structural carbohydrates with multiple biological activities (Camilli, G., Tabouret, G., and Quintin, J. The Complexity of Fungal beta-Glucan in Health and Disease: Effects on the Mononuclear Phagocyte System. Front Immunol 9, 673, (2018)). The more common yeast β-glucans, especially food-grade baker's and brewer's yeast, contain larger molecular weight (MW) and more branched β-glucans, and a variety of health benefits are attributed to these larger molecular weight (MW) and more branched β-glucans. Wellmune is a common and well-tolerated dietary food, beverage and supplement ingredient, and is derived from the cell wall of baker's yeast (Saccharomyces cerevisiae). Wellmune is a whole glucan particle (WGP), the main chain of glucose molecules is connected by a unique chemical bond (β1,3 chain), and the glucose chain is attached to the β1,3 chain by a β1,6 bond. Clinical studies have demonstrated the ability of Wellmune to enhance human immune function and has a protective effect against various respiratory infections.

[0010] Until now, conventional strategies to improve immune function and fight infections have relied on vaccination, which targets the so-called adaptive immune system and promotes immunological memory by stimulating disease-specific adaptive T and B cells. It is therefore of great interest to show how a common food supplement derived from yeast can improve our immunity against a variety of infectious agents, including bacteria and viruses.

[0011] However, prior to the present invention, such trained immune assays had not been performed with Wellmune. In particular, oral administration of Wellmune or any other β-glucan variant to animal models to reconstitute innate immune memory signatures in mature innate immune cells has never been performed and published before.

[0012] Elena De Marco et al. (Mol Nutr Food Res, 2021, 65) discusses the general state of the art related to glucans and immunity. Immune training in this publication is used in general terms, and there is no disclosure about innate immune training or programming bone marrow hematopoietic stem and progenitor cells.

[0013] The present invention is used to solve the problems of the prior art. SUMMARY OF THE INVENTION

[0015] The inventors have demonstrated for the first time that oral delivery of intact β-glucan particles derived from Saccharomyces cerevisiae (baker's yeast) (referred to herein as "β-glucans of the present invention"), in particular (1→3)-β-D glucans, induces changes in myeloid precursor cells, i.e., bone marrow hematopoietic stem & progenitor cells (HSPCs), provides expansion of the progenitor cell population at the bone marrow level, and enhances the function of mature immune cells derived from these progenitor cells when administered orally via a food supplement. The changes include a bias of myeloid progenitor cells toward myeloid precursor cells, i.e., MMP3. Myeloid cells include neutrophils, monocytes, macrophages, among others, and together constitute an important part of the immune system, responsible for innate defense.

[0016] Without being bound by theory, it is believed that oral administration of the β-glucan of the present invention induces immunometabolism changes in bone marrow progenitor cells, thereby increasing the level of inflammatory cytokine production. This pro-inflammatory microenvironment is believed to promote the expansion of multipotent progenitor cells (MPPs), which is beneficial to myelopoiesis. These innate immune cells (e.g., monocytes or neutrophils) are trained or "sensitized" and differentiated into mature innate immune cells, such as macrophages, with trained immune characteristics. Therefore, it is determined that administration can be used to promote long-term trained immunity through reprogramming of hematopoietic precursor cells.

[0017] The effect after oral administration is surprising, as it has been thought in the art that oral administration would not induce such changes. Indeed, one would expect the intestinal assimilation mechanism to degrade the active components of the β-glucan of the invention, inducing major structural changes in the molecule, or simply breaking the molecule into fragments. These results suggest that WGP particles have the ability to interact with the mucosal immune system and drive central innate immune memory.

[0018] The inventors compared the β-glucans of the present invention to other β-glucans to determine the structural and signaling requirements for training in monocytes in vitro and in mice in vivo. The inventors' work showed that the β-glucans of the present invention can drive training effects on human innate immune cells such as monocytes in the laboratory, as well as at the bone marrow level in injected mice. The inventors also showed that the β-glucans of the present invention are more effective than other β-glucans in inducing trained immune signatures in human macrophages derived from trained monocytes in vitro ( Figures 1 to 5 ). In addition, soluble and non-granular forms of beta-glucan do not have the same effect.

[0019] The inventors also showed that intraperitoneal (IP) injection of the β-glucan of the present invention induces stronger bone marrow changes than other β-glucan variants in mice. The inventors report that oral administration of the β-glucan of the present invention induces a stronger hematopoietic bias than other β-glucan variants.

[0020] The inventors have further shown that feeding a diet containing yeast-derived WGP for 4 weeks results in the expansion of myeloid immune-committed progenitor cells (MPP3), leading to an increase in more mature myeloid-committed progenitor cells (CMP, GMP). Consistent with these bone marrow changes, it was observed that mature macrophages derived from these bone marrow-derived macrophages showed enhanced responsiveness to activation ( Fig.16 ).

[0021] The present invention provides an effective amount of whole β-glucan particles derived from Saccharomyces cerevisiae, i.e., "β-glucan of the present invention", for use in a method of priming bone marrow hematopoietic stem and progenitor cells (HSPCs) in a subject. The HSPCs in the subject promote the expansion of multipotent progenitor cells (MPPs) and are beneficial to myelopoiesis.

[0022] One aspect of the invention provides an effective amount of Saccharomyces cerevisiae derived β-glucan particles for use in a method of inducing trained innate immunity in a subject. In this way, the glucan is used to elicit an enhanced immune response against invading pathogens. The glucan is a complete glucan particle.

[0023] One aspect of the invention provides an effective amount of β-glucan particles derived from Saccharomyces cerevisiae for use in a method for producing trained innate immune cells in a subject. The trained innate immune cells can be monocytes and / or mature innate immune cells, such as macrophages. The glucan is a complete glucan particle.

[0024] In embodiments of any aspect of the present invention, an effective amount of yeast-derived β-glucan particles may be formulated for oral administration or intraperitoneal injection (IP), preferably for oral administration.

[0025] The β-glucan particles of the present invention are (1→3)-β-D-glucan derived from Saccharomyces cerevisiae. It can be derived from any strain of Saccharomyces cerevisiae.

[0026] Typically, an effective amount of glucan is used to prevent, delay the onset of, or reduce the severity of a disease or infection in the subject.

[0027] Preferably, the β-glucan is administered at an early stage of a disease or infection or when a subject is suspected of having a disease or infection.

[0028] Preferably, the subject is one or more selected from the group consisting of: athletes, subjects experiencing or suffering from stress, immunocompromised subjects, subjects over 65 years old, preferably over 75 years old, subjects less than 16 years old, subjects suffering from or recovering from cancer, and subjects with defective innate immunity.

[0029] In one embodiment, the subject is an athlete, typically a high-level athlete. These athletes have an increased susceptibility to upper respiratory tract infections at certain times, such as after a marathon.

[0030] The subject can be a healthy subject. The subject can be of any age, preferably a subject over 65 years old, preferably a subject over 75 years old, and a subject less than 16 years old.

[0031] The subject may be a subject suffering from obesity. The subject may be a subject suffering from obesity and diabetes.

[0032] The subject may have post-infection immune dysregulation, such as post-COVID-19, which depletes adaptive immune memory cells and dysregulates innate immune cells. (Driving trained immunity may restore regulated innate immune responses and provide protection against severe infections).

[0033] In one embodiment, the β-glucan of the present invention is taken / administered to a subject in a cyclical cycle. This cycle can last for a period of three to four weeks. It can be administered daily, for example once a day or twice a day, for a period of three to four weeks, after which administration is interrupted or stopped.

[0034] In one embodiment, the glucan of the present invention is a β-1,6 branched β-1,3 glucan (or "β-(1,3 / 1,6)").

[0035] Preferably, the (1→3)-β-D-glucan is a β-1,6 branched β-1,3 glucan and has the following generally repeating structure:

[0036]

[0037] In one embodiment, the glucan of the invention consists essentially of β(1-6) and β(1-3) linked glucans.

[0038] In one embodiment, the 1,6 linked side chains of the glucan of the invention range from 3-8 glucose molecules, such as 3-6 glucose molecules, or 4-5 glucose molecules. In one embodiment, there are at most 8 glucose molecules.

[0039] The glucan of the present invention may have a degree of branching of 3% to 5%, for example 4%.

[0040] In one embodiment, the glucan of the present invention is a whole glucan particle (WGP).

[0041] In one embodiment, the WGP has a size of about 1 to about 6 micrometers (or μm), or about 2 to about 5 micrometers, or about 3 to about 4 micrometers.

[0042] In one embodiment, the WGP is insoluble.

[0043] Typically, the glucan is a glucan derived from or obtained from the cell walls of baker's yeast.

[0044] In one embodiment, the β-glucan of the present invention is a β-glucan preparation derived from a strain of Saccharomyces cerevisiae.

[0045] In one embodiment, the glucan of the present invention is provided as a dietary or food supplement comprising the glucan of the present invention. It can be a food supplement enriched with the glucan of the present invention. The supplement can be selected from the group including but not limited to tablets, capsules, chewing gum and powders, beverages / drinks and energy bars.

[0046] In one embodiment, the food supplement comprises ≥ 75% β1,3 / 1,6 glucan by dry weight.

[0047] In one embodiment, the supplement is Wellmune supplement or any other S. cerevisiae derived beta-glucan preparation.

[0048] The supplement comprises 80% or more beta-glucan. It may be 82% to 90%, or 82% to 84%.

[0049] Typically, supplements contain ≥75% β1,3 / 1,6 glucan by dry weight, <3.5% protein, <10% fat, <3% ash, <8% moisture, <0.1 mg / kg mercury, <0.5 mg / kg lead, <1.0 mg / kg arsenic, and <1.0 mg / kg cadmium.

[0050] A method for programming bone marrow hematopoietic stem cells and progenitor cells (HSPC) in a subject is provided, the method comprising administering the glucan of the present invention to the subject. The HSPC in the subject promotes the expansion of multipotent progenitor cells (MPP), which is beneficial to the generation of bone marrow cells.

[0051] A method of inducing trained innate immunity in a subject is provided, the method comprising administering to the subject a glucan of the present invention.

[0052] A method of producing trained innate immune cells in a subject is provided, the method comprising administering to the subject a glucan of the invention. The trained innate immune cells may be monocytes and / or mature innate immune cells, such as macrophages.

[0053] Definition and general preferences

[0054] All publications, patents, patent applications, and other references mentioned herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference and was set forth in its entirety.

[0055] When used herein and unless expressly indicated otherwise, the following terms are intended to have the following meanings, in addition to any broader (or narrower) meaning that a term may have in the art:

[0056] Unless the context requires otherwise, the use of the singular herein shall be understood to include the plural, and vice versa. The term "a" or "an" used with respect to an entity shall be understood to refer to one or more of that entity. Thus, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

[0057] As used herein, the term "comprise" or variations such as "comprises" or "comprising" should be understood to indicate the inclusion of any stated integer (e.g., features, elements, characteristics, properties, method / process steps, or limitations) or group of integers (e.g., features, elements, characteristics, properties, method / process steps, or groups of limitations), but not the exclusion of any other integer or group of integers. Thus, as used herein, the term "comprises" is inclusive or open-ended and does not exclude additional, unrecited integers or method / process steps.

[0058] As used herein, the term "disease" or "condition" is used to define any abnormal condition that impairs physiological function and is associated with specific symptoms. The term is used broadly to encompass any disorder, illness, abnormality, pathology, morbidity, condition or syndrome in which physiological function is impaired, regardless of the nature of the cause (or whether the etiological basis of the disease is actually established). Thus, it includes conditions caused by infection, trauma, injury, surgery, radiation ablation, poisoning or nutritional deficiency.

[0059] As used herein, the term "treatment" or "treating" refers to an intervention (e.g., administration of a pharmaceutical agent to a subject) that cures, ameliorates, or alleviates the symptoms of a condition or disease, or eliminates its cause (or alleviates the effects of its cause). In this context, the term is used synonymously with the term "therapy". It can be manifested by a permanent or temporary improvement in the subject's condition. In this context, it includes limiting and / or reversing disease progression.

[0060] As used herein, the term "prevention" or "preventing" refers to an intervention (e.g., administering an agent to a subject) that prevents or delays the onset or progression of a condition or the severity of a condition in a subject, or reduces (or eradicates) its incidence in a treated population.

[0061] As used herein, the term "composition" should be understood to mean something artificially prepared and does not include naturally occurring compositions. The composition can be formulated in unit dosage form, that is, in discrete portions containing a unit dose, or multiple unit doses or subunits of a unit dose.

[0062] The term "symptom" is defined as a sign that something is wrong with a disease, illness, injury, or condition.

[0063] As used herein, the term "effective amount or therapeutically effective amount" applied to the glucan of the present invention defines an amount that can be applied to a subject without excessive toxicity, irritation, allergic reaction or other problems or complications, commensurate with a reasonable benefit / risk ratio, but sufficient to provide the desired effect. Depending on the age and general condition of the individual, the mode of administration and other factors, the amount will vary from subject to subject. Therefore, although it is impossible to specify an exact effective amount, those skilled in the art will be able to determine the appropriate "effective" amount in any individual case using routine experiments and background common sense. The therapeutic outcome does not need to be a complete cure. The therapeutic outcome can be a permanent or temporary improvement in the subject's condition.

[0064] The term "subject" means a human or an animal, more typically a mammal. In one aspect, the subject is a human.

[0065] "β-glucans" are polysaccharides found in the cell walls of bacteria and fungi. They are glucose (D-glucose) polymers linked together by 1→3 linear β-glycosidic chain cores, and differ from each other in their length and branching structure. The branches derived from the glycosidic chain core are variable, and the two main branching groups are 1→4 glycosidic chains or 1→6 glycosidic chains. In the present invention, the β-glucan is a β-glucan derived from Saccharomyces cerevisiae. The Saccharomyces cerevisiae can be any suitable strain of Saccharomyces cerevisiae.

[0066] As used herein, the term "(1→3)-β-D-glucan" is a glucan comprising D-glucose units having β-1,3 bonds.

[0067] "β-1,6 branched β-1,3 glucan (or β-(1,3 / 1,6))" consists of a backbone of glucose molecules connected via unique chemical bonds β1,3 chains attached to glucose chains via β1,6 bonds.

[0068] As used herein, the term "trained immunity" refers to the immunological memory characteristics of innate immune cells mediated by epigenetic reprogramming and / or metabolic reprogramming. Trained immunity generally provides protection against infection and an enhanced immune response to infection in the absence of antibodies.

[0069] As used herein, the term "immunological memory" or "immunological memory" refers to the ability of the immune system to quickly and specifically recognize pathogen components that it recognizes as non-self and to elicit an immune response based on past exposure to that component. This can be subdivided into: conventional adaptive immune memory, which relies on recognition of specific antigens to drive antibody and cellular responses by lymphocytes; or innate immune memory / trained immunity, in which certain stimuli such as β-glucan sensitize innate immune cells, which then respond differently to a wide range of pathogen components.

[0070] The "innate immune response" is the first line of defense against invading pathogens, activated as a physical and / or chemical barrier against infectious agents. Innate immune cells include natural killer cells, macrophages, monocytes, neutrophils, dendritic cells, mast cells, basophils, and eosinophils. The innate immune system serves to activate the adaptive immune system through antigen presentation.

[0071] As used herein, the term "bone marrow hematopoietic stem and progenitor cells (HSPC)" refers to a population of precursor cells that have the ability to self-renew and produce multi-lineage differentiation. Functionally distinct hematopoietic precursor subsets include multipotent progenitor cells 2-4 (MPP2, MPP3, and MPP4). MMP3 is a myeloid cell-related lineage. MMP4 is lymphoid-related. In the bone marrow (BM), HSPCs ensure blood cell homeostasis.

[0072] As used herein, the term "trained monocytes" refers to monocytes with immunological memory mediated by epigenetic reprogramming and / or metabolic reprogramming. For example, in this context, "trained monocytes" are monocytes trained by exposure to β-glucan in the laboratory, or monocytes derived from the bone marrow of animals / subjects exposed to β-glucan. Trained monocytes typically exhibit enhanced proinflammatory cytokine responses to restimulation of TLR ligands (Saeed et al., Epigenetic programming of monocyte-to-macrophage differentiationand trained innate immunity, Science 2014).

[0073] As used herein, the term "trained macrophages" refers to macrophages (sentinel cells of the innate immune system) derived from the bone marrow (via monocytes) or from animals / subjects exposed to beta glucan / Wellmune or other drivers of trained immunity, with enhanced function mediated by epigenetic reprogramming and / or metabolic reprogramming.

