IL-1Ra blockers for treatment and prevention of sepsis

Abstract

The present invention relates to a pharmaceutical composition comprising an agent capable of inhibiting IL-1Ra activity for use in the prevention or treatment of sepsis associated with systemic fungal infection.

Description

[0001] This application claims priority to European Patent Application No. 23183633, filed on 5 July 2023, and European Patent Application No. 23215259, filed on 8 December 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the medical use of IL-1Ra blockers in the treatment of sepsis associated with systemic fungal infection. Background Technology

[0003] Sepsis is a severe, life-threatening condition caused by the dissemination of microorganisms through the bloodstream and a dysregulated systemic inflammatory response. Although fungal sepsis occurs less frequently, invasive fungal infections have a particularly high mortality rate. Candida albicans represents the most common cause of fungal bloodstream infections; despite adequate antifungal treatment, the overall mortality rate is 30–40%, exceeding 60% in critically ill patients. Candida albicans is usually immunosuppressed by the epithelial barrier and occurs as a symbiotic organism in half of the population. Innate immunodeficiency highlights the importance of the IL-17-mediated pathway in preventing mucocutaneous migration, while a functional neutrophilic response is crucial for preventing systemic infections (Puel A. (2020), HumGenet. 139, 1011–1022; Desai JV, Lionakis MS. (2018), Curr Clin Microbiol Rep. 5, 181–189). Therefore, individuals with immunosuppression due to hematologic malignancies, organ transplantation, AIDS, or prolonged intensive care hospitalization are highly susceptible to invasive candidiasis. However, previous systemic viral infections or medical interventions that impair physiological barriers, such as indwelling devices, parenteral nutrition, and abdominal surgery, may suscept other immunocompetent hosts to disseminated candidiasis. Given the high mortality rate of invasive fungal infections, the emergence of drug-resistant strains, and the increasing number of high-risk patients, there is great interest in identifying the underlying disease mechanisms to develop new treatment strategies.

[0004] Inflammation is a physiological, innate response to tissue damage, aimed at eliminating damaging agents and restoring homeostasis. To provide an appropriate defense mechanism that ensures pathogen clearance while avoiding widespread tissue damage, the dynamics and composition of its potent effector functions must be precisely adapted to the characteristics of the invading pathogen. Therefore, a network of interactions between pro-inflammatory and anti-inflammatory cytokines coordinates the differentiation and recruitment of functionally distinct immune cell populations. Furthermore, negative feedback mechanisms exist, known as immune checkpoints, which emit disproportionate signals of immune activation and suppress inflammatory processes to prevent immunopathology. However, sepsis is characterized by this dissociation from the homeostasis of both pro-inflammatory and anti-inflammatory pathways, accompanied by hyperinflammation and immune paralysis, leading to a dysfunctional systemic inflammatory response, multi-organ damage, and failure to control pathogen replication.

[0005] The proto-inflammatory cytokine interleukin-1 (IL-1) initiates and coordinates local and systemic inflammatory responses by activating IL-1 receptors (IL-1R) on both immune and non-immune cells. IL-1 cytokines, IL-1α and IL-1β, are essential components of antimicrobial immunity, particularly against Candida albicans. However, excessive IL-1 production is associated with severe acute and chronic inflammatory conditions such as autoinflammatory syndromes, rheumatoid arthritis, sepsis, or metabolic disorders. Therefore, IL-1α and IL-1β are subject to highly complex regulatory mechanisms acting at both transcriptional and post-translational levels, limiting the production of mature cytokines or affecting their ability to activate IL-1R. The inflammatory effects of biologically active IL-1α and IL-1β are controlled by IL-1R antagonists (IL-1Ra), which competitively occupy IL-1R without triggering downstream signaling. One secreted and three intracellular isoforms of IL-1Ra have been described and are derived from the same IL1RN gene. Beyond the complexity of IL-1 regulation, these individual isoforms are differentially expressed across various cell types. For example, intracellular IL-1Ra is constitutively expressed in epithelial cells, while secretory and intracellular isoforms can be induced in different leukocyte subsets in response to pro-inflammatory cytokines, microbial products, or tissue damage. The potency of IL-1Ra-mediated regulation is evident in the severe inflammatory syndrome in patients with DIRA and is being developed therapeutically for IL-1-mediated diseases. However, although the molecular pathways controlling the production and secretion of mature IL-1 are well known, much less is understood regarding regulatory mechanisms acting at the receptor-binding level, such as IL-1Ra, particularly the cell-type-specific regulation of IL-1-driven inflammation.

[0006] Based on the aforementioned prior art, the object of the present invention is to provide means and methods for preventing sepsis-related deaths associated with systemic fungal infections. This object is achieved through the subject matter of the independent claims of this specification and the further advantageous embodiments described in the dependent claims, embodiments, drawings, and general description. Summary of the Invention

[0007] The present invention provides a pharmaceutical composition comprising an agent capable of inhibiting IL-1Ra activity for the prevention or treatment of sepsis associated with systemic fungal infection.

[0008] Terms and Definitions

[0009] General

[0010] For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural, and vice versa. In the event of any conflict between any definition set forth below and any reference incorporated herein by reference, the definition set forth herein shall prevail.

[0011] As used herein, the terms “comprising,” “having,” “containing,” and “including,” as well as other similar forms and their grammatical equivalents, are intended to be equivalent in meaning and are open-ended, because one or more items following any of these words do not imply an exhaustive list of those items or that they are limited to only the listed items. For example, an article “comprising” components A, B, and C may consist of components A, B, and C (i.e., contain only), or may contain not only components A, B, and C, but may also include one or more other components. Therefore, it is intended and understood that “comprising” and its similar forms and their grammatical equivalents encompass disclosures of embodiments “consistently consisting of” or “comprises of.”

[0012] Where numerical ranges are provided, it should be understood that, unless the context explicitly specifies otherwise, every intermediate value of one-tenth of the unit of the lower limit between the upper and lower limits of the range, and any other stated value or intermediate value within the range, is covered by the invention but is subject to any specific exclusions within the range. Where the range contains one or two limits, the range excluding any one or two of those included limits is also included in this disclosure.

[0013] The “about” values ​​or parameters mentioned in this article include (and describe) changes involving that value or parameter itself. For example, a description of “about X” includes a description of “X”.

[0014] As used herein, the singular forms “a,” “or,” and “the” included in the appended claims contain plural references unless the context clearly indicates otherwise.

[0015] "And / or" is used here as a specific description of each of two particular features or components, with or without other features or components. Therefore, the term "and / or" as used in phrases such as "A and / or B" is intended to encompass "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry, organic synthesis). Standard techniques are used for molecular, genetic and biochemical methods (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.

[0017] Binding; binding agents, ligands, antibodies:

[0018] Unless otherwise specified in the “Detailed Description” of the present invention, the binders and ligands mentioned include antibodies, antibody-like molecules and aptamers as defined in the following paragraphs.

[0019] In the context of this invention, the terminology Specific binding This refers to the property of a ligand to bind to its target with a certain affinity and specificity. The affinity of this ligand is represented by its dissociation constant. Specific reactive ligands, when binding to their targets, have a dissociation constant ≤10. -8 mol / L (especially ≤10) −9 The dissociation constant is approximately mol / L, but when interacting with molecules that have a chemical composition similar to the target but a different three-dimensional structure, its dissociation constant is at least three orders of magnitude higher.

[0020] the term Non-agonist ligands This refers to ligands, especially human or humanized monoclonal antibodies, which can be used at 10 -8mol / L or lower k D Especially 10 -9 mol / L or even 10 -10 k mol / L D It specifically binds to its target. "Non-agonist" ligands interact with their targets without producing the biological effects of the target's physiological ligands. For example, non-agonist ligands of interleukin receptors bind to interleukin receptors (ILRs, the targets) without producing the effects of interleukin-ILR interactions and inhibiting interleukin binding.

[0021] In the context of this specification, the terminology dissociation constant (K) D K is used in its known sense in the fields of chemistry and physics; it refers to the equilibrium constant, which measures the tendency of a complex composed of [in most cases, two] different components to reversibly dissociate into its constituent components. The complex can be, for example, an antibody-antigen complex AbAg composed of antibody Ab and antigen Ag. D The [Ab] concentration is expressed as molar concentration [mol / L], corresponding to when half of the [Ag] binding sites are occupied; in other words, when the concentration of unbound [Ab] equals the concentration of the [AbAg] complex. The dissociation constant can be calculated using the following formula:

[0022] [Ab]: Antibody concentration; [Ag]: Antigen concentration; [AbAg]: Antibody-antigen complex concentration

[0023] In the context of this specification, the term "dissociation rate" (K) off [1 / sec]) and "binding rate" (K on [L / (sec·mol)] is used in its known sense in the fields of chemistry and physics; it refers to the measurement of the dissociation of an antibody from its target antigen (K). off ) or combination (K on The rate constant of K. off and K on It can be determined experimentally using methods known in the art. Measurement of antibody K off and K on The method utilizes surface plasmon resonance. This is the principle behind biosensor systems such as Biacore® or ProteOn®. The dissociation constant K can also be determined using the following equation. D :

[0024] Association rate K on The natural upper limit is 10 9 L / (sec·mol).

[0025] the term fit Aptamers involve oligonucleotides or peptide molecules that bind to specific target molecules. They can be generated by selecting from a large library of random sequences. Nucleic acid aptamers can be generated through in vitro selection or, equivalently, through repeated cycles of SELEX (exponentially enriched ligand system evolution) to bind molecular targets such as small molecules, proteins, or nucleic acids via non-covalent interactions. Aptamers provide molecular recognition properties comparable to antibodies.

[0026] In the context of this specification, the terminology Antibody A complete antibody refers to a glycoprotein comprising, but not limited to, immunoglobulins of type G (IgG), type A (IgA), type D (IgD), type E (IgE), or type M (IgM), any antigen-binding fragment or single chain thereof, and related or derived constructs. A complete antibody is a glycoprotein comprising at least two heavy chains (H) and two light chains (L) linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (V). H ) and heavy chain constant region (C H It consists of three domains: C10, C20, and C30. The heavy chain constant region of IgG consists of three domains: C10, C20, C30, C40, C50, C60, C70, C80, C9 ... H 1. C H 2 and C H It consists of 3 components. Each light chain is composed of a light chain variable region (abbreviated as V in this article). L ) and light chain constant region (C L It consists of a light chain constant region composed of a structural domain C. L Composition. The variable regions of the heavy and light chains include binding domains that interact with antigens. The constant regions of an antibody mediate the binding of immunoglobulins to host tissues or factors and contain various cells of the immune system (e.g., effector cells) and the first components of the classical complement system. Similarly, this term encompasses so-called nanobodies or single-domain antibodies, antibody fragments composed of a single monomeric variable antibody domain.

[0027] In the context of this specification, the terminology Humanized antibodies Antibodies originally produced by immune cells of non-human species have had their protein sequences modified to increase their similarity to naturally occurring antibody variants in humans. As used herein, the term... Humanized antibodies Antibodies containing CDR sequences derived from another mammalian species (e.g., mice) have been grafted onto human frame sequences. Further frame region modifications can be performed within the human frame sequence and within the CDR sequences derived from another mammalian species.

[0028] In the context of this specification, the terminology antibody-like molecules This refers to the ability to achieve high affinity / Kd≤10 -7 mol / L (especially ≤10) -9A molecule that specifically binds to another molecule or its target (mol / L). The binding of an antibody-like molecule to its target is similar to the specific binding of an antibody. Terminology antibody-like molecules This includes repetitive proteins, such as engineered ankyrin repeats (Zurich Molecular Partners), and engineered antibody mimics that exhibit highly specific and high-affinity binding to target proteins (see US2012142611, US2016250341, US2016075767, and US2015368302). Terminology antibody-like molecules This further includes, but is not limited to, peptides derived from armadillo repeat proteins, peptides derived from leucine-rich repeat proteins, and peptides derived from tetrapeptide repeat proteins. Terminology antibody-like molecules It also encompasses peptides derived from protein A domain, fibronectin domain FN3, shared fibronectin domain, lipid transport proteins (see Skerra, Biochim Biophys Acta 2000, 1482(1–2):337–50), peptides derived from zinc finger proteins (see Kwan et al. Structure 2003, 11(7):803–813), Src homology domain 2 (SH2) or Src homology domain 3 (SH3), PDZ domain, γ crystal proteins, ubiquitin, cysteine ​​knot peptides or knottin, cysteine ​​protease inhibitors, Sac7d, triple coiled helix (also known as α-body), Kunitz domain or Kunitz-type protease inhibitors, and carbohydrate-binding module 32-2. Terminology antibody-like molecules This also includes humanized camel antibodies. Terminology antibody-like molecules Similarly, this includes scFv fragments.

[0029] the term Peptides derived from protein A domain It refers to a molecule that is a derivative of protein A and can specifically bind to the Fc and Fab regions of immunoglobulins.

[0030] the term Armadillo repeat protein It refers to a polypeptide that includes at least one armadillo repeat, wherein the armadillo repeat is characterized by a pair of α-helices forming a hairpin structure.

[0031] In the context of this specification, the terminology Humanized camel antibodies This refers to an antibody that consists only of the heavy chain or the heavy chain variable region (V). H The H region is composed of antibodies whose amino acid sequence has been modified to increase their similarity to naturally occurring antibodies in humans, and therefore exhibit reduced immunogenicity when administered to humans. A general strategy for humanizing camel antibodies is illustrated in Vincke. et al."General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold", J Biol Chem. 2009 Jan 30;284(5):3273–3284, and US2011165621A1.

[0032] In the context of this specification, the terminology Crystallizable fragment (Fc) region It refers to a portion of an antibody, which, if applied to IgG, consists of C atoms covalently linked by disulfide bonds. H 2 and C H Two identical heavy chain segments composed of 3 structural domains.

[0033] In the context of this specification, the terminology Single-chain variable fragments (scFv) Heavy chains involving immunoglobulins (V H ) and light chains (V L The variable region of scFv is a fusion protein that results in antibody-like high affinity for targets derived from a single polypeptide chain. H and V L The chain is linked by short linker peptides of ten to approximately 25 amino acids [Huston et al. (1988). PNAS 85 (16): 5879–5883]. The linker can connect V... H The N-terminus and V L C-end connection (V L -V H ), or adopt a reverse configuration (V H -V L ).

[0034] In the context of this specification, the terminology IL-1Ra inhibitors Drugs that involve inhibiting the physiological response to interleukin-1 receptor antagonist protein (IL-1Ra; Uniprot P18510).

[0035] General molecular biology: nucleic acid sequence, expression

[0036] the term Gene expression or Express , or term Gene products "Transcription" can refer to one or both of the processes—and products—of the production of nucleic acids (RNA) or peptides or polypeptides (also known as transcription and translation, respectively), or any intermediate process that regulates the processing of genetic information to produce polypeptide products. (Term) Gene expressionIt can also be applied to the transcription and processing of RNA gene products, such as regulatory RNA or structural (e.g., ribosomal) RNA. If the expressed polynucleotide is derived from genomic DNA, expression can be contained within the splicing of mRNA in eukaryotic cells. Expression can be measured at the transcriptional and translational levels, in other words, at the mRNA and / or protein product levels.

[0037] In the context of this specification, the terminology Nucleotides This refers to oligomers of nucleic acids or nucleic acid analogues that are capable of selectively hybridizing with RNA or DNA oligomers based on base pairing. In this context, the term... Nucleotides It contains typical ribonucleotide structural units such as adenosine, guanosine, uridine (and ribosylthymidine), cytidine, and typical deoxyribonucleotides such as deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. It also contains nucleic acid analogs such as phosphate thioesters, 2′-phosphate thioesters, peptide nucleic acids (PNA; N-(2-aminoethyl)-glycine units linked by peptide bonds, wherein the nucleotides are linked to the α-carbon of glycine) or locked nucleic acids (LNA; 2′O,4′C-methylene-bridged RNA building blocks). Wherever hybridization sequences are mentioned herein, such hybridization sequences may consist of any of the above-mentioned nucleotides or mixtures thereof.

[0038] As used in this article, terminology Thiophosphates With terminology Thiophosphates and Thiophosphates Synonyms.

[0039] In the context of this specification, the terminology Able to form hybrids or Hybrid sequence This refers to sequences that can selectively bind to their target sequences under conditions present in the cytosol of mammalian cells. Such hybridization sequences may be sequentially reverse complementary to the target sequence, or may include gaps, mismatches, or other non-matching nucleotides. The minimum length of the sequence capable of forming a hybrid depends on its composition, where C or G nucleotides contribute more to the binding energy than A or T / U nucleotides, and also depends on the backbone chemistry.