[0074] As used herein, the term "hematopoiesis" refers to the formation of blood cell components. All cellular blood components are derived from hematopoietic stem cells (HSCs). HSCs are stem cells that give rise to other blood cells. In adults, hematopoiesis occurs in the red bone marrow in the core of most bones.

[0075] As used herein, the term "myeloid cell" refers to a blood cell produced by a progenitor cell of a granulocyte, monocyte, erythrocyte or platelet. The progenitor cell can be a myeloid progenitor cell or precursor cell, such as a common myeloid progenitor cell (CMP). It can be specifically from the myeloblast lineage, i.e., myelocytes and monocytes.

[0076] As used herein, the term "myelopoiesis" refers to the process by which mature innate immune cells develop from bone marrow progenitor cells.

[0077] As used herein, the term "multipotent progenitor (MPP) expansion" refers to an increase in the relative proportion and / or absolute number of the MPP cell pool in an animal / subject, typically in response to a treatment.

[0078] As used herein, the term "whole glucan particle" refers to β-glucan that is separated from a cell wall containing glucan and substantially retains the in vivo glucan morphology. WGP is insoluble and preferably spherical. The WGP of the present invention is typically pure glucan particles, such as 80% or more, 90% or more, 95%, 98% or 100% β-glucan. In one embodiment, the WGP exhibits a high water holding capacity, as shown by its viscosity in aqueous solution. For example, a WGP of about 2 to 4 microns containing 5.5 grams of glucan per deciliter has a viscosity of about 1000 centipoise. Methods for determining viscosity are known in the art, for example (US4992540).

[0079] Methods for preparing WGP are known in the art. Exemplary methods are disclosed herein. In one embodiment, the WGP is a WGP produced by one or more of these methods. Generally, the method comprises extracting alkali-soluble components from a cell wall containing glucan without prior description of the cell wall to produce whole glucan particles that retain the glucan morphology in vivo. In this way, the β-glucan layer of the cell wall is separated and remains intact, forming so-called "ghost cells" and is referred to as dispersible whole glucan particles (dWGP).

[0080] For example, Saccharomyces cerevisiae is selectively grown to obtain pure cultures and expanded in stainless steel fermentation vessels. After fermentation, the cells are lysed by maintaining them at 45°C-55°C for about 24 hours. After autolysis, the cell walls are separated from the soluble yeast extract using a continuous centrifugal separator. The collected yeast cell walls are further treated by a series of alkaline and hot water washes (70°C-90°C) to remove cell wall mannosylated proteins and any residual cellular lipids. In a subsequent acidification step, cell wall chitin is removed and the remaining purified β-1,3 / 1,6 glucan slurry is washed in hot water, concentrated and the pH adjusted as needed. The resulting product is flash pasteurized and spray dried. This process produces glucans that retain their yeast cell-like macrostructure.

[0081] Another method comprises culturing a Saccharomyces cerevisiae yeast strain in a culture medium, harvesting whole yeast cells from the culture medium, and contacting the whole yeast cells with an aqueous hydroxide solution having a pH of about 4.0 to about 12.5 or a normality of about 0.75 to about 1.5 and a temperature of about 25° C. to about 100° C. for a time sufficient to extract protein from the whole yeast cells to form water-insoluble whole glucan particles containing less than 1% protein by weight, the glucan particles substantially retaining the in vivo glucan three-dimensional structure, and typically consisting essentially of glucans having β(1-6) bonds and β(1-3) bonds (US4810646).

[0082] It is not a linear soluble beta glucan (small molecular weight, e.g. 25 kDa or less), such as disclosed by the prior art. In other words, it is intact.

[0083] As used herein, the term "β-glucan preparation" refers to β-glucan isolated from a fungal species and prepared in a specific manner that is different from other β-glucans isolated from the same or other species.

[0084] When used herein, zymosan is a β-glucan preparation derived from Saccharomyces cerevisiae to separate β-glucan-rich cell walls in granular form, i.e., ghost yeast cells. Its physical and chemical properties are described in De Graaff et al., Cancer Immunology, Immunotherapy (2021) 70: 547-561. Depleted zymosan is a zymosan preparation that is further purified to remove the outer mannan and enrich the β-glucan content and remove contaminating ligands. It is described in Gantner et al., J. Exp. Med, Vol. 197, No. 9, May 5, 2003, 1107-1117 and Ikeda et al., Biol Pharm Bull. 2008 31 (1): 13-8.

[0085] As used herein, the term "subject suffering from obesity" refers to an individual, typically a human, with a BMI>30 and a fasting glucose level <6 mmol / l and HbA1C <42 mmol / mol. Fasting glucose levels and HbA1C are calculated using methods known in the art.

[0086] As used herein, the term "subject with obesity and diabetes" refers to individuals, typically humans, with BMI>30 and fasting glucose>7mmol / l, fasting HbA1C≥48mmol / mol. Fasting glucose levels and HbA1C are calculated using methods known in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] The present invention will now be described with reference to the following drawings, in which:

[0089] Figure 1 Dectin-1-activating β-glucans differentially drive monocyte training. A) hDectin1-HEK293 NFκB-SEAP reporter cells were incubated with different concentrations of the following β-glucans (BGP, zymosan (ZYM), dispersible (dWGP), solubility of dWGP derived from heat treatment β-glucan (sWGP), schizopylan (SPG), curdlan (CURD) and Pyrrolidone Glucan (PUST), all at 1, 10 and 100 μg / mL β-glucan, 10 4 , 10 5 and 10 6 10 ng / mL of heat-killed Candida albicans (HKCA) or LPS (10 ng / mL) per 10 cells / mL. SEAP activity in the supernatant was measured 6 h after stimulation and quantified relative to untreated cells (-). B) The isolated human monocytes were exposed to β-glucan or LPS (1 ng / mL, 10 ng / mL and 100 ng / mL) as specified above for 24 h, washed and allowed to mature into monocyte-derived macrophages in the presence of MCSF. 5 days after separation, cells were restimulated with LPS (10 ng / mL, 6 h) and TNF production was measured relative to untrained cells (- / +). C) hMDMs were trained and restimulated as described above using BGP, dWGP and sWGP, and TNF production after LPS restimulation (10 ng / mL, 6 h-black bars, left axis) was measured compared to TNF produced by monocytes 24 h after training (blue line, right axis). D) Human monocytes were exposed to 5'methylthioadenosine (MTA, 1 mM) for 1 h, then trained with BGP or dWGP for 24 h, washed, matured and restimulated with LPS (10 ng / mL, 6 h) as described above. Data are mean SEAP quantification ± sem (A) of n = 3 independent experiments, mean TNF quantification ± sem (B) of n = 4 independent donors, mean TNF fold change ± sem of trained cells of n = 3 independent experiments (C) or mean TNF fold change ± sem of trained cells of n = 6 independent experiments (D). * indicates post hoc t-test for comparisons between indicated groups, P < 0.05.

[0090] Figure 2 Early proinflammatory signaling and viability in monocytes treated with β-glucan. A-C) Correlation analysis between TNF production after LPS restimulation (training) and the relative changes in NFκB-linked SEAP production in hDectin1-HEK293 reporter cells (Dectin-1 activation, A), or TNF production 24 h after treatment with the indicated β-glucans (day 1 TNF, B); (BGP, zymosan (ZYM), dispersible (dWGP), soluble (sWGP), schizophyllan (SPG), curdlan (CURD) and Pust (Pust), all 100 μg / mL β-glucan, 10 4albicans (HKCA) at 10 cells / mL) or superimposed on the graph above LPS training (10 ng / mL, C). D) hMDM surviving 5 days after training with the indicated training agents (1 μg / mL, 10 μg / mL, and 100 μg / mL of BGP, dWGP, and sWGP or 1 ng / mL, 10 ng / mL, 100 ng / mL of LPS) as measured by crystal violet blue staining and expressed relative to untrained hMDM. E) Supernatant from dWGP-trained monocytes (10 μg / mL) was transferred to untrained monocytes (S / N Txt), left for 5 days, and both WGP-trained hMDM and S / N Txt hMDM were restimulated with LPS (10 ng / mL, 6 h), and TNF production was measured. A) Plotted from Figure 1 A is the average data (n = 4-6 independent donors per condition), B is plotted from Figure 1 C shows the average data of cells treated (n=3 independent experiments), C plots the average TNF fold change (left axis) or average TNF value (right axis) ± sem on trained cells for n=3 experiments. D is the average OD relative to untrained cells for n=3 experiments. E plots the average TNF value ± sem for n=3 experiments. # indicates post hoc t-test for comparisons between the indicated groups, P < 0.05.

[0091] Figure 3 Macrophage responses to restimulated macrophages trained with β-glucan. A) Dispersibility (dWGP, 10 μg / mL, blue bars) Monocytes trained or tolerant to LPS (10 ng / mL, gray bars) were matured into hMDMs and restimulated with LPS (10 ng / mL) for the indicated times (0-24 h), and TNF production was measured. B) hMDMs derived from trained monocytes as described above were restimulated with LPS (10 ng / mL), and CXCL8 production was measured at 3 h post-stimulation, TNF production was measured at 6 h post-stimulation, and IL-10 production was measured at 24 h post-stimulation and expressed relative to untrained cells. C) hMDMs derived from trained monocytes as described above were restimulated with LPS (100 ng / mL), pam3CSK4 (100 μg / mL), or HKCA (10 6dWGP training (10 μg / mL, blue bars) or LPS tolerance (10 ng / mL, gray bars) hMDMs were treated with irradiated Mycobacterium tuberculosis H37Rv (iMtb, 500 μg / mL) for 24 h, and the production of the indicated cytokines was measured. EF) hMDMs derived from monocytes trained by dWGP (10 μg / mL, blue bars) were infected with Mtb H37Ra (MOI 5 bacteria / cell), and intracellular bacterial survival was measured by CFU analysis at the indicated time after infection (E) or TNF production was measured in the culture supernatant (F). All data are the average absolute values ​​(A, CF) of n=3 (AB, D), n=4 (C) and n=5 (EF) independent experiments or the change fold (B) ± sem relative to untrained TNF. * indicates post hoc t test for indicated between-group comparisons, P < 0.05.

[0092] Figure 4 BGP-induced training response in human monocyte-derived macrophages. A) Monocytes trained with BGP (10 μg / mL, yellow bars) or monocytes tolerated with LPS (10 ng / mL, gray bars) matured into hMDM, and restimulated with LPS (10 ng / mL) for a specified time (0-24 h) and measured TNF production. B) hMDM derived from trained monocytes as described above were restimulated with LPS (10 ng / mL), and CXCL8 production was measured at 3 h after stimulation, TNF production was measured at 6 h after stimulation, and IL-10 production was measured at 24 h after stimulation, and expressed relative to untrained cells. Data are the average absolute values ​​(A) of n=3 independent experiments or the fold change (B) ± sem relative to untrained TNF. * indicates a post hoc t-test for comparison between indicated groups, P < 0.05.

[0093] Figure 5 Dectin-1 / Syk / NFκB signaling is diffusible (dWGP)-induced monocyte training is required. A) hDectin1-HEK293 NFκB-SEAP reporter cells were incubated with the indicated concentrations of laminarin or soluble (sWGP) was incubated for 1 h and then treated with 100 μg / mL of BGP (blue) or dWGP (grey). SEAP activity in the supernatant was measured 6 h after stimulation and quantified relative to untreated cells (-). B) Monocytes were incubated with the indicated concentrations of laminarin or sWGP for 1 h and then trained with 100 μg / mL of BGP (yellow) or dWGP (blue) for 24 h. Mature macrophages were then restimulated with LPS (10 ng / mL, 6 h) and TNF production was measured. C) hDectin1-HEK293 NFκB-SEAP reporter cells were incubated with the indicated concentrations of piceatannol (PIC) for 1 h and then trained with 100 μg / mL of BGP (yellow) or dWGP (blue) for 24 h. SEAP activity in the supernatant was measured 6 h after stimulation and quantified relative to untreated cells (-). D) Monocytes were incubated with BAY-11087 (5 μM, BAY) and then trained with dWGP (100 μg / mL, blue), and TNF production was measured 24 h later and compared to the fold change of TNF production in mature macrophages restimulated with LPS. Data in A) and C) are the mean ± sem of n = 3 (A) and n = 4 (C) independent experiments. Data in B are the mean fold change of TNF relative to untrained cells from n = 2 independent experiments. Data in D are the mean TNF values ​​± sem of n = 5 independent experiments.

[0094] Figure 6 Typical Dectin-1 signaling is diffusible (dWGP) is required for the training of monocytes induced by (sWGP, 100 μg / mL) were incubated for 1 h, and then trained with dWGP (1 μg / mL, 10 μg / mL and 100 μg / mL) for 24 h. Mature macrophages were then restimulated with LPS (10 ng / mL, 6 h) and TNF production was measured. B-C) ​​Monocytes were incubated with the indicated concentrations of piceatannol (PIC, between 4 μM, 10 μM and 30 μM, B) or BAY-11087 (BAY11, 5 μM, C), and then trained with BGP (yellow), dWGP (blue) or tolerated with LPS (grey). Mature macrophages were then restimulated with LPS (10 ng / mL, 6 h) and TNF production was measured. The data in A and C are the average TNF values ​​± sem of n = 3 (A) and n = 5 (C) independent experiments. The data in B are the average fold change of TNF relative to untrained cells of n = 3 independent experiments. * indicates post hoc t-test for comparison between untrained cells, P < 0.05, and # indicates P < 0.05 for comparison between cells without inhibitors.

[0095] Figure 7 Dispersibility (dWGP) drives mTOR-mediated glycolysis for training. A) Monocytes were trained with dWGP or BGP (10 μg / mL) or tolerated with LPS (100 ng / mL) for 24 h, the medium was removed and allowed to mature. The supernatants were tested for extracellular lactate production on the indicated days after training. B) Monocytes were incubated with 2-deoxyglucose (2DG, 1 μM, 5 μM and 10 μM) and then trained with dWGP. Mature macrophages were then restimulated with LPS (10 ng / mL, 6 h) and TNF production was measured. C) Representative histograms of monocytes, which were treated or not with sWGP or dWGP (10 μg / mL, 2 h) and intracellularly stained for detection of p-S6K by flow cytometry. DE) Monocytes were pretreated with rapamycin (rapa, 10nM) for 15 minutes and then stimulated with LPS (10ng / mL) or dWGP or sWGP (both at 10μg / mL) for 2h. p-S6K activity was measured by flow cytometry. F) Monocytes were stimulated with dWGP (10μg / mL) for a specified time and p-S6K activity measured by flow cytometry. G) Monocytes were incubated with rapamycin (rapa, between 1nM, 10nM and 100nM) and then trained with dWGP. Mature macrophages were then stimulated with LPS (10ng / mL, 6h) and TNF production was measured. H) Monocytes were trained with β-glucan for 24h and allowed to mature into macrophages for 5 days. The expression of specified genes was measured by qPCR and expressed relative to untrained cells. I) Monocytes were incubated with wortmannin (Wort, between 0.1 μM, 1 μM and 10 μM) and then trained with dWGP. Mature macrophages were then restimulated with LPS (10 ng / mL, 6 h) and TNF production was measured. J) Monocytes were pretreated with piceatannol (PIC, between 4 μM, 10 μM and 30 μM) for 15 minutes and then stimulated with dWGP (10 μg / mL, 2 h). p-S6K activity was measured by flow cytometry. Data in A) are the average lactate values ​​± sem of n = 4 independent experiments. Data in B and G are the average fold change ± sem of TNF relative to untrained cells for n = 7 (B) and n = 6 (G) independent experiments. Flow cytometry data (D-F and J) are mean MFI ± sem of n = 3 independent experiments. Data in H are mean fold change ± sem of the indicated genes of trained cells relative to untrained cells of n = 3 independent experiments. Data in I are mean TNF values ​​± sem of n = 3 independent experiments. * indicates post hoc t-test for comparison between untrained cells, P < 0.05, # indicates P < 0.05 for comparison between cells without inhibitors, and ns indicates non-significant change.

[0096] Figure 8 Metabolic analysis of β-glucan-trained macrophages. A) Lactate production in monocytes treated with 10 μg / mL of zymosan (ZYM), dispersible (dWGP), soluble (sWGP), heat-killed Candida albicans (HKCA, 10 4 Cells / mL) or LPS (100ng / mL) were treated for 24h, and the supernatant was sampled on the specified days after training. B) Gating strategy for intracellular staining of monocytes for p-S6-kinase activity. C) Monocytes were stimulated with dWGP or sWGP (100μg / mL) for the specified time, or treated with LPS (100ng / mL) in the presence or absence of rapamycin (rapa, 10nM), and p-S6K activity was measured by flow cytometry. D) Monocytes were pretreated with piceatannol (PIC, between 4μM, 10μM and 30μM) for 15min and then stimulated with LPS (100ng / mL) or dWGP (10μg / mL). TNF production was measured in the supernatant after 24h. The data in A) are the average lactate values ​​± sd of n = 2 independent experiments, and D) are the average TNF values ​​± sem of n = 3 independent experiments.