[0040] In the context of this specification, the terminology Hybrid sequenceThis includes polynucleotide sequences comprising RNA (ribonucleotides), DNA (deoxyribonucleotides), phosphorothioate deoxyribonucleotides, 2′-O-methyl-modified phosphorothioate ribonucleotides, LNA and / or PNA nucleotide analogs, or substantially composed thereof. In some embodiments, the hybridization sequence of the present invention comprises 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the hybridization sequence has at least 80% identity with the reverse complementary sequence of NCBI reference sequence: XM_047444184.1, NCBI reference sequence: XM_047444185.1, or NCBI reference sequence: XM_011511121.2, more preferably 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identity. In some embodiments, the hybridization sequence comprises deoxynucleotides, phosphorothioate deoxynucleotides, LNA and / or PNA nucleotides, or mixtures thereof.

[0041] In the context of this specification, the terminology antisense oligonucleotides This involves oligonucleotides with sequences substantially complementary to and capable of hybridizing with RNA. Antisense action on such RNA will result in the regulation of RNA biological effects, particularly repression or inhibition. If the RNA is mRNA, the expression of the resulting gene product is repressed or inhibited. Antisense oligonucleotides can consist of DNA, RNA, nucleotide analogs, and / or mixtures thereof. Those skilled in the art are aware of various commercial and non-commercial sources for calculating the theoretically optimal antisense sequence for a given target. Optimization can be performed on both the nucleobase sequence and the backbone (ribose, deoxyribose, analogs) composition. Numerous sources exist for delivering actual physical oligonucleotides typically synthesized via solid-state synthesis.

[0042] the term gapmer Gapmers are short DNA antisense oligonucleotide structures with RNA-like fragments flanking their sequences. They are typically composed of locked RNA (LNA), 2′-OMe, or 2′-F modified bases. Gapmers usually consist of nucleotides modified with phosphate thioester (PS) groups, particularly at their 5′ and 3′ ends. Gapmers are engineered to hybridize with target fragments of RNA and silence genes by inducing RNase H cleavage. Due to the modified RNA flanking regions, gapmers have a higher affinity for their targets and resistance to degradation by certain nucleases. Gapmers are being developed as therapeutics for various cancers, viruses, and other chronic genetic diseases.

[0043] In the context of this specification, the terminology siRNA(Small / short interfering RNA) refers to RNA molecules capable of interfering with gene expression (in other words: suppressing or preventing expression) through a process called RNA interference, which involves nucleic acid sequences that are complementary to or hybridize with siRNA sequences. Terminology siRNA This refers to both single-stranded and double-stranded siRNAs. siRNAs are typically characterized by a length of 17–24 nucleotides. Double-stranded siRNAs can originate from longer double-stranded RNA molecules (dsRNA). According to popular theory, longer dsRNAs are cleaved by an endonuclease (called Dicer) to form double-stranded siRNAs. In a nucleoprotein complex (called RISC), the double-stranded siRNA unwinds to form single-stranded siRNA. RNA interference typically works by siRNA molecules binding to mRNA molecules with complementary sequences, leading to mRNA degradation. RNA interference can also occur by siRNA molecules binding to intron sequences of pre-mRNA (immature, unspliced ​​mRNA) in the cell nucleus, leading to pre-mRNA degradation.

[0044] In the context of this specification, the terminology shRNA (Hairpin RNA) refers to artificial RNA molecules with tight hairpin turns that can be used to silence target gene expression via RNA interference (RNAi).

[0045] Any patent references cited in this article shall be deemed to be incorporated herein in their entirety by reference.

[0046] As used in this article, terminology Pharmaceutical Composition The term refers to the compounds of the present invention, or pharmaceutically acceptable salts thereof, together with at least one pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions of the present invention are provided in a form suitable for topical, parenteral, or injectable administration.

[0047] As used in this article, terminology Pharmaceutically acceptable carriers This invention includes any solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antimicrobial agents, antifungal agents), isotonic agents, absorption delay agents, salts, preservatives, pharmaceuticals, pharmaceutical stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, and combinations thereof (see, for example, Remington: the Science and Practice of Pharmacy, ISBN 0857110624). The invention also includes... Pharmaceutically acceptable carriers Nanoparticles, liposomes, or cell carriers within the definition.

[0048] In the context of this specification, the terminology InhibitorsThis refers to any pharmaceutically acceptable agent or compound that can interact with and specifically interfere with the biological activity of its designated target (IL-1Ra in this case). Inhibitors comprise small molecule drugs that satisfy Lipinski's five rules (the drug must satisfy at least three of the following rules: number of hydrogen bond donors ≤ 5; number of hydrogen bond acceptors ≤ 10; molecular weight < 500 Da; octanol / water partition coefficient ≤ 5). Specific examples of such inhibitors are mentioned herein.

[0049] As used in this article, terminology treat or treat In one embodiment, "treatment" or "curing" refers to improving the disease or condition (e.g., slowing, stopping, or reducing the development of the disease or at least one of its clinical symptoms). In another embodiment, "treatment" or "curing" refers to alleviating or improving at least one bodily parameter, including those that a patient may not be able to discern. In yet another embodiment, "treatment" or "curing" refers to regulating the disease or condition physically (e.g., stabilization of identifiable symptoms), physiologically (e.g., stabilization of bodily parameters), or both. Unless specifically described below, methods for assessing the treatment and / or prevention of disease are generally known in the art. Detailed Implementation

[0050] This invention relates to a pharmaceutical composition comprising an agent capable of inhibiting IL-1Ra activity for the prevention or treatment of sepsis associated with systemic fungal infection.

[0051] The inventors propose that the treatment presented herein is particularly suitable for acute sepsis in patients with a confirmed primary or secondary fungal infection, especially one caused by Candida albicans, or patients considered to be at particularly high risk of developing sepsis associated with Candida albicans or other fungal pathogens. It is noteworthy that inhibition of IL-1Ra may be contraindicated in patients with bacterial sepsis.

[0052] Ideally, patients suspected of having a systemic fungal infection should be treated when symptoms first appear.

[0053] In some implementations, the systemic fungal infection is a Candida albicans infection.

[0054] In some embodiments, the agent capable of inhibiting IL-1RA is an IL-1RA ligand selected from monoclonal antibodies and antibody-like molecules.

[0055] Antibodies have an advantage in serum half-life, thus providing long-term protection.

[0056] However, the inventors believe that the data obtained indicate that shorter-lived protein agents, such as protein-based binders like designed ankyrin repeats or antibody-like molecules, such as nanobodies, variable fragments, or camel antibodies, can also be used for useful purposes, given that rapid clearance after bolus administration may be sufficient to disrupt the immune response triggered by infection.

[0057] In a specific embodiment, the ligand for IL-1Ra is a neutralizing antibody or a neutralizing antibody-like molecule. The term "neutralizing antibody or neutralizing antibody-like molecule" refers to an antibody or antibody fragment that can specifically bind to the target IL-1RA in a manner that inhibits the biological activity of the target. In the context of this patent, the term "neutralizing antibody" includes monoclonal and polyclonal antibodies, as well as any fragment, variant, or derivative thereof that retains the ability to neutralize the target.

[0058] Alternatively, peptides derived from soluble IL-1 receptors may be considered, where the epitopes binding to IL-1α and IL-1β have been altered to prevent the soluble receptor from clearing these cytokines, while binding to IL-Ra remains as an effective inhibitor of IL-1Ra.

[0059] In some embodiments, the agent capable of inhibiting IL-1RA is an oligonucleotide agent capable of inhibiting IL-1RN gene expression.

[0060] In some implementations, the oligonucleotide agent is capable of hybridizing with mRNA encoding IL-1Ra.

[0061] In some embodiments, the oligonucleotide agent is selected from antisense oligonucleotides, gapmers, siRNA, and shRNA. Using siRNA transcribed from an expression vector controlled by a macrophage-specific promoter may allow for specific targeting of the origin of IL-1Ra discovered by the inventors.

[0062] Medical treatment

[0063] Similarly, within the scope of this invention is a method for treating sepsis associated with systemic fungal infection in patients in need, comprising administering to the patient an agent capable of inhibiting IL-1Ra activity as described above.

[0064] Pharmaceutical compositions, administration / dosage forms and salts

[0065] According to one aspect of the compounds of the present invention, the compounds of the present invention are provided as pharmaceutical compositions, pharmaceutical administration forms, or pharmaceutical dosage forms, said pharmaceutical compositions, pharmaceutical administration forms, or pharmaceutical dosage forms comprising at least one compound of the present invention or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier, diluent, or excipient.

[0066] The present invention also includes pharmaceutical compositions comprising the compounds of the present invention or pharmaceutically acceptable salts thereof and pharmaceutically acceptable carriers. In a further embodiment, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein.

[0067] Some embodiments of the present invention relate to dosage forms for enteral administration, such as nasal, oral, rectal, transdermal, or oral administration, or as inhalation forms or suppositories. Furthermore, the pharmaceutical compositions of the present invention can be formulated in solid forms (including, but not limited to, capsules, tablets, pills, granules, powders, or suppositories) or liquid forms (including, but not limited to, solutions, suspensions, or emulsions).

[0068] Some embodiments of the present invention relate to dosage forms for parenteral administration, such as subcutaneous, intravenous, intrahepatic, or intramuscular injection. Optionally, a pharmaceutically acceptable carrier and / or excipient may be present.

[0069] Some embodiments of the present invention relate to dosage forms for topical administration. Those skilled in the art will recognize a wide range of possible formulations for providing topical preparations, as illustrated in, for example, Benson and Watkinson (Eds.), Topical and Transdermal Drug Delivery: Principles and Practice (1st Edition, Wiley 2011, ISBN-13: 978-0470450291); Guy and Handcraft: Transdermal Drug Delivery Systems: Revised and Expanded (2nd Ed., CRC Press 2002, ISBN-13: 978-0824708610); and Osborne and Amann (Eds.): Topical Drug Delivery Formulations (1st Ed. CRC Press 1989; ISBN-13: 978-0824781835). In embodiments of the invention relating to the topical use of the compounds of the invention, pharmaceutical compositions are formulated in a manner suitable for topical administration, such as aqueous solutions, suspensions, ointments, creams, gels, or sprayable formulations, for example for delivery by aerosols, and include active ingredients and one or more of solubilizers, stabilizers, tension enhancers, buffers, and preservatives known to those skilled in the art.

[0070] The dosage regimen of the compounds of this invention will vary based on known factors, such as the pharmacodynamic characteristics of the specific agent and its mode and route of administration; the recipient's species, age, sex, health, medical condition, and weight; the nature and severity of symptoms; the types of concurrent treatments; the frequency of treatment; the route of administration, the patient's renal and hepatic function, and the desired effect. In some embodiments, the compounds of this invention may be administered in a single daily dose, or the total daily dose may be administered in divided doses twice, three, or four times daily.

[0071] The manufacturing method and treatment method of the present invention

[0072] In another aspect, the present invention also includes the use of agents identified herein capable of inhibiting IL-1Ra activity in methods of preparing medicaments for treating or preventing sepsis associated with systemic fungal infections.

[0073] Similarly, the present invention includes a method for treating a patient diagnosed with sepsis associated with a systemic fungal infection. This method requires administering to the patient an effective amount of an agent capable of inhibiting IL-1Ra activity, as detailed herein.

[0074] Whenever alternatives to a single separable feature, such as a homologous protein or coding sequence, or cancer, are listed herein as “implementations,” it should be understood that such alternatives may be freely combined to form discrete implementations of the invention disclosed herein.

[0075] The invention is further illustrated by the following embodiments and accompanying drawings, from which further implementation methods and advantages can be derived. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Example

[0076] Here, we investigated the expression of IL-1Ra in different myeloid cell subsets in a mouse model of invasive candidiasis and examined the effects of IL-1Ra produced by specific cell types on antifungal immunity. We showed that macrophage-secreted IL-1Ra acts as an innate immune checkpoint, preventing effective pathogen clearance; and its targeted clearance protects against lethal Candida albicans sepsis. Furthermore, we found that macrophage-secreted IL-1Ra is positively regulated by type I interferon (IFN), reflecting an association between secondary invasive candidiasis and prior viral infection. In summary, these results provide a mechanistic explanation for high susceptibility to Candida bloodstream infections and suggest IL-1Ra-targeted therapy as a novel treatment approach.

[0077] result

[0078] IL-1Ra is rapidly expressed in bloodstream infections caused by Candida albicans. To determine the correlation of IL-1 signaling during invasive fungal infections, we tested 2.5 × 10⁻⁶ cells in intravenous infections. 5 Expression of IL-1 family cytokines in the kidneys following CFU infection with Candida albicans. IL-1β and IL-1Ra proteins were strongly expressed throughout the infection focus on day 3 post-infection, while IL-1α-positive cells were located at the outer edge of the lesion. Transcripts of IL-1β were already present in the kidneys of juvenile animals, but IL-1R1 and IL-1Ra were only detectable at very low levels. However, fungal infection rapidly induced an increase in IL-1β and IL-1Ra gene expression in the kidneys, while IL-1R1 mRNA expression remained unchanged compared to juvenile mice. Figure 1 A). Furthermore, a considerable level of IL-1Ra protein was present in the serum of infected mice, peaking on day 2 post-infection. Figure 1 B). Conversely, mature IL-1β cytokines remain undetectable in peripheral blood and may be produced locally in infected tissues. IL-1Ra protein expression in the kidneys is consistent with leukocyte recruitment and is limited to inflammatory infiltration, suggesting that immune cells are the primary producers of IL-1Ra. In fact, primary neutrophils, monocytes, dendritic cells, and bone marrow (BM)-derived macrophages secrete IL-1Ra in response to two Candida morphotypes in vitro; monocytes with the highest IL-1Ra levels are exposed to Candida hyphae (…). Figure 1 C). These data confirm that the IL-1β / IL-1R1 axis is an important component of early antifungal immune defense and suggest that IL-1Ra produced by bone marrow cells regulates it.

[0079] Ablation protection against invasive fungal infections is provided by IL-1Ra produced by macrophages. Therefore, we sought to analyze the effect of IL-1Ra produced by specific leukocyte subsets on the Candida albicans immune response by selectively and conditionally deleting IL-1Ra from neutrophils, macrophages, or dendritic cells. We used IL-1Ra... fl / fl Mice were crossed with the corresponding Cre-driven strain to target neutrophils and macrophages (LysM-Cre, referred to as IL-1Ra). LysM In macrophages (Mafb-Cre, denoted as IL-1Ra) Mafb In cells expressing CD11c (CD11c-Cre, referred to as IL-1Ra) and in cells expressing CD11c (CD11c-Cre, referred to as IL-1Ra) CD11c Knock out IL-1Ra ( Figure 1 (D, 8A, and 8B). With IL-1Ra fl / fl Compared with littermate control mice, macrophage-specific knockout of IL-1Ra resulted in IL-1Ra... LysM and IL-1Ra MafbThe early immune control ability of mice against Candida albicans infection was significantly improved. Figure 1 E and 1H). Fungal titers in the kidneys of both strains decreased by the morning of day 3 post-infection and controlled for Candida albicans below the detection limit in most animals within 7 days. Despite IL-1Ra LysM Mice lacked IL-1Ra in both macrophages and neutrophils, but their phenotype was almost identical to that of mice with selective IL-1Ra deficiency only in macrophages. Mafb The same in mice. Furthermore, IL-1Ra... LysM Mouse macrophages and neutrophils showed residual IL-1Ra expression both in vivo and in vitro, while IL-1Ra... Mafb The loss of IL-1Ra in mouse macrophages is highly specific and efficient. Figure 8 (A, 8B). This makes it unlikely that IL-1Ra expressed by neutrophils will make a major contribution, and macrophage-produced IL-1Ra was identified as an important negative regulator of IL-1R signaling during systemic Candida infection. However, elimination of IL-1Ra in CD11c-expressing cells provides weak and only transient protection. Although IL-1Ra CD11c The kidneys of mice showed improvement compared to IL-1Ra on day 3 post-infection. fl / fl Litterctes had lower fungal loads, and this difference disappeared by day 7. Figure 1 G). Therefore, IL-1Ra produced by cells expressing CD11c affects initial antifungal defense but not long-term controls.

[0080] These observations were confirmed by histopathological examination of the infected kidneys, demonstrating their superior ability to initiate early fungal replication. Figure 1 E and 1F), IL-1Ra LysM and IL-1Ra Mafb Mice showed a very limited distribution of Candida albicans and less inflammation in the kidneys on day 3 post-infection. Figure 1 H-1I). Furthermore, compared to the control, IL-1Ra LysM and IL-1Ra Mafb The mice lost weight, which suggests a decrease in their general morbidity. Figure 8 C). In contrast, IL-1Ra CD11c Mice exhibited an intermediate phenotype with reduced Candida spread and inflammation on day 3, but weight loss remained unchanged. Figure 1 G, 1J, and 8C). Consistent with their improved Candida albicans immune control, we found significantly fewer infectious foci and reduced IL-1Ra positive areas in kidney sections of all three IL-1Ra deficient strains. Figure 1H–K). The beneficial effects of IL-1Ra ablation in macrophages were most pronounced in IL-1RaMafb mice, such as its reduction in creatinine and blood urea nitrogen levels (H–K). Figure 1 Lower expression of L) and kidney injury markers Lcn2 and Kim-1 ( Figure 1 As shown in M). In summary, our results confirm that the elimination of IL-1Ra produced by macrophages strongly enhances the ability to control invasive Candida infections, leading to rapid pathogen clearance, reduced disease severity, and better preservation of organ function.