[0097] Fig. 9 Complete dispersibility (dWGP) phagocytosis drives mTOR-mediated glycolysis. AB) Monocytes were treated with the indicated β-glucans (10 μg / mL) for 15 min (A) or pretreated with cytochalasin D (CytD, 10 μM) for 1 h and then dWGP (100 μg / mL) for 15 min (B). Cells were immediately fixed and stained to measure surface Dectin-1 expression by flow cytometry. C) Monocytes were incubated with cytochalasin D (CytD, 10 μM) and subsequently trained with the indicated β-glucans (10 μg / mL) for 24 h. Mature macrophages were then restimulated with LPS (10 ng / mL, 6 h) and TNF production was measured. DE) Monocytes were pretreated with cytochalasin D (CytD, between 1 μM, 10 μM and 100 μM) or bafA1 (bafA1, between 1 μM, 5 μM and 10 μM) for 15 min and then stimulated with dWGP (10 μg / mL) for 2 h. p-S6K activity was measured by flow cytometry. FG) Monocytes were trained with 10 μg / mL (orange bars), 50 μg / mL (grey bars) or 100 μg / mL (black bars) of dWGP or unsonicated WGP (uWGP) for 24 h or left untreated (blue bars), and TNF production in the supernatant was measured (F). Mature macrophages were restimulated with LPS (10 ng / mL, 6 h) and TNF production was measured (G). H) Surface Dectin-1 staining of monocytes incubated with the indicated β-glucan (10 μg / mL) for 15 min was measured by flow cytometry. I) Extracellular lactate production of monocytes trained with dWGP or uWGP (100 μg / mL) for 24 h, allowed to mature, and sampled at the indicated times after training. J) hDectin1-HEK293 NFκB-SEAP reporter cells were incubated with 1 μg / mL, 10 μg / mL, and 100 μg / mL of dWGP, sWGP, or defined fractions F1-F4 of sWGP (MW range as indicated below, kDa). SEAP activity was measured in supernatants 6 h after stimulation and quantified relative to untreated cells (-). K) dWGP (dis), soluble (sWGP, sol) or indicated soluble fractions F1-F4 (10 μg / mL) trained monocytes for 24 h. Mature macrophages were then restimulated with LPS (10 ng / mL, 6 h) and TNF production was measured. L) Monocytes were stimulated with 200 μg / mL aminated polystyrene particles (AM-PS) conjugated to sWGP (sWGP-PS) alone or (equal amounts of sWGP roughly correspond to 200 μg / ml of sWGP-PS (1 μg / ml), dWGP (100 μg / ml)) or left untreated for 3 days, and extracellular lactate production was measured. The data in AB, DE, and H are the average MFI ± sem of n = 3 independent experiments. The data in B are the average fold change ± sem of TNF relative to untrained cells for n = 7 independent experiments. The data in FG and K are the average TNF values ​​± sem of n = 3 independent experiments. Data in I and L are mean lactate values ​​± sem of n = 3 independent experiments. Data in J are mean ± sem of n = 5 independent experiments. * indicates post hoc t test for comparison between untrained cells, P < 0.05, # indicates P < 0.05 for comparison between cells without inhibitors, and ns indicates non-significant change.

[0098] Fig.10 Phagocytosis of particulate β-glucan. A) Representative histogram of monocytes, monocytes were expressed using dispersibility (dWGP, 10 μg / mL, 2 h) or not and stained for surface Dectin-1 expression by flow cytometry. B) Monocytes were stimulated with dWGP or BGP (100 μg / mL) for the indicated time and surface Dectin-1 expression was detected by flow cytometry. C) Dispersibility of “unsonicated” from aggregates by sonication (uWGP) Preparation of single particle "dispersion" (d) Schematic diagram of WGP. Light micrographs show monocyte culture medium containing each formulation. D) hDectin1-HEK293 NFκB-SEAP reporter cells with 10 4 , 10 5 and 10 6 cells / mL of heat-killed Candida albicans (HKCA), zymosan (ZYM), dWGP, uWGP or soluble (sWGP) (all 1 μg / mL, 10 μg / mL and 100 μg / mL), or LPS (1 ng / mL, 10 ng / mL and 100 ng / mL). SEAP activity in the supernatant was measured 6 h after stimulation and quantified relative to untreated cells (-). E) Sugar content of sWGP preparations after incubation with 2 μg of aminated polystyrene particles (AM-PS) in the presence or absence of the conjugate CDI and then filtered. The data in D are the mean ± sem of n = 2 independent experiments. The data in E are the mean ± sd determined in triplicate.

[0099] Fig.11 Dispersibility in primary human monocytes, mouse BMDM, and mouse bone marrow (dWGP) response. A) As shown, monocytes were sorted based on CD14 / CD16 expression (left figure) and further analyzed by flow cytometry based on CCR2 expression (right figure). B) The monocytes isolated in A were trained with dWGP for 24h and TNF production was measured. C) BMDMs at 5 days after maturity were trained with dWGP (10μg / mL-100μg / mL) for 24h or left untrained. Lactic acid production was measured 5 days after dWGP treatment. DE) C57 / BL6-JOlaHsd mice were injected intraperitoneally with dWGP (0.2mg / mouse and 0.4mg / mouse), BGP (1mg / mouse) or vehicle PBS. After 7 days, bone marrow (BM) was isolated, quantified, stained and analyzed by flow cytometry for the following designated populations: Lin-x, c-ki, Sca1 (LKS), long-term hematopoietic stem cells (LT-HSC), short-term HSC (ST-HSC), multipotent progenitor cells 1-3 (MPP) or CD150- / CD48- cells. C) shows the gating strategy (left figure) for identifying LT-HSC, ST-HSC and MPP and the absolute number of each population. D) shows the gating strategy (left figure) for identifying the MPP subpopulation and the absolute number of each population. The data in A are the average MFI±sem of n=3 donors. The data in BC are the average values±sem of n=3 independent experiments. The flow cytometry of representative samples and the average total population±sem of n=4-5 mice per group are shown in CD.

[0100] Fig.12 Dispersibility (dWGP) sensitizes monocytes and drives myeloid bone marrow reprogramming. AB) As shown, monocytes were sorted based on CD14 / CD16 expression and further analyzed by flow cytometry based on Dectin-1 expression (A), or trained with dWGP, allowed to recover and restimulated with LPS (10ng / mL, 6h, B). C) BMDMs at 5 days after maturation were trained with dWGP (1μg / mL, 10μg / mL-100μg / mL), BGP (10μg / mL) or LPS (5ng / mL) for 24h. 11 days after separation, mature macrophages were restimulated with LPS (10ng / mL) and TNF production was measured. DG) C57 / BL6-JOlaHsd mice were injected intraperitoneally with dWGP (0.2mg / mouse and 0.4mg / mouse), BGP (1mg / mouse) or vehicle PBS. After 7 days, bone marrow (BM) was isolated, quantified, stained and analyzed by flow cytometry for the following indicated populations: Lin-x, c-kit, Sca1 (LKS), long-term hematopoietic stem cells (LT-HSC), short-term HSC (ST-HSC), multipotent progenitor cells 1-3 (MPP) or CD150- / CD48- cells. H) Splenic macrophages were isolated by plastic adherence and stimulated with LPS (10 ng / mL) or heat-killed Mycobacterium tuberculosis H37Ra (hk-Mtb, 500-1000 μg / mL) for 6 h. Data in A are the mean MFI ± sem of n = 6 donors. Data in BC are the mean TNF values ​​± sem of n = 3 independent experiments. Data in D are the LKS cell % (left panel) or absolute LKS cell number (right panel) in total BM cells. Data in E are the absolute cell number of the indicated subpopulations. Data in F are the relative percentages of each subpopulation in the total LKS population. Data in G are the relative frequencies of each population in the total MPP pool, which does not include MPP1 (separately gated, see Fig.11 ). Data in H are mean TNF values. All data in DH are mean ± sem of n = 4-5 mice per group.

[0101] Fig.13 Orally dispersible (dWGP) reprograms bone marrow macrophage responses. AG) ​​Mature C57 / BL6-JOlaHsd mice were fed a control (0dWGP diet) for 2 weeks before starting a diet supplemented with increasing concentrations of dWGP (0, 0.003%, 0.025%, 0.050% / kg food). Mice were killed 1 or 3 weeks after dWGP feeding and bone marrow (BM) was examined by flow cytometry (A, C, and E) or matured into BMDM and stimulated for 6 h with LPS (10 ng / mL, B and FG) or heat-killed Mycobacterium tuberculosis H37Ra (hk-Mtb, 500-1000 μg / mL, FG), and TNF was measured by ELISA. The data in A) are the absolute LKS cell numbers in total BM cells. The data in B) are the relative percentages of each subpopulation in the total LKS population. The data in C) are the relative frequencies of the MPP3 population in the total MPP3 / 4 population. All data in A, C, and E are mean ± sem of n = 6 mice per group. Data in B, D, and FG are mean TNF values ​​± sem of n = 4 mice per group.

[0102] Fig.14 Orally dispersible Effect of (dWGP) on bone marrow HSPC spectrum. AH) Mature C57 / BL6-JOlaHsd mice were fed a control (0dWGP diet) for 2 weeks before starting to feed a diet supplemented with increasing concentrations of dWGP (0, 0.003%, 0.025%, 0.050% / kg food). Mice were killed 1 or 3 weeks after dWGP feeding, and bone marrow (BM) was examined by flow cytometry (AF) or matured into BMDM and stimulated with LPS (10ng / mL) or heat-killed Mycobacterium tuberculosis H37Ra (hk-Mtb, 500-1000μg / mL) for 6h, and TNF (GH) was measured by ELISA. The data in A) are the relative percentages of the indicated subpopulations in the total LKS population. The data in C) are the relative frequencies of MPP3 or MPP4 populations in the total MPP3 / 4 population. The data in AF) are the mean ± sem of n = 6 mice per group. Data in GH) are mean TNF values ​​± sem from n = 4 mice per group.

[0103] Fig.15Long-term feeding of Wellmune reveals a transient increase in trained immunity. Mature C57 / BL6-JOlaHsd mice were fed a diet supplemented with 0.05% dWGP / kg food for 12 weeks, 8 weeks or 4 weeks and compared with a 12-week no WGP control. Mice were sacrificed and the % of total bone marrow LKS+ cells was examined by flow cytometry (A), and the MPP3 subpopulation is shown in B. In addition, the bone marrow was matured into BMDM and stimulated with LPS (10 ng / mL), PAM3CYS (10 μg / mL) or heat-killed Mycobacterium tuberculosis H37Ra (hk-Mtb, 500 μg / mL) for 3 h, and TNF production was measured by ELISA (C). The data in AB) are the relative percentages of the subpopulations shown in the total LKS population. The data in C) are the average TNF values ​​± sem of n = 5 mice per group.

[0104] Fig.16 Oral delivery of Wellmune WGP results in an increase in the proportion of myeloid-directed progenitor cells, resulting in an increase in cytokine responses in mature cells. C57 / BL6 mice were given Wellmune WGP (200 mg / mouse) by intraperitoneal injection (IP) or via oral gavage (OG) or PBS (-) by oral gavage and placed for 7 days before sacrifice. Bone marrow was extracted and LKS+HSPC cells were isolated, counted and expressed as the total number of cells per leg (A) or multi-parameter flow cytometry was performed to identify the proportion of myeloid-directed MPP3 subpopulations or lymphoid-directed MPP4 subpopulations in the LKS+ population (B). Bone marrow from the other leg grew into mature BMDM. Seven days after separation, the cells were replated and stimulated with low-dose LPS (1 ng / mL) for 3-24 h the next day, as shown. TNF production of cell-free supernatant was measured by ELISA (C). Data are expressed as total LKS+ cell number per leg (A), % of each subpopulation in the total LKS+ population (B), or as [TNF] in pg / mL (C), and represent mean ± sem of n = 5 mice per group. *P < 0.05, **P < 0.01 for the indicated comparisons from one-way ANOVA analysis followed by post hoc multiple comparison tests.

[0105] Fig.17Feeding a diet containing Wellmune-WGP for 4 weeks resulted in an increase in the frequency of more abundant and committed myeloid progenitor cells, independent of fiber content. C57 / BL6 mice were fed 3 different mouse diets (regular mouse chow [Walachowski, #60], WGP (0.050% w / w) and a conventional mouse diet supplemented with the dietary fiber inulin (0.050% w / w) as a control (CTRL) for the additional fiber in the diet supplemented with Wellmune. After 4 weeks of feeding, bone marrow was extracted and LKS+ HSPC cells were isolated and counted and expressed as total cells per leg (A) or subjected to multiparameter flow cytometry to identify the proportion of myeloid-directed MPP3 subsets or lymphoid-directed MPP4 subsets in the LKS+ population (B). LKS(-) cells were retained and further multiparameter flow cytometry was performed to identify total myeloid progenitors [MP], granulocyte-macrophage progenitors [GMP], common myeloid progenitors [CMP], and common lymphoid progenitors [CMP]. Figure 20. The ratio of LKS+ cells to LKS+ progenitor cells [CLP] in each leg (C). The bone marrow from the other leg was grown into mature BMDM. Seven days after separation, the cells were re-plated and stimulated with low-dose LPS (10 ng / mL) for 24 h on the second day. The levels of specified cytokines in the cell-free supernatant were measured by ELISA (D). Data are expressed as the total number of LKS+ cells per leg (A), the % of each subpopulation in the total LKS+ population (B), the % of each subpopulation in the total Lin- population (C), or [cytokines] in pg / mL (F), and represent the mean ± sem of n = 4 mice per group. * P < 0.05 for the indicated comparisons from one-way ANOVA analysis and subsequent post hoc multiple comparison tests.

[0106] Fig.18Intestinal immune sensing of Wellmune WGP β-glucan. A) C57 / BL6 mice were fed a standard fat diet (SFD) or a diet supplemented with 0.05% WGP (WGP) for 4 weeks. Intestinal tissue was separated, and immune cells were separated by Percoll centrifugation and analyzed by flow cytometry. The ratio of total immune cells (CD45+) and recruited monocytes (Ly6C+, CD11b+) was measured. The data are the % of viable cells (left figure) or the % of non-lymphocyte populations (right figure), and are the mean ± sem of n = 10 mice per group. * indicates P value> 0.05 (Student t test). B) C57 / BL6 mice were injected intraperitoneally (IP) or orally gavaged (OG) with 250mg DTAF-conjugated WGP for 3 days. Tissues were separated from designated organs, and immune cells were separated by Percoll centrifugation and analyzed by flow cytometry. Macrophages were identified as F4 / 80+, CD11b_, Ly6C_ and CD45+ cells. The percentage of this population that was positive for DTAF-WGP for 1 mouse per treatment is shown.

[0107] Fig.19 Wellmune WGP β-glucan restores defective innate immune responses in obese mice. C57 / BL6 mice were fed a high fat diet (containing 45% calories from palmitate, HFD) for 12 weeks or fed a standard fat diet (10% fat, SFD) or HFD supplemented with 0.05% Wellmune WGP (HFD-WGP). Bone marrow was isolated and BMDM was generated. Mature BMDM were stimulated with LPS (10 ng / mL, 4 h) and inflammatory cytokine levels (TNF (left) or IL10 (right)) in the supernatant were measured (A) or infected with Mycobacterium tuberculosis H37Ra strain (MOI 5 bacteria / macrophage) for 3 h, and then extracellular bacteria were removed. Intracellular Mtb was measured by colony formation assay between 3h-72h (B). Data are mean ± sem of n = 5 mice per group. * indicates P value > 0.05, ** indicates P value > 0.01, and *** indicates P value > 0.005 (one-way ANOVA analysis for multiple comparisons as indicated).