[0081] IL-1Ra controls neutrophil recruitment and fungal killing ability. To investigate how the removal of macrophage-produced IL-1Ra can effectively suppress systemic candidiasis, we characterized antifungal immune responses in corresponding IL-1Ra-deficient strains. Macrophage-specific loss of IL-1Ra increased IL-1Ra levels. Mafb and IL-1Ra LysM Early mobilization and maturation of neutrophils in mice, such as a larger proportion of mature CD101 cells in the intraperitoneal blood on day 2. + Ly-6G high neutrophils as shown ( Figure 2 A–B). This effect occurs in IL-1Ra. Mafb The strongest effect was observed in mice, which also showed a reduced proportion of circulating immature neutrophils and inflammatory monocytes. Figure 2 B).

[0082] The absence of IL-1Ra produced by macrophages also accelerated the recruitment of neutrophils to inflammatory sites. To rule out feedback mechanisms triggered by differential microbial control, we examined the inflammatory response to cell wall components of non-replicating fungi. Yeast glycan-induced IL-1Ra... Mafb and IL-1Ra LysM The peritoneal lavage fluid from mice contained three times and more than twice the number of neutrophils, respectively. Figure 2 (C, 9A, and 9B). Relative to IL-1Ra fl / fl Control, IL-1Ra Mafb and IL-1Ra LysM In mice, tissue-infiltrating neutrophils exhibited significantly enhanced antifungal activity and secreted higher levels of IL-1β, indicating increased activation. Figure 2 (D and 2E). Furthermore, the absence of IL-1Ra in CD11c-expressing cells did not affect the number of neutrophils in the blood, only slightly enhancing their activity in IL-1Ra. CD11c Peritoneal recruitment in mice ( Figure 2 (B and 2C); however, it confers enhanced antifungal ability on tissue-infiltrating neutrophils, similar to that of IL-1Ra. Mafb and IL-1RaLysM Observed in mice ( Figure 2 D).

[0083] Characterization of neutrophil phenotypes in blood, spleen, and kidney revealed a correlation with IL-1Ra. fl / fl Compared with the control group, post-infection IL-1Ra Mafb CD101, which produces high levels of ROS in mouse kidneys + Neutrophils were significantly enriched, and the proportion of neutrophils expressing IL-1β and MPO was even higher. Figure 2 F–I). IL-1Ra LysM and IL-1Ra CD11c Neutrophils in mouse kidneys showed an intermediate phenotype. Figure 2 F–I). CD63 expression is associated with Candida albicans in infected organs, but is not affected by IL1-Ra (F–I). Figure 2 H). IL-1Ra removal did not affect either the neutrophil phenotype or the fungal titer in the spleen (H). Figure 3 F–I and 10D). Therefore, the absence of IL-1Ra produced by macrophages promotes the rapid recruitment of highly fungicidal neutrophils to infected kidneys.

[0084] IL-1Ra Mafb Mice, IL-1Ra LysM Mice and weaker IL-1Ra CD11c In mice, fungal replication in the kidneys was limited on day 3 post-infection (pi). Figure 1 E–1G). As a result, it did not show up in IL-1Ra. fl / fl Excessive inflammatory response to functional impairment detected in controls Figure 1 H–1J), and expresses low levels of pro-inflammatory cytokines (H–1J). Figure 2 K). Furthermore, at this time IL-1Ra Mafb With IL-1Ra LysM The mice had fewer neutrophils and inflammatory monocytes in their kidneys, which may reflect a lower fungal load and faster resolution of inflammation in these mice. Figure 2 J, 4C, and 10A). Extensive tissue RNA-seq and gene set enrichment analysis (GSEA) of infected kidneys indicated that IL-1Ra Mafb The lack of IL-1Ra expression in mouse macrophages suppresses inflammatory pathways, including IL-6 / Jak / Stat3 signaling, IFN-α response, and TNF-α signaling. Besides IL1rn IL-1Ra Mafb Reduced transcripts in the kidneys of mice contain key chemical attractants. Ccl2 , Cxcl2 , Cxcl1 Adhesion molecules Icam1 NFkB signal transduction modulator Nfkbia and antifungal effectors Cybb (Not shown). We infer that the absence of macrophage-specific IL-1Ra promotes rapid neutrophil-mediated pathogen clearance and limits harmful excessive inflammation induced by persistent fungal proliferation.

[0085] Serum IL-1Ra is generated by CD169 in the marginal zone of the spleen. + Macrophage production. Minimal residual Cre activity in neutrophils and monocytes prompts us to focus on the potential intrinsic cellular functions of IL-1Ra. However, partial removal of IL-1Ra from mixed BMCs also affects the recruitment and effector functions of cells derived from bone marrow with IL-1Ra expression and IL-1Ra gene modification. Figure 11 A–11G). This suggests that the extracellular effect may be mediated by IL-1Ra secreted by macrophages. Given these results highlighting the important role of IL-1Ra secreted by macrophages, and hypothesizing that we detected baseline levels of circulating serum IL-1Ra in infected mice ( Figure 1 (B) Next, we attempted to assess the effect of IL-1Ra secreted into the bloodstream and identify macrophage subsets that contribute to the production of serum IL-1Ra.

[0086] Bloodstream infection with Candida albicans stimulates the release of serum IL-1Ra in wild-type mice; and IL-1Ra LysM IL-1Ra Mafb and IL-1Ra CD11c This response was not observed in mice. Figure 3 A). Similarly, lipopolysaccharide (LPS) infusion induced dramatic serum IL-1Ra production, which was significantly reduced in all tested conditionally IL-1Ra-deficient strains (A). Figure 3 B). Intravenous injection of Candida or LPS induced the expression of IL-1Ra protein in the splenic marginal zone. The production of splenic IL-1Ra is equivalent to the presence of serum IL-1Ra, and is significantly different from that IL-1Ra induced by Candida or LPS. LysM IL-1Ra Mafb and IL-1Ra CD11c Significantly reduced in mice (not shown). EYFP expression confirms the presence of EYFP. LysM EYFP Mafb and EYFP CD11c The history of Cre activity in mouse marginal zone cells. Mafb-Cre activity was most extensively observed in EYFP-labeled cells in the spleen marginal zone, and also affected macrophages in the red pulp. Figure 3C). This is related to IL-1Ra Mafb The effective loss of IL-1Ra in the spleen and serum of mice is highly consistent with its strong protective effect against invasive candidiasis. Immunofluorescence co-staining was used to identify CD169 in the marginal zone of the spleen. + Macrophages are the main producers of IL-1Ra, while MARCO + Macrophages were negative for IL-1Ra.

[0087] We directly examined the correlation between splenic IL-1Ra production and serum IL-1Ra response and antifungal immune defense in splenectomized wild-type mice infected with Candida albicans. Compared with a simulated Candida-infected control, preoperative spleen removal reduced Candida-induced serum IL-1Ra levels ( Figure 3 D). Furthermore, splenectomy confers resistance to increased Candida replication, such as through reduced fungal load ( Figure 3 E) The limited spread of Candida and lower levels of inflammation in the kidneys indicate this. To confirm that serum IL-1Ra is indeed generated by splenic CD169... + Macrophage-secreted chlorophosphonate was cleared using a chlorophosphonate clearance protocol, which allows for the utilization of its unique reproliferative kinetics.36 Figure 11 H) to selectively target macrophages residing in the splenic marginal zone. Depletion of splenic marginal zone macrophages and marginal metallophilic macrophages reduced serum IL-1Ra levels to those of uninfected mice ( Figure 3 H), and CD101 + Neutrophils and IL-1β + Increased recruitment of neutrophils to the kidneys is associated with ( Figure 3 I, and 11I–11L). In summary, these findings establish the CD169 in the splenic marginal zone. + A direct correlation was found between IL-1Ra expression in macrophages and serum IL-1Ra production after blood transmission of Candida albicans.