[0108] Fig. 20Depleted zymosan β-glucan particles drive in vitro trained immunity. A) HEK-Blue reporter cell lines and RAW-Blue macrophages overexpressing hDectin-1b, hTLR2 or hTLR4 were stimulated with zymosan (ZYM) or depleted zymosan (ZYM-d, 10 μg / mL) for 6 h, and NFκB-linked SEAP activity was measured. Data are the mean ± sd of n = 3 independent experiments and are expressed relative to unstimulated cells. B) Mature mouse BMDM were trained with the specified β-glucan preparations (100 μg / mL) for 24 h and left to recover for 6 days. BMDM were restimulated with LPS (10 ng / mL) and TNF production was measured 6 h later. Data are the mean ± sd of n = 3 independent experiments and are expressed in ng / mL. CD) Human monocyte-derived macrophages or mouse BMDMs trained with depleted zymosan (ZYM-d) were restimulated with LPS (10 ng / mL) and TNF production was measured between 0-24 h. Data are mean ± sd of n = 3 independent experiments and are expressed in ng / mL. *P < 0.05 for the indicated comparisons from one-way ANOVA analysis followed by post hoc multiple comparison tests.

[0109] Fig.21 Schematic overview of the flow cytometry gating strategy to quantify LKS+ HSPCs and more committed hematopoietic progenitors in mouse bone marrow. DETAILED DESCRIPTION OF THE INVENTION

[0111] The present invention provides an effective amount of whole Saccharomyces cerevisiae (1→3)-β-D-glucan particles for use in a method for programming bone marrow hematopoietic stem and progenitor cells (HSPC) in a subject. The HSPC in the subject provides multipotent progenitor cell (MPP) expansion to facilitate myelopoiesis, i.e., the generation of innate immune cells, particularly monocytes, after exposure. Mature cells derived from the expanded population have altered functions.

[0112] Therefore, the subject produces more MPP3, the myeloid precursor. Typically, the ratio of MPP4 to MPP3 in a subject is about 60:40 ( Fig.12 G). However, administration of the glucan of the present invention results in a larger MPP3 population provided by the subject's HSPC and biases the ratio toward an increase in MPP3. The ratio may be 50:50 or 40:60 or 30:70. It should be understood that this may depend on the condition, dosage and / or subject.

[0113] Without being bound by theory, it is believed that β-glucan triggers its effects by engulfing the C-type lectin receptor Dectin-1 (encoded by Clec7a) (Brown, GD et al. Dectin-1 mediates the biological effects of beta-glucans. J Exp Med 197, (2003)) to drive metabolic and epigenetic changes in innate immune cells, particularly monocytes, which promote enhanced macrophage responses to restimulation, i.e., trained immunity.

[0114] Training of innate immune cells by epigenetic modification results in enhanced induction of innate immune genes, leading to quantitatively greater and earlier cytokine responses, i.e., a general strengthening of immune signaling. It provides enhanced induction of chemokines by monocyte-derived macrophages. It provides enhanced induction of proinflammatory cytokines (TNF). It provides enhanced anti-inflammatory immunomodulation (IL-10).

[0115] The inventors have shown that whole fungal particles are recognized by Dectin-1 and internalized by phagocytic synapses, thereby inducing an optimal antimicrobial response. Dectin-1 has signaling functions and has been shown to activate NFκB and pro-inflammatory gene expression through SYK / CARD10. Different β-glucans can bind Dectin-1 differently depending on the mode of presentation. Although soluble low molecular weight (MW) β-glucans can bind Dectin-1 and drive NFκB, recognition of larger β-glucan chains (such as the β-glucans of the present invention) present on intact fungal particles is necessary to drive surface Dectin-1 receptor localization and form phagocytic synapses associated with antimicrobial actives such as ROS.

[0116] β-glucan is a commonly used and tolerated food ingredient and is therefore particularly suitable as a food supplement. In use, when a subject consumes the β-glucan of the present invention, for example as a food supplement or food, the subject's HSPCs are biased towards the production of MPP3 and innate immune cells with immune memory, thereby enhancing the subject's response to pathogen infection.

[0117] Interestingly, a key feature of trained innate immune cells is that their recall capacity is independent of the nature of the training stimulus, i.e., they can respond efficiently to restimulation with a wide variety of nonspecific ligands.

[0118] The β-glucan of the present invention may be formulated as a composition comprising the β-glucan of the present invention. In one embodiment, the β-glucan of the present invention is dispersed.

[0119] The β-glucan of the present invention may be a dietary or food supplement. The supplement may be selected from the group consisting of beverages (including "shots" or small drink portions), bakery products, dairy products, snack products, powdered products, milk powder, confectionery products, yogurt, breakfast cereals, bread products, nutritional supplements, sports nutrition supplements. The supplement may be a powdered supplement incorporated into a food or beverage product.

[0120] The β-glucan of the present invention can be formulated in the form of capsules or tablets.

[0121] It is noteworthy that the β-glucan of the present invention is a Saccharomyces cerevisiae whole glucan particle (WGP). In one example, the whole glucan particle is a β-glucan sphere further purified from yeast (Saccharomyces cerevisiae) cells based on alkaline solubility and substantially retains the in vivo glucan morphology / three-dimensional structure. WGP is insoluble. Methods for preparing WGP are known in the art. Exemplary methods are disclosed herein. In the publication and the following study, Li B et al. Yeastglucan particles activate murine resident macrophages to secreteproinflammatory cytokines via MyD88-and Syk kinase-dependent pathways. Clin Immunol. 2007 August; 124(2): 170-81. WGP differs from other β-glucan preparations in terms of β-glucan purity, but importantly, WGP maintains the intact yeast cell wall shape in the form of a non-viable ghost. The WGP of the present invention is commercially available for research purposes and is known to activate conventional innate immune signaling pathways ( https: / / www.invivogen.com / wgp-dispersible ; Goodridge HS. et al., 2011. Activation of the innate immune receptor Dectin-1upon formation of a'phagocytic synapse'. Nature. 472(7344):471-5.), but has not yet been described to activate trained immunity, especially when administered orally.

[0122] In one embodiment, the particles have a particle size, i.e., diameter, of about 1 micron to about 5 microns (or μm), about 2 microns to about 4.5 microns, about 2.5 microns to about 4 microns, or about 3 microns to about 3.5 microns. This can be an average diameter. At least 80% to 99%, or >85% or 90% of the particles may have a diameter within this range. Particle size is measured using a laser differential particle size analyzer, and such methods are known in the art.

[0123] The glucan of the present invention may be a preparation comprising more than one β-glucan. The WGPs in the preparation have an average size or a size distribution wherein the average is between 1 micron and 6 microns, preferably between 2 microns and 4 microns.

[0124] The composition of the invention may have more than one intact β-glucan particle derived from different Saccharomyces cerevisiae strains or preparations.

[0125] Glucans are derived from or isolated from the cell walls of baker's yeast (Saccharomyces cerevisiae).

[0126] It is noteworthy that the glucan of the present invention is

[0127] It is a dietary food, beverage and supplement ingredient. It is an insoluble large whole glucan particle (WGP) derived from the cell wall of baker's yeast (Saccharomyces cerevisiae), in which the β-1,3 / 1,6-glucan consists of a backbone of glucose molecules connected by unique chemical bonds β1,3 chains, which are attached to glucose chains via β1,6 bonds. The dextran may be as disclosed in US4810646, US4992540, US5037972, US5082936, US5028703, US5250436 and US5506124, each of which is incorporated herein by reference.

[0128] It should be understood that those skilled in the art will be able to determine the appropriate dosage for administering the glucan of the present invention or a supplement comprising the glucan of the present invention without undue experimentation. The amount and frequency are those that are best suited for the purpose. The frequency of application or administration can vary widely, depending on the needs of each subject, with the recommended range for application or administration being once a month to ten times a day, preferably once a week to four times a day, more preferably three times a week to three times a day, and even more preferably once or twice a day.

[0129] In one embodiment, the daily dose is about 250 mg (0.003% w / w of dietary intake). In one embodiment, the daily dose may be in the range of 250 mg (0.003% w / w of dietary intake) to 5 g / day (0.05% w / w of dietary intake). In one embodiment, the daily dose is 500 mg to 4 g per day, or 1 g to 3 g per day.

[0130] In one aspect of the invention, an effective amount of the β-D-glucan of the present invention is provided to prevent the subject's disease or infection, delay its onset or reduce its severity. It is noteworthy that the subject can be a subject susceptible to infection after certain events or at certain times. For example, the subject can be a high-level athlete with increased susceptibility to upper respiratory tract (URT) infections. Using the beta-D-glucan of the present invention as a supplement can reduce the incidence of URT within the time window of increased vulnerability (e.g., after a marathon). It is believed that their innate immune system has been trained to resist these infections more effectively.

[0131] In one embodiment, the β-glucans of the invention are co-administered or used together with one or more antigens to drive antigen-specific immune memory.

[0132] In one aspect, the present invention provides an effective amount of saccharomyces cerevisiae derived β-glucan of the present invention or for treating the purpose of post-infection immune disorder in a subject. The subject is a subject suffering from post-infection immune disorder, for example, a subject after COVID-19 (SARS-CoV-2 infection). This depletes adaptive immune memory cells and dysregulates innate immune cells. Driving trained immunity can restore regulated innate immune responses and provide protection against severe infections. Immune dysfunction associated with SARS-CoV-2 infection is discussed in Phetsouhanh et al. (Nature Immunology, Vol. 23, February 2022, 210-216).

[0133] Another aspect of the present invention provides a composition, such as a food supplement, comprising two or more types of β-glucans derived from different strains of Saccharomyces cerevisiae. In other words, the composition comprises more than one β-glucan type, each β-glucan type being derived from a different strain of Saccharomyces cerevisiae.

[0134] It will be appreciated that features of the embodiments of the invention may be combined in any combination.

[0135] The present invention will now be described with reference to specific embodiments. These are merely exemplary and are for illustrative purposes only: they are not intended to limit the scope of the claimed patent or the invention described in any way. These embodiments constitute the best mode currently contemplated for practicing the present invention. Example

[0136] Method Overview

[0137] The inventors tested the ability of the food-grade yeast WGP of the invention and a panel of different β-glucans to drive trained responses in human monocyte-derived macrophages, and a HEK-based reporter cell assay to study Dectin-1 signaling. The inventors also tested the ability of the WGP to promote myelopoiesis of the bone marrow, which is associated with the long-term memory effect of trained immunity.

[0138] like Figure 1-Figure 5 The first part of the presented study describes the in vitro innate immune training of human monocytes. Leukocytes were isolated from healthy anonymous donors and monocytes were isolated. These precursor cells were treated with training agents, such as Saccharomyces cerevisiae. (dWGP) or control treatment lasts for 24h, rest and allow it to mature into macrophages. After this rest period, mature macrophages are stimulated again with common innate immune activators (mainly Gram-negative component LPS), and cytokine production is measured using TNF ELISA to the supernatant from these cultures. Training is indicated by an enhanced response to LPS restimulation, which is measured by the TNF production increased in the cells trained by dWGP relative to untrained controls.

[0139] In the second part of the study, bone marrow was removed from all mice and grown into mature macrophages (BMDM) using L929 conditioned medium in the laboratory. At maturity (6 days after differentiation), BMDM were stimulated with different doses of microbial agonists, such as lipopolysaccharide (LPS) of Escherichia coli (E.coli) cells or heat-killed Mycobacterium tuberculosis (Mtb), which activated BMDM to produce proinflammatory cytokines, such as TNF. This serves as a readout of innate immune function enhanced by trained immunity.

[0140] The third part of the study ( Figure 15-Figure 21 ) to further support parts one and two, and show:

[0142] Delivery of yeast WGP by oral gavage resulted in enhanced responsiveness to activation of these bone marrow derived mature macrophages (BMDM).

[0143] Feeding a diet containing yeast WGP for 4 weeks resulted in the expansion of myeloid / innate immune committed progenitors, as well as an increase in more mature and common myeloid committed progenitors, consistent with an enhanced responsiveness to activation of these bone marrow-derived mature macrophages (BMDM). It also altered the intestinal innate immune cell composition and restored the defective innate response of obese mice.

[0144] Beta-glucan sold under the trade name Wellmune was used in this study.

[0145] This is important because the changes previously observed in mouse bone marrow after yeast WGP diet feeding involve low-abundance HSPC progenitor cell populations. This new data detected more downstream directed progenitor cells of myeloid lineages, and confirmed that the small changes previously observed in low-abundance HSPC progenitor cell populations became larger and more significant changes in more directed cells. When measuring proinflammatory cytokines TNF and IL6, mature cells from these new studies (4 weeks after WGP feeding) also showed enhanced responsiveness to restimulation, beyond the observations previously limited to TNF alone. The new data also presents a comparison with a fiber-matched diet and regular mouse food, as a control for any fiber effect in the Wellmune diet.

[0146] Materials and methods

[0147] Detailed method:

[0148] Cell Isolation and Culture Reagents

[0149] hDectin1B-HEK293 NFκB-SEAP reporter cells were obtained from Invivogen and cultured according to the manufacturer's instructions. Puromycin and HEK-Blue TM CLR Selection (Invivogen) was used as the selective antibiotic, and cells were cultured using DMEM (4.5 g / L glucose), 10% (v / v) fetal bovine serum (FBS), 100 U / ml penicillin, 100 μg / ml streptomycin, 100 μg / ml Normocin, and 2 mM L-glutamine. Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats obtained from Irish Blood Transfusion Services (Dublin, Ireland) using density gradient centrifugation with Lymphoprep (Stem Cell Technologies) and then lysed using Hybri-Max buffer. TM(Sigma-Aldrich) for red blood cell lysis. PBMCs were resuspended in differentiation medium (cRMPI, 10% FBS, 10 ng / mL M-CSF) and monocytes were enriched by adhesion to plastic and used for subsequent training assays or stimulation. Clinical indemnity approved the supply of human blood products from IBTS. Bone marrow derived macrophages (BMDM) were isolated by flushing the tibia and femur of both legs from C57BL / 6J male mice with DMEM (Gibco), which were generated and maintained at Comparative Medicine Unit, Trinity College Dublin (Dublin, Ireland). The obtained bone marrow cell suspension was filtered on a 40 μm nylon mesh cell retainer (Biolegend). After washing, the pellet was resuspended and lysed with red blood cell (RBC) lysis buffer Hybri-Max TM (Sigma-Aldrich) for 2 minutes to lyse red blood cells. After washing and counting, the cells were resuspended in DMEM, 10% FBS, 20% L929 conditioned medium and seeded to differentiate into BMDM within 1 week. Mature BMDM were scraped off and the cells were re-seeded in DMEM, 10% FBS, 5% L929 conditioned medium at the desired density and allowed to stand overnight before stimulation.

[0150] Beta-Glucan Preparations and Training Stimulants

[0151] Saccharomyces cerevisiae-derived whole glucan particles were provided by Dr. Sonja Nodland, Kerry Health & Nutrition, MN, USA. These include Whole glucan particles (WGP), in which cell wall β-glucan is preserved (producing non-aggregated ghost yeast cells), or soluble preparations derived from heat-treated WGP. For all experiments, WGP powder was resuspended in PBS and sonicated to de-agglomerate and obtain dispersible "single-cell" ghost particles or dispersible WGP (dWGP). Although the soluble preparation consisted of a heterogeneous mixture of soluble β-glucans of different molecular weights, we referred to this preparation as soluble WGP (sWGP) to emphasize that dWGP and sWGP consist of β-glucans from the same source. Dr. Nodland also provided β-glucan fractions of different molecular weights separated from sWGP by size exclusion chromatography followed by filtration (1.5-100 nm filtration step). The fractions are as follows: F1: <100 kDa, F2: 100-400 kDa, F3: 400-800 kDa and F4: >800 kDa. The sonication procedure for generating dWGP from WGP powder was as follows: WGP was weighed and dissolved in sterile endotoxin-free water (Invitrogen) to produce 10-15 mL of 25 mg / mL WGP. The solution was left overnight (8h-16h) at room temperature and then sonicated using a 150VT ultrasonic homogenizer with a 5 / 32" micro tip. The solution was sonicated for 5 minutes at 50% power and 50% time pulse rate while immersing the tip approximately 5 mm below the surface of the liquid. Due to the heat caused by sonication, the tube containing the solution was kept in ice. Following this sonication step, dWGP was precipitated by centrifugation (1,000G, 10 minutes at room temperature) and the water was removed by careful decantation and replaced with a certain volume of 0.2M NaOH aqueous solution to reach 25 mg / mL WGP. After 20 minutes, the dWGP was washed three times with sterile water using the same conditions as described for precipitation, decantation, and solvent replacement. Finally, two final washes were performed to replace the sterile water with sterile PBS and stored at 4°C for up to 6 months. As the dWGP settled out of solution, it was vortexed before each use. Unsonicated intact glucan particles (uWGP) were used in some experiments and were directly resuspended in PBS without sonication. Saccharomyces cerevisiae β-glucan-derived particles, crude yeast polysaccharide, and depleted yeast polysaccharide were obtained from Invivogen.