[0088] Serum IL-1Ra represents an innate immune checkpoint mediating impaired pathogen control and dysfunctional hyperinflammatory activity during invasive fungal infections. Our findings to date reveal that macrophage-produced IL-1Ra hinders effective early containment of disseminated candidiasis by limiting neutrophil tissue recruitment and antifungal capacity. It also indicates that IL-1Ra from the spleen CD169... + The substantial contribution of serum IL-1Ra released from macrophages. To examine the effect of circulating IL-1Ra on the Candida immune response, we then administered intravenous IL-1Ra protein containing group IL-1Ra. Mafb The collection of serum IL-1Ra was reconstructed in mice. We infer that due to IL-1Ra... MafbMice are deficient in both circulating serum IL-1Ra and IL-1Ra produced by macrophages in infected tissues, so this method will allow for selective study of the contribution of serum IL-1Ra. Injection of recombinant IL-1Ra elicited IL-1Ra in Candida infections. Mafb Serum IL-1Ra levels in mice were initially higher than those in IL-1Ra competent mice, but remained at wild-type levels for up to 18 hours. Figure 4 (A and 12A). Therefore, we administered injections twice daily throughout the experiment to fully replenish IL-1Ra. Mafb Serum IL-1Ra in mice. Deficiency of IL-1Ra in the splenic marginal zone following Candida infection or in peritoneal exudate during yeast-glycan peritonitis. Figure 4 B) It was confirmed that this regimen only restored serum IL-1Ra, but not the locally secreted cytokines. The reconstruction of serum IL-1Ra almost completely restored IL-1Ra. Mafb The protected phenotype in mice was restored to IL-1Ra fl / fl The protected phenotype in the control group. For example, in IL-1Ra Mafb The enhanced neutrophil recruitment induced by yeast polysaccharide observed in mice was eliminated upon supplementation with serum IL-1Ra. Figure 4 C). Similarly, the reconstitution of serum IL-1Ra will... Mafb The antifungal and phagocytic activities of neutrophils in mice were limited to the levels found in IL-1Ra-competent wild-type mice. Furthermore, neutrophils derived from IL-1Ra... Mafb IL-1Ra from mice rather than IL-1Ra reconstructed Mafb The function of effectors in mouse neutrophils in vitro was expanded by in vitro IL-1β stimulation, indicating that the lack of serum IL-1Ra in vivo increased the sensitivity of neutrophils to local IL-1β stimulation. Figure 4 D and 4E). In fact, when primary neutrophils are pre-pulsed with IL-1Ra in vitro, this eliminates IL-1R signaling upon subsequent exposure to IL-1β, even after the removal of unbound extracellular IL-1Ra. Figure 4 F). From these results, it can be expected that IL-1Ra-reconstructed IL-1Ra Mafb Mice no longer resemble IL-1Ra Mafb The mice showed that Candida replication in their kidneys was controlled as effectively as in mice. Instead, they exhibited higher fungal loads, increased Candida spread, and more severe kidney inflammation. Figure 4 G–4I), therefore showing similarity to IL-1Ra fl / fl The comparison showed similar phenotypes. This indicates that IL-1Ra MafbThe significant immediate resistance to Candida in mice was primarily attributed to the lack of serum IL-1Ra in these mice, highlighting the impact of serum IL-1Ra on early immune control against Candida albicans. Furthermore, this suggests that targeted removal of IL-1Ra may provide an effective strategy to leverage the endogenous antifungal response during disseminated candidiasis.

[0089] Therefore, we tested whether in vivo neutralization of IL-1Ra before infection increased the ability of wild-type mice to contain fungal infections. Figure 12 B). Daily injection of neutralizing antibodies against mouse IL-1Ra effectively depleted serum IL-1Ra but did not affect the expression of IL-1Ra protein in the splenic marginal zone. Figure 4 J). Furthermore, prophylactic IL-1Ra neutralization significantly improved immune control of Candida infections, as evidenced by reduced fungal titers in infected kidneys, limited Candida transmission, and lower tissue inflammation. Figure 4 K and 4L). We observed that serum IL-1Ra concentrations peaked on day 2 post-infection and returned to naïve levels by day 7 post-infection. Figure 1 C and 4M). Therefore, we examined in detail whether therapeutic IL-1Ra neutralization protects against lethal Candida albicans infection (C and 4M). Figure 12 C). In fact, IL-1Ra neutralization initiated on day 2 post-infection reduced fungal titers and significantly improved the survival rate of wild-type mice after high-dose Candida albicans challenge (C). Figure 4 M, 4N, and 4O). IL-1Ra LysM IL-1Ra Mafb and IL-1Ra CD11c The phenotype of mice has revealed that early inhibition of fungal replication can prevent infection in wild-type IL-1Ra. fl / fl Functional inflammatory response observed in controls ( Figure 2 These observations were confirmed after serum IL-1Ra depletion and reconstitution. Mafb Mice exhibited very low levels of pro-inflammatory cytokines in the kidneys, and the reconstitution of serum IL-1Ra in these mice not only hindered its ability to limit Candida proliferation ( Figure 4 G), and also caused by IL-1Ra fl / fl Similar excessive kidney inflammation observed in mice ( Figure 4 Conversely, neutralization of IL-1Ra promoted rapid immune control of Candida in wild-type mice and was associated with a significant reduction in the inflammatory spectrum in their kidneys. Figure 4These data directly link macrophage-produced IL-1Ra to ineffective immune control and resulting dysfunctional inflammatory responses in invasive fungal infections; thus suggesting that serum IL-1Ra serves as a biomarker and potential therapeutic target for disseminated candidiasis.

[0090] IL-1Ra secreted by infiltrating macrophages limits pathogen clearance from infected tissues. Our findings demonstrate that serum IL-1Ra has a significant impact on immune defense against Candida. However, in IL-1Ra... CD11c The transient protection observed in mice suggests that the later stages of the response are influenced by IL-1Ra, which is secreted by a second population of macrophages sensitive to Mafb-Cre rather than CD11c-Cre gene deletion. Therefore, we investigated the relevance of locally generated IL-1Ra in infected tissues and characterized the IL-1Ra response of renal infiltrating leukocytes. Neutrophils, inflammatory monocytes, and renal resident macrophages comprised the major leukocyte populations present in the kidney on day 3 post-infection (…). Figure 2 J). Analysis of FACS purified cells from infected mice showed that IL-1Ra mRNA expression was highest in neutrophils, while IL-1β and IL-1R1 were expressed at comparable levels in all three populations. Figure 5 A). Nevertheless, in vitro exposure of untreated primary cells to Candida stimulation resulted in monocytes secreting significantly more IL-1Ra protein than neutrophils. Simultaneous stimulation with IL-1β cytokines did not affect IL-1Ra secretion in either monocytes or neutrophils. Figure 5 B). However, we found that type I IFN significantly increased Candida-induced IL-1Ra secretion in monocytes, but did not affect IL-1Ra release from neutrophils (B). Figure 5 B). Similarly, neutrophils isolated from infected kidneys secrete low levels of IL-1Ra, regardless of in vitro exposure to Candida or type I IFN (…). Figure 5 C). Conversely, inflammatory mononuclear responsive Candida albicans isolated from the same animal released large amounts of IL-1Ra, which was further increased by additional IFNβ stimulation (C). Figure 5 C). Western blot analysis revealed that neutrophils primarily express the 16 kDa intracellular isoform of IL-1Ra, which could explain the differential mRNA expression and cytokine secretion detected in these cells in response to Candida. Figure 5 D). In summary, these data identify inflammatory monocytes as the primary early IL-1Ra producers in infected tissues and suggest a positive regulation of their response by type I IFN.

[0091] Intracellular mRNA staining, performed using Prime Flow analysis, confirmed that inflammatory monocytes represented more than 50% of total IL-1Ra mRNA-positive leukocytes on day 2 post-infection. Figure 5 E). However, in IL-1Ra Mafb or IL-1Ra CD11c The contribution of IL-1Ra secreted by monocytes observed in mice to the enhanced protective effect appears unlikely, since both strains contain IL-1Ra mRNA-positive monocytes at a wild-type ratio. Figure 5 F), and assume EYFP Mafb and EYFP CD11c The report mice showed negligible Cre activity in kidney-infiltrating mononuclear cells. Figure 5 G and 5H). However, we observed a second Ly-6Cl MHC II on day 5 post-infection. + F4 / 80 + The IL-1Ra producing population, presumably composed of monocyte-derived macrophages, comprised 40% of IL-1Ra producers by day 5 post-infection. Figure 5 I and 5J). This subgroup is in EYFP. Mafb China and Belgium at EYFP CD11c Higher cre activity was observed in the report mice, indicating that IL-1Ra Mafb IL-1Ra will be more effectively lost in these cells of mice. Figure 5 J). Assuming that type I IFN increases IL-1Ra secretion from monocytes, we examined its effect on the IL-1Ra response produced by macrophages. Type I IFN does not require inducing a serum IL-1Ra response to Candida infection because IFNAR signaling-deficient mice (IFNAR...) - / - Wild-type IL-1Ra was expressed in the spleen and serum. Figure 5 K), and controlled fungal replication in the kidneys compared to the control ( Figure 5 The same applies to L and 12E. Type I IFN preferentially enhances IL-1Ra secretion in a subset of MafbCre-tagged monocyte-derived macrophages, which we have identified as key IL-1Ra producers. Specifically, from EYFP on day 5 post-infection... Mafb All CD11c isolated from infected kidneys of mice + In macrophages, only EYFP + Populations labeled with MafbCre showed a response to massive IL-1Ra secretion from Candida, which amplified upon accompanying IFN-β stimulation. Figure 5In summary, these results highlight the influence of IL-1Ra secreted by monocyte-derived macrophages in infected tissues. Furthermore, they also suggest that IL-1Ra... Mafb The particularly effective gene targeting of this type I IFN-sensitive population in mice contributes to its sustained long-term protection against Candida.

[0092] Type I IFN amplifies macrophage IL-1Ra responses and exacerbates fungal sepsis. Previous viremia predisposes hospitalized patients to secondary invasive fungal infections, and our data demonstrate that type I IFN signaling enhances Candida-induced macrophage-derived IL-1Ra secretion; therefore, we evaluated whether IFN-I responses induced in viral infections could induce IL-1Ra as a permissive factor for subsequent invasive candidiasis. Intravenous injection of IFN-β confirmed its ability to directly elicit serum IL-1Ra responses (…). Figure 6 A). Furthermore, intravenous administration of the synthetic TLR3 agonist polyinosinic-polycytidylic acid (PIC)—an established method for inducing robust IFN-I production in mice—rapidly triggered IL-1Ra mRNA and protein expression in the spleen and induced significant levels of circulating IL-1Ra in serum. Figure 6 B and 6C). However, PIC in IL-1Ra Mafb The absence of such IL-1Ra production in mice suggests that fungal infection and IFN-I stimulation of the same macrophage population release IL-1Ra ( Figure 6 C). Furthermore, PIC treatment significantly amplified the Candida-induced IL-1Ra response in the spleen and serum (C). Figure 6 D), resulting in mice becoming highly susceptible to fungal infections. Specifically, PIC-treated mice, when treated with 2.5 × 10⁻⁶ mg / L, showed improved susceptibility to fungal infections. 5 CFU Candida infection showed a significantly increased morbidity and required premature removal from the experiment, even though the fungal dose was tolerated in the absence of PIC-induced IFN-I. Figure 6 E). Conversely, PIC-induced IFN-I failed to trigger detectable IL-1Ra production and did not exacerbate disease severity in Candida-infected IL-1Ra Mafb mice. Figure 6 E). This confirms that IFN-driven expansion of IL-1Ra produced by macrophages is a potential mechanism for increased susceptibility to Candida infection in wild-type mice after PIC injection. Co-injection of PIC also exacerbated IL-1Ra... + / + Disease severity in mice infected with lower levels of Candida inoculum ( Figure 6 F). Although we only accept 10 5Lowest levels of serum IL-1Ra were detected in mice with CFU Candida albicans. Simultaneous induction of IFN-I strongly enhanced the serum IL-1Ra response, interfered with early immune control of Candida albicans, and increased the fungal titer in the kidneys by two orders of magnitude. Figure 6 F). These observations were confirmed by infection with microbial pathogens known to stimulate IFN-I production in vivo, such as lymphocytic choriomeningitis virus (LCMV), vesicular stomatitis virus (VSV), vaccinia virus (VV), or Listeria monocytogenes, which induced IL-1Ra expression in the spleen, albeit to varying degrees (not shown). In particular, potent IFN-I inducers LCMV and VSV induced strong serum IL-1Ra production in MafbCre-labeled macrophages. Figure 6 G).

[0093] LCMV represents a well-characterized experimental model that encapsulates relevant aspects of systemic viral infection in human patients; therefore, we further investigated the effect of IFN-enhanced IL-1Ra production on susceptibility to fungal bloodstream spread in LCMV-WE infection. Similar to our findings on PIC-induced IFN-I, high-dose LCMV co-infection significantly exacerbated the morbidity of Candida-infected mice; and no longer contained the fungal dose tolerated in control mice without LCMV co-infection, leading to a worsening morbidity rate. Figure 6 H). Although low-dose LCMV infection in mice was able to control lower Candida inoculum levels (10). 5 CFU) until day 3 post-infection, however, it showed very high levels of serum IL-1Ra (CFU). Figure 6 I) and showed uncontrolled fungal spread in its kidneys ( Figure 6 J). The aggravating effect of viral infection was strictly type I IFN-dependent, as co-infected IFNAR-deficient mice completely lacked the increased IL-1Ra response in the spleen and serum; and their fungal titers were comparable to those of mice infected with Candida albicans alone. Figure 7 (A and 7B). Therefore, the kidneys of co-infected IFNAR-deficient mice contained isolated foci of infection, thus resembling those of controls infected with Candida albicans alone, while the kidneys of co-infected wild-type mice showed unrestricted fungal spread. IFN-regulated genes Ipit1 , Isg15 and Mx1 The transcripts revealed that Candida albicans infection itself induces considerable IFNAR signaling in the spleen and kidneys, which is further enhanced by virus-induced IFN-I. Figure 7 C and 7D). In the case of Candida albicans infection. IL1rn Expression remains IFN-independent, while viral amplification is partially IFNAR-dependent. Figure 7C and 7D). IL-1Ra Mafb Co-infection in mice confirmed that LCMV-induced IFNAR signaling led to the production of serum IL-1Ra by MafbCre-labeled macrophages. Figure 7 E). Furthermore, this reveals that virus-induced IFN-I exacerbates Candida infection, partly by amplifying the IL-1Ra response produced by macrophages, and also through an IFNAR-dependent mechanism unrelated to IL-1Ra produced by macrophages. Figure 7 In summary, these observations suggest that type I IFN induced during viral infection strongly increases susceptibility to Candida bloodstream infection, has detrimental effects on host survival, and suggests the production of IL-1Ra by macrophages during this process.

[0094] discuss

[0095] Invasive fungal infections remain an urgent and underresolved medical problem due to their high mortality rates and limited treatment options. Our study reveals the disease mechanisms contributing to high susceptibility to disseminated candidiasis and their known amplification through viral co-infection. We identified serum IL-1Ra produced by macrophages as an innate immune checkpoint that promotes disease and can be inhibited to defend against fatal candidal sepsis in a mouse model. These findings are crucial for our understanding of the pathogenesis of invasive fungal infections and may open new avenues for their treatment.

[0096] Our results highlight the crucial role of serum IL-1Ra during invasive candidiasis. Serum IL-1Ra produced by hepatocytes has been described as present in various inflammatory conditions. Conversely, we identified splenic CD169... + Macrophages are the major producers of serum IL-1Ra during fungal infections. While serum IL-1Ra may be beneficial by preventing excessive IL-1 signaling in bacterial sepsis, we found it to be detrimental during invasive candidiasis. Genetic ablation of macrophage-produced IL-1Ra and liposome consumption by marginal macrophages inactivate serum IL-1Ra levels and enhance protective neutrophil responses, thereby highlighting CD169. + Effects of macrophage-derived IL-1Ra. Marginal zone macrophages sense blood-borne pathogens, including Candida, to guide the induction of innate and adaptive immunity. Following spleen colonization, CD169... + G-CSF released by macrophages promotes neutrophil dysfunction in fungal sepsis. We could not detect any effect on splenic neutrophils in IL-1Ra-deficient mice. Figure 2(F, 2G, 2H, 2I, and 10A), but can modulate neutrophil functional and excessive renal inflammation by remodeling or neutralizing IL-1Ra. This suggests that CD169 + Macrophage-derived IL-1Ra suppresses antifungal immunity by limiting the rapid tissue recruitment of IL-1-driven maturing and highly fungicidal neutrophils. Figure 2 and 10 A). We detected a second wave of IL-1Ra produced by macrophages in infected tissue; and effective targeting of this population may contribute to IL-1Ra. Mafb Excellent resistance to Candida in mice. Besides macrophages, renal-infiltrating neutrophils and monocytes showed considerable IL-1Ra expression, while expression in dendritic cells or renal-resident macrophages appeared negligible. Neutrophils primarily contain the intracellular IL-1Ra isoform and secrete limited amounts of IL-1Ra in vitro. This suggests that IL-1Ra expressed by neutrophils is primarily biologically active after neutrophil death and release. Unlike neutrophils, monocytes secrete large amounts of IL-1Ra, which is further enhanced by IFN-I stimulation. The Cre-driven vector used here does not delete IL-1Ra in monocytes (…). Figure 5 Furthermore, we could not conclude the extent to which IL-1Ra secreted by monocytes affects immune defense against disseminated Candida. However, despite the presence of IL-1Ra expressed by monocytes, we observed evidence of a strong protective effect against macrophage-expressed IL-1Ra, demonstrating the primary role of macrophage-secreted IL-1Ra.