[0152] For some experiments, 3 μm aminated polystyrene particles (AM-PS; Magsphere) were conjugated to sWGP based on previously published methods (Tam JM et al., Use of fungal derived polysaccharide-conjugated particles to probe Dectin-1 responses in innate immunity. Integr Biol (Camb) 4, 220-227 (2011)). Using a centrifugal filter containing a 0.65 μm PVDF membrane (Ultrafree, Millipore), 2 mg of AM-PS was washed three times with anhydrous DMSO (Sigma) and then incubated with 250 μL of 2M 1,1′-carbonyldiimidazole (CDI; Sigma) freshly dissolved in anhydrous DMSO for 1 hour at room temperature while shaking. The particles were then washed twice with anhydrous DMSO to remove excess CDI (again using a centrifugal filter) and then incubated with 250 μL of 0.1 mg / mL sWGP dissolved in anhydrous DMSO for 1 hour at room temperature while shaking. After this conjugation step, sWGP was collected by centrifugation, a centrifugal filter was used to block particles, and the conjugation efficiency was evaluated by measuring the loss of sWGP using a filtered sWGP solution without AM-PS or CDI as a control. Based on the reported protocol (Masuko, T.Carbohydrate analysis by a phenol-sulfuric acid method in microplate format.Anal Biochem.339, 69-72 (2005)), sWGP was measured using the phenol-sulfuric acid method. On a 96-well plate, 130 μL of concentrated sulfuric acid (Sigma) was added to a 50 μL sWGP sample, followed by 30 μL of 5% w / v phenol (Sigma) in sterile water, and then pipetting up and down quickly to fully mix the solution. The plate was placed on a 90 ° C hot plate for 5 minutes and then cooled to room temperature with a water bath. The absorbance at 492 nm was measured, and the concentration of sWGP in each sample was determined using a standard curve of known amounts of sWGP to quantify how much sWGP had been conjugated to AM-PS.

[0153] Other β-glucans were obtained from Invivogen and included: β-glucan peptide (BGP), a high molecular weight polysaccharide extracted from the macrofungus Trametes versicolor; zymosan, a cell wall preparation derived from Saccharomyces cerevisiae; schizophyllan, a high molecular weight β-glucan derived from the fungus Schizophyllum commune; curdlan, a β-1,3 linked glucan derived from the bacterium Alcaligenes faecalis; and pyroglucan, a medium molecular weight linear β-1,6 linked glucan from the algae lichen Lasallia pustulata. All β-glucans were used in concentrations ranging from 1, 10, or 100 μg / mL. Heat-killed Candida albicans (HKCA) was also obtained from Invivogen and was concentrated at 1 × 10 4 cells / mL to 1×10 6 Use concentrations between 10 cells / mL.

[0154] Macrophage activation / re-stimulation

[0155] Ultrapure lipopolysaccharide (LPS) from E. coli O111:B4 was obtained from Invivogen and used at concentrations of 1 ng / mL, 10 ng / mL, and 100 ng / mL for tolerance induction in human monocytes or at 10 ng / mL or between 10 ng / mL and 100 ng / mL in most experiments for restimulation of trained macrophages. The synthetic triacylated lipopeptide TLR2 / TLR1 agonist Pam3CSK4 was obtained from Invivogen and used for restimulation at 100 μg / mL. 6 The trained macrophages were restimulated with HKCA at 100 cells / mL. The trained monocyte-derived macrophages were restimulated with non-surviving irradiated mycobacterium tuberculosis (iMtb) obtained from the American Type Culture Collection (ATCC) (Manassas, VA) and prepared according to the manufacturer's instructions and used at 500 μg / mL. The BMDM of mice trained in vivo were stimulated with heat-inactivated mycobacterium tuberculosis (hk-Mtb) from Invivogen and used at a concentration between 500 μg / mL and 1000 μg / mL. The trained monocyte-derived macrophages were also infected with live mycobacterium tuberculosis (Mtb) strain H37Ra obtained from ATCC and proliferated to logarithmic phase in Middlebrook 7H9 culture medium. On the day of infection, the logarithmic phase bacteria were precipitated by centrifugation and resuspended in DMEM. The bacterial precipitate was de-agglomerated several times by passing through a syringe with a 25G needle. The single cell suspension was isolated by centrifugation of the bacterial suspension at 800 rpm for 3 minutes. The supernatant of the centrifugation was analyzed by spectrophotometry (OD600nm ) were quantified and used to infect macrophages. Macrophages were infected with an MOI of 5 bacilli / cell for 3 h (as previously described by Hackett EE et al., Mycobacterium tuberculosis Limits Host Glycolysis and IL-1β by Restriction of PFK-M via MicroRNA-21. Cell Rep. 2020 Jan 7; 30(1): 124-136).

[0156] Further purification was performed by centrifugation at 13,000 rpm for 10 minutes to pellet extracellular bacteria.After washing in DMEM to remove extracellular bacteria, bacteria-free medium was returned to the macrophages and the cultures were grown for up to 72 h post infection.

[0157] Training assay and readout

[0158] For the human monocyte training assay, PBMCs isolated from human blood were seeded in 96-well plates (100,000 cells per well in 180 μL RMPI, 10% FBS, 50 ng / mL M-CSF) and 20 μL of training stimulants were immediately added. The cells were incubated overnight at 37°C. The culture medium was removed and the cells were washed twice very gently with 100 μL warm PBS to remove the training stimulants, and 200 μL of fresh culture medium was added. The cells were washed and given fresh culture medium every 2-3 days, and the cells were restimulated in fresh culture medium on the 5th day for the specified time. For the training inhibition assay, PBMC cells were incubated with the desired inhibitor for the specified time before adding the training stimulants. Unless otherwise specified, inhibitor concentrations were as follows: 1 mM 5'methylthioadenosine, excess sWGP (10 μg / mL-100 μg / mL), 10 μg / mL-100 μg / mL laminarin (Invivogen), 4 μM, 10 μM and 30 μM piceatannol, 5 μM BAY-11087, 2-deoxyglucose between 1 mM, 2.5 mM, 5 mM and 10 mM, rapamycin between 1 μM, 10 μM and 100 nM, wortmannin between 0.1 μM, 1 μM and 10 μM, cytochalasin D between 2 μM, 10 μM and 100 μM, and bafilomycin A1 between 1 μM, 5 μM and 10 μM. Unless otherwise specified, all inhibitors were from Sigma-Aldrich. TNF secretion from trained cells is measured by ELISA (human TNFELISA kit, Invitrogen) of supernatant as a readout of training. Alternatively, CXCL8 or IL-10 are produced by ELISA. For metabolic analysis of trained cells, the lactic acid concentration in the supernatant is measured using a colorimetric lactate assay kit (MAK064, Sigma-Aldrich). ELISA is also used to analyze the TNF production of the culture medium taken out from trained monocytes after 24h to assess the effects of training stimulants on monocyte activation or for measuring extracellular lactic acid production. In some experiments, RNA was isolated from trained monocytes using RNeasy kit (Qiagen).For gene expression analysis, cDNA was prepared with a high-capacity cDNA Archive kit (Applied Biosystems) according to the manufacturer's instructions, and individual mRNAs were monitored using the following human TaqMan assays (Applied Biosystems): 18s (Hs03003631_g1), hexokinase-2 (HK2, Hs00606086_m1), GLUT-1 transporter (SLC2A1, Hs00892681_m1), lactate dehydrogenase A (LdhA, Hs01378790_g1), and PKM-2 (Hs00987255_m1). The AB7900HT platform (Applied Biosystems) was used for all PCRs, which were performed in triplicate in rapid mode. 18S was used as an endogenous control, and changes in expression were calculated by the threshold change (ΔΔCT) method and normalized to the results obtained in untreated cells. For experiments with trained macrophages infected with Mycobacterium tuberculosis (Mtb), baseline growth was assessed by lysing the 3h time point in 0.1% Triton-X for 10 minutes. Serial dilutions were plated in triplicate on 7H10 Middlebrook agar, and colony forming units were counted after incubation at 37°C for 14-21 days. For later growth measurements, the lysate was combined with the extracellular bacteria of the precipitation obtained by centrifugation of the supernatant, and the change fold of bacterial colony forming units (CFU) was expressed relative to the baseline time point. For supernatant transfer experiments, supernatants from 24h human monocytes after training were harvested and applied to initial, untrained monocytes and monocytes trained with dWGP, matured for 5 days, and then re-stimulated with LPS to assess whether the soluble factors induced by the training stimulus confer enhanced responsiveness to re-stimulation.

[0159] The BMDM training assay was performed by stimulating BMDM with training stimuli 6 days after isolation and allowing them to recover and mature for another 6 days in DMEM, 10% FBS, 5%-7% L929 conditioned medium, changing the medium every 3 days, and then restimulating with LPS 12 days after isolation. TNF production in the supernatant was analyzed using the Invitrogen mouse TNF ELISA kit according to the manufacturer's instructions, or lactate production was measured as described above.

[0160] QuantiBlue Assay

[0161] hDectin1b-HEK293 NFκB-SEAP reporter cells (Invivogen) were cultured according to the manufacturer's instructions. The reporter cell assay was performed by inoculating 50,000 cells per well in 180 μL in a 96-well flat-bottom plate and incubating the cells with 20 μL of the specified agonist overnight. SEAP activity was measured using QUANTI-Blue (Invivogen) according to the manufacturer's instructions. In short, 20 μL of supernatant was added to 180 μL of Quanti-Blue solution and incubated at 37 ° C for 15 minutes. The optical density at 620 nm was then measured using a plate reader.

[0162] Animal work

[0163] For induction of trained immunity in vivo, C57BL / 6J-OlaHsd male mice were generated and maintained at the Comparative Medicine Unit, Trinity College Dublin (Dublin, Ireland). Mice were bred and maintained under specific pathogen-free conditions with free access to food and water. Mice aged 8-12 weeks were used. All experiments were performed with the approval of the Irish Health Products Regulatory Authority and the Trinity College Dublin Animal Research Ethics Committee. Trained immunity was induced in mice by a single intraperitoneal injection of 200 μL PBS as a control or 200 μL of 1 mg / ml or 2 mg / ml dWGP resuspended in PBS. To induce trained immunity in vivo by oral supplementation, mice were divided into four main experimental groups. The control group was fed a slightly modified standard diet (reference D11112201) containing 25 g / kg inulin (2.5% inulin / kg food) as a fiber source. The other three groups of mice were fed with a diet supplemented with 0.003% (0.03g / kg food), 0.025% (0.25g / kg food) or 0.05% (0.5g / kg food) dietary fiber dWGP, including a proportionally reduced amount of inulin to balance the amount of dWGP fiber incorporated into the supplemented diet. All groups of mice were allowed to adapt to a standard diet rich in inulin for two weeks, and then switched to a diet supplemented with dWGP for 1 to 3 weeks. At the end of the experiment, animals were euthanized by CO2 inhalation, and tissues were collected for analysis. Bone marrow cells were harvested by flushing the tibia and femur from both legs with DMEM (Gibco). Bone marrow progenitor cell staining was used for flow cytometry analysis as described below or for generating BMDM as described above. After erythrocyte lysis, washing and counting, 3 million cells were retained for flow cytometry analysis as described below, and the remaining cells were resuspended in DMEM, 10% FBS, 20% L929 conditioned medium and inoculated to differentiate into BMDM within 1 week. To isolate splenocytes, the spleen was carefully dissected after euthanasia, collected and stored in RPMI / FBS 0.1% medium on ice. The spleen was then minced into small pieces, gently crushed through a 70 μm nylon mesh cell retainer and diluted with PBS. After washing, the pellet was resuspended and lysed with red blood cell (RBC) lysis buffer Hybri-Max TM(Sigma-Aldrich) for 2 minutes. The spleen cells were then washed again, resuspended in DMEM, 10% FBS, 5% L929 conditioned medium and inoculated. The cells were stimulated with LPS (10ng / mL) or heat-killed Mycobacterium tuberculosis H37Ra (hk-Mtb, 500μg / mL-1000μg / mL). To isolate immune cells from the intestine and other tissues, mice were dissected after euthanasia. Tissue blocks were isolated after cleaning and removal of feces. Single cell suspensions were produced after collagenase digestion and centrifugation through a 40 / 80% Percoll gradient. The isolated immune cells were stained for analysis by flow cytometry, as outlined below. For experiments with labeled Wellmune WGP, fluorescent DTAF was conjugated to WGP, as described in Geller et al. Nat Commun. 2022 9; 13(1): 759. For obesity modeling experiments, a diet-induced obesity model was used. Male C57 / BL6 mice aged 8 weeks were fed a high fat diet containing 40% of calories from palmitate (research diet) or a control standard fat diet (10% fat), or a modified high fat diet supplemented with 0.05% w / w Wellmune. The mice were fed for 12 weeks, and the induction of obesity was confirmed by weekly monitoring of body weight and fat at sacrifice and by measuring metabolic regulation using a glucose tolerance test. Wellmune WGP supplementation did not alter the induction of obesity or metabolic dysregulation (data not shown).

[0164] Multiparameter Flow Cytometric Analysis of Human Monocytes

[0165] To analyze phospho-S6 ribosomal protein activity after β-glucan treatment, PBMCs were isolated as described above and cultured at 2 × 10 6 / mL was resuspended in RPMI-1640 medium supplemented with 10% human AB serum (Sigma-Aldrich). As described above, cells were first incubated with metabolic inhibitors for 15 minutes. Cells were then stimulated with LPS (10ng / mL, Invivogen), sWGP (10μg / mL), uWGP (10μg / mL) or dWGP (10μg / mL) for 30min, 1h, 2h, 6h or 24h. After the incubation time, the cells were resuspended by rapid vortexing, washed in flow buffer [PBS-1X (Gibco) supplemented with 5% heat-inactivated FBS (Gibco) and 0.1% sodium azide (Sigma)], and the following flow cytometry staining protocol was applied to all samples, with a maximum of 100,000 cells per sample. The cells were stained with the fixable viability stain ZombieAqua at a concentration of 1:500. TM(Biolegend) staining for 15 minutes. Subsequently, the sample was washed with flow buffer and incubated with anti-Dectin-1-PE (clone 15E2, Biolegend) antibody for Dectin-1 surface staining, then washed and fixed with IC fixation buffer (Invitrogen) for 20 minutes. Alternatively, the cells were incubated at 4 ° C for 30 minutes with anti-CD14-APC (clone M5E2, Biolegend), anti-CD16-PE-Cy7 (clone 3G8, Biolegend), and anti-HLA-DR-BB515 (clone G46-6, BD Bioscience) at a concentration of 1:100 in flow buffer. All cells were subsequently washed with flow buffer and resuspended with IC fixation buffer (Invitrogen) for 20 minutes for fixation. The cells were then washed with flow buffer, incubated with 1:200 anti-phospho-S6-PE (Ser235 / 236) (clone D57.2.2E, Cell Signaling) in Perm / Wash buffer (BD Biosciences) at 4°C for 30 min for intracellular pS6 staining, and finally washed again with flow buffer. TM Compensation controls were obtained after staining with compensation beads (Invitrogen). Cells were collected on a BD flow cytometer Canto II with FACSDiva software. Data analysis and flow cytometry plots were performed using FlowJo software v.7.6 (TreeStar).

[0166] To sort human monocyte subsets, PBMCs were isolated from peripheral blood buffy coats as described above. After separation, PBMCs were resuspended in flow buffer and the following flow cytometry staining protocol was applied to all samples, up to 30,000,000 cells per donor. The cells were stained with a 1:500 concentration of the viability stain propidium iodide (Biolegend) for 15 minutes. Subsequently, the samples were washed with flow buffer and incubated with anti-CD14-APC (clone M5E2, Biolegend), anti-CD16-PE-Cy7 (clone 3G8, Biolegend), anti-HLA-DR-BB515 (clone G46-6, BD Bioscience), and anti-CCR2-APC-Cy7 (Biolegend) at a concentration of 1:100 in flow buffer at 4°C for 30 minutes. All cells were subsequently washed and resuspended in flow buffer. UltraComp eBeads were stained with appropriate antibodies. TMCompensation controls were obtained after staining with compensation beads (Invitrogen). Cells were collected and sorted on a BD FACSAria Fusion cell sorter with FACSDiva software. Data analysis and flow cytometry were performed using FlowJo software v.7.6 (TreeStar). After sorting, monocyte subsets were plated in 96-well plates at 1 × 10 6 / mL was inoculated in RPMI-1640 medium supplemented with 10% human AB serum (Sigma-Aldrich), left to rest for 24 h, and then stimulated and trained according to the above-mentioned protocol.

[0167] Multiparameter Flow Cytometric Analysis of Mouse Bone Marrow Cells

[0168] To analyze the HSPC population in the bone marrow of trained immunized mice after in vivo induction, isolated bone marrow cells were resuspended in flow buffer [PBS-1X (Gibco) supplemented with 5% heat-inactivated FBS (Gibco) and 0.1% sodium azide (Sigma)] and the following flow cytometry staining protocol was applied to all bone marrow lines with a maximum of 3,000,000 cells per sample. Cells were fixed with the fixable viability stain ZombieAqua at a concentration of 1:500. TM(Biolegend) for 15 minutes. Subsequently, the samples were washed with flow buffer and incubated with anti-CD16 / 32 (Biolegend) at a concentration of 1:100 in flow buffer at 4°C for 20 minutes. The following antibodies were then used for staining Lin-c-Kit+Sca-1+ cells (LKS), hematopoietic stem cells (HSC), and multipotent progenitor cells (MPP): anti-Ter-119, anti-CD11b (clone M1 / 70), anti-CD5 (clone 53-7.3), anti-CD4 (clone RM4-5), anti-CD8a (clone 53-6.7), anti-CD45R+ (clone RA3-6B3), anti-Ly6G / C+ (clone RB6-8C5), all biotin conjugates (all Biolegend) were added at a concentration of 1:50 for 30 minutes at 4°C. The cells were then washed with flow buffer. Streptavidin-APC-Cy7 (Biolegend), anti-c-Kit-APC (clone 2B8, Biolegend), anti-Sca-1-PE-Cy7 (clone D7, eBioscience), anti-CD150-eFluor450 (clone mShad150, eBioscience), anti-CD48-PerCP-eFluor710 (clone HM48-1, BD Bioscience), anti-CD34-FITC (clone RAM34, eBioscience), anti-Flt3-PE (clone A2F10.1, Biolegend) were added and incubated at 4°C for 30 minutes. Figure 15-17 , LKS+ HSPCs were enriched using Milteny cKit+ beads on a MACS column. Fig.17 cKIT negative cells were used to measure mature myeloid progenitors (MP, CMP, GMP and CLP). LKS+ cells were stained as outlined above. Fluorescence Minus One (FMO) controls were performed using 1,000,000 cells obtained by mixing equal volumes of samples from different experimental conditions and stained with appropriate antibodies. Fig.21 An exemplary gating is shown in FIG. 1 . All cells were then washed with flow buffer and resuspended in IC fixation buffer (Invitrogen). TM Compensation controls were obtained after staining with compensation beads (Invitrogen). Cells were collected on a BD flow cytometer Canto II with FACSDiva software. Data analysis and flow cytometry plots were performed using FlowJo software v.7.6 (TreeStar).

[0169] Experimental design, statistical analysis, and presentation

[0170] The data shown represent the average data of the experiments carried out on human monocytes / macrophages, which are derived from the independent donor quantity specified, the number of animals specified for each group of in vitro studies, or the independent BMDM preparations for in vitro studies of mice. Data were analyzed by Graph Pad Prism or Excel, and graphs were annotated in Adobe Illustrator to generate figures. Multiparameter experiments were analyzed by ANOVA and post hoc tests to indicate the significant differences between the treatment groups or treatment conditions, and these were indicated in the figures.

[0171] Results and Conclusions

[0172] In a preliminary screen, the inventors first tested the ability of various commonly available β-glucans to drive canonical Dectin-1 / NFκB signaling using HEK-Dectin-1b overexpressing reporter cells. Well-described Dectin-1 activators, including Saccharomyces cerevisiae zymosan (a cell wall β-glucan preparation) and Trametes versicolor-derived β-glucan peptide (BGP), drove NFκB signaling in a dose-dependent manner, as did similar concentrations of fungal-derived β-glucans (Schizopylan), bacterial-derived β-glucans (Curdlan), and lichen-derived β-glucans (Auricularia auricularia) ( Figure 1 A) Dispersibility A granular form of Saccharomyces cerevisiae β-glucan (in which the β-glucan layer of the yeast cell wall is isolated and intact to form "ghost cells"), referred to herein as dispersible whole glucan particles (dWGP), also drives NFκB activation via Dectin-1b. However, a derivative soluble form of the same Saccharomyces cerevisiae β-glucan (referred to herein as soluble WGP (sWGP), prepared by thermal degradation of dWGP, resulting in a heterogeneous mixture of β-glucan chains of varying molecular weights, and lacking the intact single-cell structure of dWGP) failed to drive NFκB activation via Dectin-1b.

[0173] Then, freshly isolated human monocytes were exposed to the same β-glucan concentration for 24 h to test their ability to induce monocyte training. After washing and maturing into human monocyte-derived macrophages (hMDM) for 5 days, the cells were re-stimulated with the TLR4 ligand lipopolysaccharide (LPS), and extracellular TNF production was measured as a readout of reactivation (Dominguez-Andres, J. et al., In vitro induction of trained immunity in adherent human monocytes. STAR Protoc 2, (2021)). Both BGP and LPS were used as positive and negative controls for macrophage re-stimulation, respectively ( Figure 1 B, far left and far right), dose-dependent augmented and impaired TNF responses indicative of both training and tolerance were observed. A trend toward some training was observed with higher concentrations of other β-glucans including curdlan, while both zymosan and heat-killed Candida albicans (HKCA) drove enhanced restimulation responses at the lower concentrations tested. Significant training was observed in monocytes treated with higher concentrations of dWGP (10 μg / mL-100 μg / mL), while the soluble form of this β-glucan (sWGP) did not enhance the response relative to control-treated cells. Interestingly, the ability of the various tested β-glucans to drive training appeared to be independent of their ability to drive canonical Dectin-1b NFκB signaling ( Figure 1 A and Figure 2 A) Therefore, the inventors decided to use the soluble synthetic peptide BGP with large molecular weight and the ghost yeast cell pellet dWGP and its soluble form sWGP in future studies to characterize the requirements of training.

[0174] The ability of various β-glucan preparations to promote trained responses was independent of their ability to drive proinflammatory signaling during the training phase, as demonstrated by the Dectin-1b reporter studies. BGP treatment during the monocyte phase (24 h post-training) drove little TNF production, with more modest levels observed in dWGP-treated monocytes ( Figure 1 C, right axis), although both promote similar macrophage training ( Figure 1 C left axis, and Figure 2 B) LPS at the concentrations used to drive a tolerance response in macrophages stimulates the production of large amounts of TNF in the monocytic stage ( Figure 2 C). On the other hand, BGP treatment at the monocyte stage increased the viability of the resulting macrophages (5 days after treatment, Figure 2 D), while dWGP, sWGP or LPS treatment had no effect on this.

[0175] Thus, the inventors demonstrated that dWGP β-glucan drives intrinsic properties within trained cells and found that by transferring supernatant from monocytes trained with dWGP for 24 h to untrained monocytes, they were unable to drive an enhanced restimulation response to LPS when mature ( Figure 2 E), supporting that the training is not mediated by soluble factors. However, inhibition of intracellular DNA methyltransferases by pre-treating monocytes with 5' methylthioadenosine (MTA) prior to β-glucan training was able to block the enhanced responsiveness of β-glucan-trained macrophages to both BGP and dWGP ( Figure 1 D) This confirms that these different β-glucans are training monocytes to enhance responsiveness to restimulation as a result of epigenetic modifications to cell fate.

[0176] Training of innate immune cells through epigenetic modifications resulted in enhanced induction of innate immune genes, leading to quantitatively greater and earlier cytokine responses. Relative to untrained macrophages, dWGP-induced training of monocytes resulted in increased TNF production 3 h after restimulation and significantly increased TNF production 6 h after restimulation, whereas LPS-tolerant macrophages showed impaired TNF response to restimulation ( Figure 3 A). Similar results were observed with BGP-trained macrophages ( Figure 4 A). While training has been shown to drive the production of pro-inflammatory cytokines, β-glucan-induced training resulted in a general enhancement of immune signaling, with both dWGP and BGP training enhancing macrophage induction of chemokines (CXCL8), pro-inflammatory cytokines (TNF), and anti-inflammatory immunomodulatory cytokines (IL-10) relative to controls ( Figure 3 B, Figure 4 B). This suggests that trained cells respond faster and more efficiently, yet still maintain regulation of their activation.

[0177] A key feature of trained innate immune cells is that their recall capacity is independent of the nature of the training stimulus, as they can respond efficiently to restimulation with different nonspecific ligands. The inventors confirmed this in dWGP-trained macrophages, observing increased TNF production in macrophages restimulated with LPS as described previously, but also in macrophages restimulated with the TLR2 bacterial lipopeptide ligand PAM3CSK4 or when stimulated with the heat-killed fungus Candida albicans (HKCA, Figure 3 C) was observed to increase TNF production. Similarly, challenge of trained macrophages with irradiated bacteria Mycobacterium tuberculosis H37Rv (iMtb) resulted in increased production of a range of cytokines (TNF, IL-1β and IL-10) 24 h after treatment, whereas tolerizing with LPS inhibited these responses ( Figure 3 D). The concept of enhancing early innate immune responses without triggering unregulated, long-term inflammation is attractive for strengthening responses to infections, where impairment of early containment can lead to disease progression, such as COVID-19 and tuberculosis (TB). Therefore, the inventors tested the ability of dWGP-trained macrophages to contain intracellular infection. The inventors found that after infection with Mtb H37Ra (72h), dWGP-trained macrophages had superior containment ( Figure 3 E). Interestingly, although Mtb-infected dWGP-trained macrophages produced more TNF shortly after infection ( Figure 3 F, 3 h), but TNF levels normalized at a later time point (72 h) when mycobacterial growth was suppressed, supporting the notion that dWGP-trained macrophages possess superior innate antimicrobial capabilities.

[0178] After characterizing the enhanced response to restimulation in macrophages trained with dWGP, the inventors sought to determine how such β-glucan particles, such as dWGP, drive training processes in monocytes. Because both BGP and dWGP drive comparable levels of Dectin-1-mediated NFκB activation, the inventors examined whether Dectin-1 is required for training downstream of these different β-glucans. It has been previously reported that soluble β-glucans can block activation of Dectin-1 signaling by larger β-glucan particles by occupying sites on the receptor and preventing binding and subsequent signaling. Therefore, the inventors employed soluble β-glucan particles to The researchers investigated the effects of BGP (referred to here as sWGP)—a heterogeneous mixture of high-MW soluble β-glucans and low-MW soluble β-glucans released from its granular structure—and laminarin, a soluble low-MW β-glucan derived from macroalgae. Both compounds blocked Dectin-1b-mediated NFκB activation in reporter cells driven by both BGP and dWGP, with sWGP being slightly more effective in both cases ( Figure 5 A). Pretreatment of monocytes with sWGP prior to training was able to block the enhanced restimulation effect observed with various concentrations of dWGP ( Figure 6 A), and similar results were obtained when laminarin and sWGP were compared side by side in both BGP-trained cells and dWGP-trained cells ( Figure 5 B), where laminarin was more effective at all doses tested.

[0179] Therefore, blocking Dectin-1 receptor occupancy can prevent monocyte training driven by larger molecular weight β-glucan. Typical Dectin-1 signaling uses the adapter protein SYK, and although there are SYK-independent pathways, β-glucan-driven NFκB activation is SYK-dependent. The inventors blocked NFκB activation driven by dWGP and BGP by pretreatment with increasing concentrations of the SYK-kinase inhibitor piceatannol (PIC), which was confirmed in reporter cells ( Figure 5 C). Similarly, PIC pretreatment inhibited monocyte training driven by both BGP and dWGP, as measured by LPS restimulation response ( Figure 6 B), although it was more potent than the observed inhibition of NFκB activation at higher concentrations. Similar results were observed when NFκB activation downstream of dWGP was blocked using the IKKb inhibitor BAY11-7082, which abolished LPS restimulation-driven TNF production in trained macrophages ( Figure 6 C). These data, together with previous observations that while a range of β-glucans activate NFκB, not all β-glucans drive monocyte training, suggest a model in which early NFκB activation is necessary but not sufficient to reprogram myeloid responses to restimulation. Indeed, blocking early NFκB activation enhanced early TNF production despite attenuating training in dWGP-treated cells ( Figure 5 D), indicating a bifurcation of the β-glucan / Dectin-1 response associated with training. Therefore, the inventors sought to identify additional cellular pathways and processes affected by the β-glucan driver of training.

[0180] Candida albicans derived β-glucans have been shown to drive significant metabolic reprogramming in trained monocytes, which is associated with epigenetic modifications required for enhanced macrophage responsiveness. In particular, upregulation of cytosolic glycolysis driven by HIF1α emerges as a key signal activated by the Dectin-1 / PI3K / mTOR pathway. The inventors measured the ability of a series of β-glucans to drive glycolysis in trained monocytes by measuring changes in extracellular lactate production over time. The results showed that dWGP was a strong inducer of this process, which was maintained as monocytes differentiated over time (up to 6 days after treatment), while similar soluble β-glucan preparations (BGP, sWGP) were not ( Figure 7 A and Figure 8 A). Blocking the switch to glycolysis by targeting the gate-keeper enzyme hexokinase with 2DG abolished the ability of dWGP to increase the response to LPS restimulation ( Figure 7B). The inventors found that monocytes treated with dWGP drove the phosphorylation of the mTOR substrate S6 in a rapamycin-dependent manner ( Figure 7 C- Figure 7 E, gating strategy such as Figure 8 B), indicating mTOR activation. Although LPS also drives this phenotype, treatment with soluble sWGP does not. This upregulated mTOR activity is particularly evident early after treatment (2 h, Figure 8 C), but when untreated monocytes upregulated mTOR as part of the normal differentiation process, this was maintained until 24 h after treatment ( Figure 7 F). This early mTOR activity promotes β-glucan training, as pretreatment of dWGP-trained macrophages with rapamycin to block mTOR activity limited the trained response to LPS restimulation ( Figure 7 G). mTOR activation downstream of Dectin-1 supports glycolytic reprogramming in trained monocytes by enhancing the expression of rate-limiting glycolytic genes such as SLCA1, HK2, PKM2, and LDHA, which we observed in β-glucan-trained cells 3 days after training ( Figure 7 H). Previous data on C. albicans β-glucan showed that this glycolytic pathway is driven by PI3K signaling, and indeed, the addition of wortmannin during the training phase abolished the WGP-driven training response ( Figure 7 I). Interestingly, β-glucan-driven Dectin-1 signaling leading to SYK activation is not required for mTOR activation, as pretreatment of dWGP-trained monocytes with PIC did not affect mTOR upregulation ( Figure 7 J), despite its ability to block conventional pro-inflammatory signaling and monocyte TNF production ( Figure 8 D) This data suggests differences in conventional Dectin-1 signaling and suggests that intrinsic properties of particulate β-glucan may support the increased metabolic response required for training.

[0181] Previous comparisons of both soluble and granular yeast β-glucan indicated that the granular structure drives antimicrobial responses, including phagocytosis via the involvement of Dectin-1 clusters at the cell surface. By measuring cell surface Dectin-1 expression by flow cytometry, the inventors found that dWGP resulted in a loss of Dectin-1 surface expression shortly after treatment (15 min), a trend not observed with soluble BGP β-glucan ( Fig. 9 A. Fig.10 A). This loss of Dectin-1 surface expression induced by dWGP was dependent on phagocytosis, as pretreatment with the actin polymerization inhibitor cytochalasin-D (cytD) attenuated this effect ( Fig. 9 B). Similarly, cytD pretreatment prior to dWGP training, while affecting the responsiveness of untrained cells to LPS, preferentially blocked the training effect observed after LPS restimulation in dWGP-treated macrophages ( Fig. 9 C). CytD pretreatment also blocked dWGP-driven mTOR activation in monocytes ( Fig. 9 D). These data support a role for phagocytosis and internalization of the dWGP-Dectin-1 receptor complex in triggering mTOR and subsequent metabolic responses. β-glucan phagocytosis triggered by internalization of bound Dectin-1 clusters leads to phagosome-lysosome fusion. Lysosomes are also reported sites of mTOR activation in many other contexts, so the inventors hypothesized that β-glucan phagocytosis (preferentially driven by particles) delivers Dectin-1 complexes to lysosomes to engage in the signaling mechanisms necessary for activation of mTOR. Therefore, the inventors blocked lysosomal maturation using bafilomycin A1 (which blocks the lysosomal membrane VATPase pump required for acidification) and observed a significant reduction in monocyte mTOR activation by dWGP similar to experiments using cytD pretreatment ( Fig. 9 E).

[0182] Because soluble β-glucan does not trigger mTOR activation, lactate production, or training in the same way, the inventors tested whether other forms of yeast β-glucan particles could drive metabolic signaling and training. Dispersible Preparation of yeast ghost cells results in crude particle collections as clusters. These are disrupted by sonication to produce single dispersible particles, which are referred to as dWGP (eg. Fig.10 C). Dispersibility without ultrasonic treatment Preparation (uWGP) can drive NFκB activation in Dectin-1b reporter cells ( Fig.10 D), and triggered significant early monocyte pro-inflammatory signaling, as measured by TNF production 24 h after treatment, to a similar extent as observed with dWGP ( Fig. 9 F). However, uWGP treatment failed to drive monocyte training as observed with dWGP, and in fact, dWGP training drove characteristically enhanced TNF production in response to LPS restimulation, whereas uWGP-treated macrophages showed a diminished TNF response in response to restimulation ( Fig. 9 G). Similarly, uWGP treatment of monocytes did not result in a loss of cell surface Dectin-1 expression comparable to dWGP ( Fig. 9H), indicating that these crude β-glucan clusters are too large to drive efficient phagocytosis. As a result, uWGP-treated monocytes did not upregulate extracellular lactate production ( Fig. 9 I), confirming the importance of Dectin-1 / β-glucan complex internalization and phagocytosis in driving training-associated metabolic reprogramming.

[0183] The insolubility of intact β-glucan particles is determined by the glucose chain length and linkages. Dispersibility The particles (dWGP) are heterogeneous complexes with multiple cross-linked chain lengths of varying molecular weight. The data suggest that the size and preservation of β-glucan particles are key properties that control recognition, uptake, internalization, and participation in intracellular metabolic mechanisms. Therefore, the inventors sought to determine the optimal chain length required for training. Although soluble (sWGP) is not able to drive training, but it represents a chemically identical form of dWGP β-glucan in which the β-glucan chains have been released from yeast ghost cells to allow solubilization. It represents a heterogeneous mixture of glucan chains and MW in a soluble non-granular form. Therefore, the inventors isolated specific MW fractions of sWGP preparations such that each fraction represented pure β-glucans of different chain lengths defined by their MW, and compared these to dWGP and sWGP preparations in signaling studies. Although some of these fractions could drive NFκB activation in Dectin-1 reporter cells, particularly the lower MW fractions (F1, >100kDa), none of these drove training to the same extent as dWGP ( Fig. 9 J). Notably, these soluble yeast-derived β-glucans of defined MW enhanced the LPS restimulation response when compared to sWGP preparations ( Fig. 9 K ), and taken together with the finding of increased signaling capacity in Dectin-1b reporter cells, suggests that overall solubility, determined by the extraction method rather than chain length and MW of β-glucan, determines optimal training by Dectin-1.

[0184] Because soluble forms of yeast-derived β-glucan can drive NFκB activation but not training, unlike dWGP particles, we hypothesized that particle recognition is a critical step in training monocytes, ensuring proper activation of metabolic reprogramming. To test this, we injected soluble Yeast β-glucan (sWGP) conjugated to 3 μm aminated polystyrene particles (AM-PS) as described in Supplementary Fig.10E is shown by the loss of sWGP in the conjugated formulation. Human monocytes were then stimulated with the resulting particles or with controls: equal amounts of sWGP, AM-PS alone, or dWGP. Comparison of the increase in extracellular lactate production after 3 days, indicating upregulation of glycolysis, revealed that neither sWGP alone nor AM-PS particles alone altered cellular metabolism ( Fig.10 However, sWGP conjugated with AM-PS gained the ability to drive lactate production, albeit to a lesser extent than pure dWGP particles ( Fig. 9 L). These data suggest that while β-glucan is required for activation of conventional Dectin-1 signaling, recognition of β-glucan present on microbial-sized particles is required to trigger metabolic reprogramming and commit cells to a trained phenotype.

[0185] Many training papers have used in vitro stimulation of monocytes and therefore, having established the relevance of the β-glucan / Dectin-1 pathway in humans where various monocyte subsets exist, the inventors examined the expression of Dectin-1 in these cell populations. Compared to CCR2, a marker for inflammatory migratory monocytes ( Fig.11 A), Dectin-1 has low total expression but is present on classical inflammatory and intermediate macrophages, gated for CD14 / CD16 expression. It is also present on non-inflammatory CD14- / CD16+ monocytes, albeit at a lower level ( Fig.12 A). Although all three subpopulations responded to the initial treatment with WGP as measured by TNF production 24h after stimulation ( Fig.11 B), but only classical and intermediate monocytes showed an enhanced LPS response upon restimulation, indicating training ( Fig.12 B) Since the presence or absence of Dectin-1 on monocytes was not predictive of training, the inventors considered other cellular substrates for in vivo training.

[0186] HSPCs exhibited sensitivity to training stimuli delivered systemically by various methods, including intravenous delivery of BCG, IP injection of Candida albicans β-glucan, or hypercholesterolemia-induced NLRP3-inflammation driven by feeding a Western diet. Specifically, the resulting inflammation led to an increase in the total number of bone marrow HSPCs, as well as an increase in the ratio of multipotent progenitor cells (MPPs) skewed toward myeloid-committed MPP3 and away from the more predominant lymphoid-lineage MPP4 cells.

[0187] The inventors determined whether mouse bone marrow-derived macrophages are amenable to training: training with dWGP and similarly with BGP resulted in an enhanced LPS restimulation response in BMDM that had been trained 5 days prior to restimulation ( Fig.12 C). This was associated with enhanced extracellular lactate production by these cells ( Fig.11C), supporting the model that WGP reprograms mature mouse macrophages through in vitro metabolic reprogramming. The inventors determined whether similar long-term immune training could be observed in vivo by examining HSPC progenitors in mouse bone marrow. Importantly, mouse HSPCs express low levels of Clec7a mRNA, so changes in the bone marrow compartment after systemic delivery of training agents via IP delivery were examined.

[0188] The inventors found that IP injection of WGP resulted in total bone marrow c-Lin expression 1 week after administration. - ,ckit + ,Sca-1 + The increase of LKS+ HSPC cells ( Fig.12 D), in a manner similar to that observed previously with BGP injections, and this expansion was significantly stronger at lower amounts of dWGPβ-glucan. The absolute numbers of LT-HSPCs and ST-HSPCs were significantly expanded (representative flow cytometry plots and quantification are shown in Fig.11 D), demonstrating increased metabolic and proliferative activity required to provide HSPC turnover, as well as significant expansion in more lineage-directed MPPs ( Fig.12 E- Fig.12 F), although the overall frequency of each subpopulation did not change significantly. However, MPP cells were observed (gating and quantification are shown together in Fig.11 E) (which is the more abundant subpopulation), the inventors observed a significant expansion of myeloid-committed MPP3 cells in mice injected with both concentrations of dWGP used, while lymphoid progenitor MPP4 was reduced, as observed with equivalently high concentrations of BGP ( Fig.12 G). The abundance of megakaryocyte-related progenitor cells MPP2 was not significantly altered in the treatment. In addition to increasing myelopoiesis, training stimuli were shown to reprogram HSPCs to enhance activity when mature, which is consistent with what was observed with peripheral monocyte training. Spleens were isolated shortly after sacrifice to generate splenic macrophages, and these splenic macrophages were stimulated with the TLR4 ligand LPS or various concentrations of heat-inactivated Mtb (hk-Mtb). Splenic macrophages from animals trained in vivo with BGP or lower concentrations of dWGP showed significantly enhanced TNF production ( Fig.12 H). These data demonstrate that IP delivery of particulate β-glucan can lead to reprogramming of mouse bone marrow, quantitatively increasing myeloid cell turnover and qualitatively altering their phenotype such that the resulting mature cells have enhanced responsiveness to activation.

[0189] A major question in the field of trained immunity remains the durability of training effects. Therefore, the inventors performed a time-course analysis of the effect of IP injection of dWGP on bone marrow myelopoiesis. Consistent with previous results, when 0.2 mg of dWGP was injected into C57 / BL6-JOlaHsd mice, it was found that the percentage of MPP3 cells increased significantly 1 week after injection, while MPP4 cells decreased ( Fig.13 A). This was not observed 1 day after dWGP injection and was maintained until 2 weeks after injection, but resolved at 3 weeks. Importantly, splenocytes extracted from these mice showed an enhanced response to LPS stimulation that was particularly pronounced and maintained 3 weeks after injection ( Fig.13 B) These results are consistent with previous studies with β-glucan injections, which demonstrated dynamic remodeling of bone marrow progenitors over time.

[0190] β-Glucans represent a key class of non-digestible dietary fibers found in many foods and supplements. Yeast β-glucan is particularly well tolerated and safe. The inventors analyzed the dispersibility Whether dietary consumption of (dWGP) can drive in vivo training effects. A dose equivalent to the amount injected in IP (0.2 mg dWGP) was delivered, but by oral gavage (OG). Bone marrow was harvested 1 week after OG and HSPC subsets were examined. Although no expansion of total HSPC numbers similar to that observed with previous IP injections was observed with OG administration (data not shown), the ratio of myeloid-committed MPP3 cells did increase in mice given dWGP by both IP and OG routes ( Fig.13 C). Splenocytes from these mice were stimulated with LPS and increased TNF production was observed from mice given dWGP via oral gavage ( Fig.13 D) These qualitative and quantitative differences in bone marrow effects may be a result of different routes of administration of the training agents, however the data support the notion that consumption of β-glucan can enhance innate immune function.

[0191] To examine this in a more relevant setting, the inventors designed a feeding study in which increasing doses of dWGP incorporated as a commercial ingredient Wellmune were fed to groups of mice for up to 3 weeks together with a control diet equivalently enriched with inert non-digestible dietary fiber (inulin) to match dietary energy and fiber intake. Again, as observed in the previous OG study, no expansion of total bone marrow LKS+ cell numbers occurred in the dWGP-fed mice ( Fig.14C). Nevertheless, dynamic changes in the LKS population over time were observed, with an increase in the frequency of LT-HSCs in mice after 1 week of feeding followed by a decrease in this population relative to controls 3 weeks after feeding, while at the same time the percentage of more committed MPP cells increased ( Fig.14 D- Fig.14 E). Crucially, the ratio of myeloid-committed MPP3 cells to lymphoid-committed MPP4 cells was significantly increased in mice fed a diet containing dWGP relative to a control diet, particularly 3 weeks after the start of feeding ( Fig.13 E). Importantly, mature BMDM derived from the bone marrow of these mice showed differences in functional responses to stimulation. BMDM generated from mice after 1 week of feeding showed enhanced TNF production in response to stimulation with LPS or hk-Mtb, which was not evident in BMDM taken from mice fed for 3 weeks ( Fig.13 F- Fig.13 G). In particular, in terms of dispersibility In mice fed with , cytokine production was enhanced 6 h after stimulation and was less evident 24 h after stimulation ( Fig.14 G- Fig.14 H), consistent with a reprogramming effect of training that enhances innate immune dynamics. Notably, differences in cytokine production were more pronounced in mice fed the highest amount of dWGP, whereas effects on HSPC expansion were more pronounced in mice fed lower amounts of dWGP, suggesting that qualitative and quantitative effects of training on bone marrow reprogramming can be decoupled and that oral training agents can differentially engage these. is a yeast-derived β-glucan particle that can drive an educated response in innate immune cells (both monocytes and myeloid progenitor cells).

[0192] To do this, the inventors designed various mouse food diets with increasing doses of dWGP, which is present as a composition under the trade name "Wellmune" (Carpenter, KC et al., Baker's yeast beta-glucan supplementation increases monocytes and cytokines post-exercise: implications for infection risk? Br J Nutr 109, 478-486, (2013)); and a matched control diet with inert, non-digestible dietary fiber (inulin) to match caloric consumption and roughage effects. C57 / BL6OwaJ mice were fed these Wellmune-containing diets for up to 4 weeks, and the bone marrow population was examined. Unlike mice injected with BGP or dWGP, the inventors did not observe an expansion in the number of total bone marrow LKS+ cells in WGP-fed mice ( Fig.12 A).

[0193] However, when we examined the relative frequencies of specific HSPC populations, we did observe an increase in the frequency of LT-HSCs in mice after 1 week of feeding, followed by a decrease in this population relative to controls at 3 weeks post-feeding, with an increase in the percentage of more committed MPP cells at this same time point ( Fig.12 B). The data suggest that although the apparent expansion of the HSPC population after inflammatory challenge (IP injection) did not occur in mice fed the training agent, there was still a dynamic remodeling of HSPC progenitors in these mice. Although there was no significant change in the ratio of myeloid-committed MPP3 cells to lymphoid-committed MPP4 cells ( Fig.12 C. Fig.13 B), but when the inventors generated mature BMDM from these bone marrows, we observed differences in functional responses to stimulation. BMDM generated from mice after 1 week of feeding showed enhanced TNF production in response to stimulation with LPS or hk-Mtb, which was less obvious in BMDM taken from mice fed for 3 weeks ( Fig.12 D- Fig.12 E). In particular, the difference in cytokine production was enhanced 6 h after stimulation and was less obvious 24 h after stimulation ( Fig.13 C- Fig.13D), which is consistent with the reprogramming effect of training that enhances innate immune dynamics. Notably, the differences in cytokine production were more pronounced in mice fed the highest amounts of WGP, while the slight effect on HSPC expansion was more pronounced in mice fed lower amounts of WGP, suggesting that the qualitative and quantitative effects of training on bone marrow reprogramming can be decoupled and that oral training agents can differentially engage these. In summary, WGP is a yeast-derived β-glucan particle that can drive training responses in innate immune cells (both monocytes and bone marrow progenitors).

[0194] In the control diet (no dispersibility Additional feeding studies were conducted in C57 / BL6 mice fed a diet containing either 0.05% dWGP or a diet high in Wellmune (0.05% dWGP) for between 0 and 12 weeks. The mice were sacrificed and bone marrow was extracted. HSPC populations were measured by flow cytometry ( Fig.15 A- Fig.15 B), and showed a significant increase in the percentage of myeloid-directed MPP3 population after 4 weeks of WGP feeding. However, after 12 weeks of feeding, this decreased to control levels. In addition, these bone marrow cells were matured into BMDM and restimulated with TLR ligands. In mice fed Wellmune for 4 and 8 weeks, BMDM from mice fed Wellmune showed enhanced TNF production after restimulation, but this was not the case in mice fed Wellmune for 12 weeks. This data is consistent with previous data showing that while Wellmune feeding does not drive expansion of HSPC numbers, it does affect the frequency and turnover of HSPC cells in the bone marrow, and optimally over a 3-4 week period, drives myeloid-directed MPP3 skewing, indicating in vivo hub training. This effect was not maintained after 8 weeks of feeding.

[0195] Thus, mice consuming Wellmune via dietary supplementation displayed features of trained immunity, with reprogramming of the bone marrow HSPC population over time and enhanced innate immune function in mature macrophages derived from these cells.

[0196] Delivery of WGP by oral gavage resulted in enhanced responsiveness of these bone marrow-derived mature macrophages (BMDM) to activation

[0197] The present inventors have previously demonstrated that IP injection of WGP drives a significant expansion in the number of LKS+HSPC cells in the bone marrow of mice, as well as an increase in the proportion of the myeloid-committed MPP3 subset. However, delivery of an equivalent dose of WGP by oral gavage did not result in the same expansion in the number of LKS+HSPC cells observed with IP injection ( Fig.16A). However, within the LKS+ population, the proportion of the myeloid-committed MPP3 subset was increased in mice injected with WGP and in mice given WGP by oral gavage ( Fig.16 B). Going beyond previous data, the inventors now show that when the bone marrow containing these expanded MPP3 populations grow into mature macrophages (bone marrow-derived macrophages), they display enhanced function. When stimulated with low concentrations of the TLR4 ligand LPS, less proinflammatory cytokine TNF production was observed in BMDM from control mice. However, this was enhanced and accelerated in BMDM from mice given WGP by IP ( Fig.16 C), consistent with enhanced function as a result of trained immunity. BMDM from mice given WGP by oral gavage showed increased levels of TNF production relative to control BMDM, albeit with slower kinetics than observed with IP-trained mice ( Fig.16 C) These data suggest that oral delivery of WGP results in some features associated with trained immunity, although it does not share all features observed with IP injection, but has an increased sensitivity to low-dose activation consistent with delivery through a more tolerable intestinal environment.

[0198] Feeding a diet containing Wellmune WGP for 4 weeks resulted in an increase in more mature and more common myeloid-committed progenitor cells, consistent with an increased responsiveness of these bone marrow-derived mature macrophages (BMDM) to activation

[0199] The inventors' previous data support that after WGP feeding, early immune cell progenitors expand rapidly, which begin to shrink and return to baseline levels despite continued feeding. To determine whether feeding WGP leads to long-term responses in more committed and mature progenitors, the inventors conducted a new feeding study, this time focusing on the highest concentration of WGP supplementation for 4 weeks. Since the previous feeding study used inulin as a control fiber, to confirm that some of the dose-dependent effects previously observed were not due to inulin, the inventors included an additional inulin-free control diet and compared the same amount of WGP to the same amount of inulin (in terms of fiber content). As previously mentioned, no increase in the number of total LKS+ progenitor cells was measured ( Fig.17 A). Consistent with previous results, an increase in the proportion of myeloid-committed MPP3 cells was measured within the LKS+ population ( Fig.17 B), indicating increased myelopoiesis. To determine whether changes in this low-abundance population led to an increase in cells further downstream in the hematopoietic tree, the inventors measured myeloid progenitors in the LKS(-) fraction of bone marrow cells. The inventors observed a significant increase in total myeloid progenitors as well as increased GMP and CMP of granulocyte and macrophage progenitors, and a significant decrease in CLP of lymphoid lineage progenitors ( Fig.17C). Mature BMDM from WGP-supplemented mice showed increased TNF and IL6 production after LPS stimulation ( Fig.17 D), supporting the idea that, although reprogramming at the level of early progenitor cells returned to baseline after 4 weeks of feeding, functional changes in more mature cells persisted, consistent with innate immune memory. Importantly, all of these trained immune-like effects were limited to the Wellmune WGP-supplemented group, with inulin-fed mice behaving similarly to mice fed a control diet.

[0200] Orally delivered Wellmune WGP β-glucan alters intestinal mucosal immune composition.

[0201] Studies from intraperitoneal delivery of β-glucan or intravenous delivery of BCG to drive trained immunity indicate that local innate immune cells drive cytokines that affect bone marrow hematopoiesis. Because oral delivery of Wellmune WGP β-glucan, both by oral gavage and incorporation into the mouse diet, results in myelopoiesis in the bone marrow and supports innate immune responses in progeny macrophages, the effects on intestinal immune cells were examined. Mice fed a Wellmune WGP β-glucan supplemented diet for 4 weeks (WGP) showed no changes in total CD45 positive immune cells in the small intestine relative to mice fed a standard diet (SFD, Fig.18 A, right). However, when the proportion of monocytes was measured, this was significantly enhanced in WGP-fed mice ( Fig.18 A, left). This suggests that following WGP delivery to the intestine, monocytes traffic to this site and this is indicative of an inflammatory response. To determine if these cells contain β-glucan, we employed a strategy to fluorescently label Wellmune WGP β-glucan using DTAP. This was used in a recent report examining intraperitoneally delivered WGP β-glucan, which demonstrated accumulation of WGP β-glucan-positive macrophages in the pancreas. We also observed this in preliminary experiments following IP delivery ( Fig.18 B, blue dots). Interestingly, this was not observed when labeled WGP β-glucan was delivered by oral gavage. However, at this time point (3 days after delivery), WGP β-glucan-positive macrophages were measured in the colonic lamina propria. These data support that β-glucan is sensed by resident or recruited innate immune cells in the intestine, initiating an inflammatory response that can affect myelopoiesis of the bone marrow.

[0202] Applicability of WGP to restore defective innate immune function in obesity models.

[0203] Dysregulated inflammation is now thought to contribute to the development of obesity-related diseases including diabetes, but also increases the risk of cancer and serious infections. Obesity and associated metabolic dysfunctions are modeled in mice using a diet-induced obesity model in which C57 / BL6 mice are fed a high-fat diet (HFD) for 12 weeks. Bone marrow-derived macrophages (BMDM) obtained from these HFD mice display an altered LPS-induced cytokine pattern ( Fig.19 A), with significantly more pro-inflammatory TNF and decreased immunomodulatory IL-10 production. This is consistent with the loss of immunomodulatory responses and chronic inflammation in the obese state. However, when the HFD was supplemented with Wellmune WGP β-glucan (HFD-WGP), BMDM cytokine responses showed a more regulated response, similar to that of SFD mice ( Fig.19 A), with less TNF and elevated IL-10 levels relative to BMDM from HFD-fed mice. Because people with obesity are susceptible to infection, and diabetes in particular is emerging as a risk factor for tuberculosis (TB), we tested the response to infection with the pathogen Mycobacterium tuberculosis (Mtb) in BMDM from these mice. Consistent with the loss of innate immune function, macrophages from HFD-fed mice were unable to contain intracellular Mtb, and intracellular Mtb levels were found to be significantly higher at later times (72h) after infection than those observed in BMDM from SFD-fed mice ( Fig.19 B). However, mice fed a HFD supplemented with Wellmune WGP β-glucan, showed improved Mtb suppression as observed in SFD-fed macrophages. These data support the notion that obesity leads to a dysregulated state of inflammation that compromises host defense function. In this dysregulated state, oral supplementation with WGP β-glucan may correct at least some of these immune deficiencies and restore normal innate immune responses.

[0204] Other yeast-derived β-glucan particles can drive trained immunity in vitro

[0205] To test whether the trained immune responses reported with Wellmune WGP were specific to this molecule or a conserved feature of β-glucan particles, the inventors employed a commonly available research grade β-glucan, zymosan (a β-glucan-rich cell wall preparation from Saccharomyces cerevisiae). Fig. 20A shows that crude zymosan (obtained from Invivogen) activates the β-glucan receptor Dectin-1, but also other innate immune receptors including TLR 2 and TLR 4. However, a purer form of zymosan from which contaminating non-β-glucan ligands have been removed, termed depleted zymosan (ZYM-d), does not trigger TLR2 / TLR4. In training assays, crude zymosan leads to a tolerance-like phenotype, whereas pure ZYM-d should trigger training immunity more similar to the training immune response observed with dWGP ( Fig. 20 B). In human monocytes and mouse macrophages ( Fig. 20 C- Fig. 20 D), Pure ZYM-d drives the trained response, and a kinetic enhancement of TNF production is observed after LPS restimulation.

[0206] discuss

[0207] The inventors have uncovered a pathway whereby recognition of intact fungal particles and subsequent internalization via the phagocytic synapse is intimately linked to the target cell's metabolic investment in a trained phenotype by engaging mTOR. While these studies used artificial β-glucan-enriched particles to engage Dectin-1 in this manner, it is tempting to speculate that this represents a conserved pathway for the innate immune system to avoid inappropriate and wasted resources in long-term memory responses to soluble ligands or non-viable pathogens, and instead promote a trained phenotype in response to intact fungal particles only when necessary. This involves reprogramming cellular metabolism to adopt a glycolytic mode only upon successful engagement of lysosomal mTOR following engulfment of an intact particle. This commitment to glycolysis is associated with epigenetic changes that emphasize the trained phenotype and accelerated response to nonspecific restimulation. This pathway has been observed in very short-lived monocytes, except that very short-lived monocytes migrate into tissues as macrophages, and the inventors have also observed that artificial β-glucan-enriched particles (dWGP) affect myeloid progenitors in the bone marrow and the ability to enhance the functional responses of mature macrophages. These functional responses include cytokine responses. Therefore, the physical and chemical requirements for optimal training responses can be used to improve innate immune function for therapeutic methods such as vaccination or immunotherapy, or to promote increased resistance to new pathogens for which we have no pre-existing acquired memory (such as the current COVID19 disaster). In the post-COVID era, where long-term immune dysregulation has been portrayed, we hypothesize that β-glucan training that produces balanced innate responses can prevent the increased severity of other infections.

[0208] Dispersibility (dWGP) itself represents an artificial β-glucan preparation, enriched with β-glucans from which contaminating TLR2 ligands and other mannans have been removed. Outer cell wall mannans have been observed in Candida albicans to block β-glucan interaction with the innate immune system and drive phagocytosis, and thus mannans may appear as an immune evasion strategy to block the training of the host by β-glucan exposure. Crude zymosan, representing a granular Saccharomyces cerevisiae cell wall preparation containing β-glucans masked by a mannan layer, cannot drive the same level of trained immunity in vitro, but instead promotes an acute inflammatory response. Removal of these mannans confers the ability to metabolically reprogram and train cells to the depleted zymosan preparation. Therefore, the discovery that Wellmune WGP and other yeast-derived β-glucan granular preparations represent strong drivers of trained immunity may actually be an artificial observation that does not occur in nature due to mannan masking. However, this is knowledge that we can use to promote immune function.

[0209] This work reveals that the MW of the β-glucan chains in the mixture does not affect their interaction with the innate immune system, but ultimately affects the physical properties and manner in which these ligands are presented to innate immune cells, namely their insoluble nature, which is determined by the extraction method that retains a highly pure and intact ghost yeast cell wall. Soluble yeast β-glucan has been used in both immunotherapy approaches and nutritional supplementation strategies. Although these data suggest that soluble Although it is not possible to train, but rather to block occupancy of the receptor by higher molecular weight particulate β-glucans, other soluble β-glucans can drive training responses in vitro and in vivo, including Trametes versicolor BGP. The mechanisms behind this remain uncertain, although they may involve enhanced proliferation without significant glycolytic activity to epigenetically alter cell fate ( Fig.12 A), as shown in the training model using C. albicans (β-1,3)-glucan, or may involve enhanced growth factor signaling associated with differentiation as recently shown in other yeast β-glucan preparations.

[0210] The WGP used here is the dispersibility The invention discloses a novel compound WGP, which is a kind of oral WGP, for sale, and its effect on upper respiratory tract infection of elite marathon runners after oral consumption, and the effect on circulating cytokine levels and LPS-induced mononuclear cell production in immunocompromised groups. The inventors found that oral WGP has biological and training-like effects in mice, consistent with the in vitro training study of human monocytes, which has opened up new ways for commercial and therapeutic development of such molecules. It is worth noting that the effect on myelopoiesis of mouse bone marrow bone by the two different approaches examined is discreet. Here, the inventors show that WGP affects bone marrow hematopoiesis by increasing HSPC in quantity and qualitatively biasing myelopoiesis.

[0211] Since WGP-trained macrophages exhibited both increased clearance of Mtb and upregulated glycolytic metabolism, metabolically reprogrammed trained macrophages may be metabolically predisposed to be more antagonistic to intracellular pathogen replication. In fact, although WGP-trained macrophages produced more proinflammatory cytokines early after infection with Mtb, by the later stages when macrophages cleared more Mtb, cytokine levels had normalized, supporting the idea that training is more than just epigenetic sensitization and altered cytokine dynamics. The inventors' work places mTOR activation at the center of training induced by β-glucan particles. The inventors demonstrate that it is upregulated by dWGP in a rapamycin-sensitive manner. S6K itself is an important ribosomal protein that drives protein synthesis activated by mTOR in response to changes in lysosomal activity and its activation during the training phase driven by dWGP, indicating that large-scale proteomic and metabolic changes occur in trained macrophages that may persist throughout maturation.

[0212] In summary, the inventors identified a novel immunomodulatory effect of yeast-derived particulate β-glucans by driving metabolic reprogramming in target cells required for trained immunity. This may impact strategies for using these and similar β-glucans to boost innate immune function and could be used to promote innate immune resistance to infection.

Claims

1. An effective amount of Saccharomyces cerevisiae derived (1→3)-β-D-glucan for use in a method for programming bone marrow hematopoietic stem and progenitor cells (HSPCs) to promote multipotent progenitor cell (MPP) expansion to facilitate myelopoiesis in a subject, wherein the β-glucan is a substantially spherical intact glucan particle, and wherein the β-glucan is to be administered orally.

2. An effective amount of Saccharomyces cerevisiae-derived (1→3)-β-D-glucan for use according to claim 1, wherein myelopoiesis produces trained innate immune cells. 3 . The effective amount of Saccharomyces cerevisiae-derived (1→3)-β-D-glucan for use according to claim 2 , wherein the immune cells are monocytes and / or monocyte-derived macrophages.

4. An effective amount of Saccharomyces cerevisiae derived (1→3)-β-D-glucan for use according to any one of the preceding claims, for preventing a disease or infection, delaying the onset of a disease or infection, or reducing the severity of a disease or infection in the subject.

5. An effective amount of Saccharomyces cerevisiae-derived (1→3)-β-D-glucan for use according to any one of the preceding claims, wherein the subject is in the early stages of a disease or infection, is at risk of a disease or infection, or when the subject is suspected of having a disease or infection.

6. An effective amount of Saccharomyces cerevisiae-derived (1→3)-β-D-glucan for use according to any of the preceding claims, wherein the subject is one or more selected from the group consisting of: healthy subjects, athletes, subjects experiencing or suffering from stress, immunocompromised subjects, subjects with obesity, subjects with diabetes, subjects over 65 years old, preferably over 75 years old, subjects less than 16 years old, subjects suffering from or recovering from cancer, and subjects with defective innate immunity. 7 . The effective amount of Saccharomyces cerevisiae-derived (1→3)-β-D-glucan for use according to claim 6 , wherein the subject is a healthy subject. 8 . The effective amount of Saccharomyces cerevisiae-derived (1→3)-β-D-glucan for use according to claim 6 , wherein the subject suffers from obesity.

9. An effective amount of Saccharomyces cerevisiae-derived (1→3)-β-D-glucan for use according to any one of the preceding claims, wherein the subject suffers from a post-infectious immune disorder.

10. An effective amount of Saccharomyces cerevisiae-derived (1→3)-β-D-glucan for use according to claim 9, wherein the infection is a SARS-CoV-2 infection.

11. An effective amount of a Saccharomyces cerevisiae derived (1→3)-β-D-glucan according to any one of the preceding claims, wherein the (1→3)-β-D-glucan is a β-1,6 branched β-1,3 glucan (or "β-(1,3 / 1,6)").

12. An effective amount of Saccharomyces cerevisiae-derived (1→3)-β-D-glucan for use according to claim 11, wherein the (1→3)-β-D-glucan is a β-1,6 branched β-1,3 glucan and has the following structure:

13. An effective amount of Saccharomyces cerevisiae derived (1→3)-β-D-glucan for use according to any one of the preceding claims, wherein the WGP has a size of about 1 to about 6 micrometers (or μm), or about 2 to about 5 micrometers, or about 3 to about 4 micrometers.

14. An effective amount of Saccharomyces cerevisiae derived (1→3)-β-D-glucan for use according to any one of the preceding claims, provided as a food supplement comprising the Saccharomyces cerevisiae derived (1→3)-β-D-glucan.

15. An effective amount of Saccharomyces cerevisiae derived (1→3)-β-D-glucan for use according to any one of the preceding claims, wherein the food supplement comprises ≥ 75% β1,3 / 1,6 glucan by dry weight.

16. An effective amount of Saccharomyces cerevisiae derived (1→3)-β-D-glucan for use according to any one of the preceding claims, wherein the Saccharomyces cerevisiae derived (1→3)-β-D-glucan is 17. An effective amount of Saccharomyces cerevisiae derived β-glucan for use according to any one of the preceding claims, wherein the β-glucan is administered on a cyclic schedule.

18. The effective amount of Saccharomyces cerevisiae-derived (1→3)-β-D-glucan for use according to claim 17, wherein the cycling schedule is a period of three to four weeks.

19. An effective amount of Saccharomyces cerevisiae-derived (1→3)-β-D-glucan for use according to claim 18, wherein the cycling schedule is a four-week period.

20. An effective amount of Saccharomyces cerevisiae-derived (1→3)-β-D-glucan for use in a method of inducing trained innate immunity in a subject, wherein the β-glucan is a substantially spherical whole glucan particle, and wherein the β-glucan is to be administered orally.

21. An effective amount of Saccharomyces cerevisiae derived (1→3)-β-D-glucan for use in a method of eliciting an enhanced immune response against an invading pathogen in a subject, wherein the β-glucan is a substantially spherical intact glucan particle, and wherein the β-glucan is to be administered orally.

22. An effective amount of Saccharomyces cerevisiae-derived (1→3)-β-D-glucan for use according to claim 21, wherein the β-glucan is to be administered at an early stage of a disease or infection, when the subject is at risk of a disease or infection, when the subject is suspected of having a disease or infection, and / or when the subject exhibits symptoms of a disease or infection.

23. An effective amount of (1→3)-β-D-glucan derived from Saccharomyces cerevisiae for use in treating post-infectious immune disorders in a subject.

24. An effective amount of Saccharomyces cerevisiae derived (1→3)-β-D-glucan for use according to claim 23, wherein the infection is a SARS-CoV-2 infection.

25. A composition comprising more than one β-glucan type, each β-glucan type being derived from a different strain of Saccharomyces cerevisiae.

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