[0097] Our study identified neutrophils as a key defense mechanism regulated by IL-1Ra secreted by macrophages and demonstrated that neutrophil maturation, tissue recruitment, and enhanced function mediate the protective effect of IL-1Ra removal. Neutrophil effector pathways, including phagocytosis, ROS production, and NET formation, are essential for systemic antifungal immunity. Our data suggest that neutrophil exposure to blood-derived IL-1Ra determines their IL-1β responsiveness after recruitment to inflamed tissues. Figure 4 IL-1 controls neutrophil aggregation behavior and the execution of their defense mechanisms during antifungal responses. Therefore, in the absence of IL-1Ra, neutrophils are observed to have higher sensitivity to IL-1β stimulation. Figure 4 Increased production of IL-1β and IL-1β Figure 2 This can enhance the neutrophil-directed activity of IL-1. IL-1 signaling is likely also increased in other cell types, including endothelial cells, which increases IL-1Ra. MafbSuperior neutrophil response in mice. Enhanced IL-1 signaling also protects against Candida albicans by promoting granulocyte production and through a neutrophil-independent mechanism.

[0098] We found that macrophage-secreted IL-1Ra is positively regulated by IFN-I. Type I IFN signaling is involved in host defense against Candida in human patients. However, its role in protective immunity remains controversial, as positive and negative disease outcomes have been reported in IFNAR-deficient mice. We did not observe a significant effect of IFNAR deficiency on Candida monoinfection, which may signal differences in genetic background or microbiota composition between these studies. Candida stimulates the secretion of IFN-I from conventional dendritic cells, which increases its antifungal capacity and allows monocytes to promote protective NK cell and neutrophil responses. Conversely, type I IFN exacerbates the severity of Candida infection by inhibiting inflammatory activation and the production of bioactive IL-1, as well as by promoting inflammatory kidney injury. Furthermore, the IFN-induced factor IFIT2 limits ROS production and the antifungal activity of leukocytes. We demonstrate that IFN-I inhibits protective IL-1 signaling by increasing macrophage-secreted IL-1Ra (…). Figure 6 and 7 This suggests that IL-1Ra interferes with both type I IFN-driven and IL-1-driven inflammation in invasive candidiasis, as proposed for bacterial infection. Candida induces IFNAR-independent IL-1Ra production, while additional IFN-I signaling significantly amplifies the Candida-induced IL-1Ra response. IFN-I is induced by many pathogens, and prior viremia constitutes a risk factor for invasive candidiasis. We found that viral co-infection significantly exacerbated disease mortality through IFN-dependent increases in IL-1Ra production in Mafb-Cre-labeled macrophages, providing a potential pathogenesis for secondary candidiasis or multimicrobial sepsis. Targeting IL-1Ra in such IFN-I / IL-1 crosstalk may prove advantageous, as it would enhance protective IL-1R signaling by increasing the potency of physiologically secreted endogenous IL-1 without impeding type I IFN-induced beneficial defense mechanisms.

[0099] IL-1-mediated inflammation is absolutely essential for controlling Candida bloodstream infections, as clearly demonstrated by the fact that defects along the IL-1 pathway dramatically increase disease mortality. However, a lack of IL-1Ra to maintain IL-1 balance due to genetic defects or neutralizing anti-IL-1Ra autoantibodies leads to uncontrolled IL-1 signaling and multi-organ inflammatory syndrome. Impaired IL-1 / IL-1Ra balance is associated with an excessive inflammatory state, and anakinin is thought to be used in bacterial sepsis. Conversely, we see IL-1Ra deficiency...Mafb No exacerbated multi-organ inflammation was observed in mice or during Candida infection following IL-1Ra neutralization. Instead, IL-1Ra removal not only led to faster pathogen clearance but, surprisingly, also resulted in a rapid and paradoxical reduction of the pathogen-induced hyperinflammation observed in mice expressing functional IL-1Ra. Therefore, the dysfunctional inflammatory response during Candida sepsis was not caused by excessive IL-1 signaling but by the inability to eliminate the pathogen. Therapeutic neutralization significantly improved survival in wild-type mice, which will facilitate the development of more effective IL-1Ra-targeting approaches. We hypothesize that enhancing IL-1-driven mechanisms by neutralizing IL-1Ra may be beneficial for patients with active fungal replication but may not be suitable for inflammatory conditions triggered by residual fungal antigens. For example, IL-1α-induced neutrophilic inflammation is required to clear pulmonary Aspergillus infection but exacerbates the disease consequences of Aspergillus-induced asthma. Thus, increased neutrophil recruitment following IL-1Ra neutralization may promote resistance to invasive fungal infections but may also maintain pathogenic inflammation against non-replicating fungal components. Whether such mechanisms contribute to immune reconstitution inflammatory syndrome (IRIS) in chronic disseminated candidiasis remains to be investigated.

[0100] In summary, by removing the endogenous IL-1 inhibitor IL-1Ra instead of the IL-1 cytokine or its receptor, our study allowed for two aspects: highlighting the effects of the physiological response by enhancing the potency of the IL-1 response, and gaining valuable insights into its regulation by IL-1Ra expressed by different immune cell subsets. While this approach confirms the beneficial role of IL-1 in antifungal defense, it also exposes the detrimental consequences of macrophage-secreted IL-1Ra in suppressing bloodstream Candida infection. Moreover, it reveals that the increased inflammation observed during invasive candidiasis does not reflect excessive signaling via IL-1R, yet the fungal pathogen was not successfully eliminated. In conclusion, these observations suggest that serum IL-1Ra serves as a future biomarker and a potential therapeutic target for invasive candidiasis. Attached Figure Description

[0101] Figure 1 Ablation protection against invasive fungal infections from IL-1Ra produced by macrophages.

[0102] (A) mRNA expression of IL-1β, IL-1Ra and IL-1R in the kidneys at the indicated number of days post-infection (n=4–8 mice / time point, pooling in two experiments).

[0103] (B) Quantification of serum IL-1Ra at the indicated number of days post-infection (n=12–20 mice / time point).

[0104] (C) When stimulated in vitro with LPS, heat-inactivated Candida albicans yeast or hyphae, IL-1Ra is produced by the indicated myeloid cell subset.

[0105] (D) Conditional IL-1Ra defective strains were produced.

[0106] (E–G) IL-1Ra on days 3 and 7 post-infection LysM IL-1Ra Mafb IL-1Ra CD11c Mice and wild-type IL-1Ra fl / fl Candida titer in mouse kidneys (n≥7 mice / group, three pooling experiments).

[0107] (H–K) Histopathological analysis (HJ) and quantification of IL-1Ra positive areas (K).

[0108] (L) Plasma creatinine and BUN concentrations of the strain shown on day 2 post-infection. Dashed lines indicate the initial baseline. (n = 7–13 mice / group, two pooling experiments).

[0109] (M) Quantification of renal Kim-1 and Lcn2 mRNA expression. (n=4–9 mice / group, two pooling experiments).

[0110] Error bars represent the mean ± SEM.

[0111] Figure 2 IL-1Ra produced by macrophages prevents rapid neutrophil recruitment and inflammation resolution.

[0112] (A–B) On day 2 post-infection, the Ly-6G fluorescence intensity of neutrophils in blood leukocytes of the strains shown was analyzed (A), and the proportions of mature neutrophils, immature neutrophils, and monocytes were analyzed (B). (n=6–8 mice / group, two pooling experiments).

[0113] (C) Absolute counts of neutrophils and monocytes in the peritoneal lavage fluid of the mice shown 18 hours after injection of yeast polysaccharide.

[0114] (D, E) Antifungal activity (D) and IL-1β secretion (E) of renal neutrophils purified from the mice shown on day 2 post-infection. (n=6 mice / group).

[0115] (F–I) CD101 in the blood, spleen, and kidney of the strain shown on day 2 post-infection. + ROS hi (F), pro-IL-1β + (G), C63 + (H), and MPO+ (I) The proportion of neutrophils. (n=4 mice / group, two representative experiments).

[0116] (J) Kidney inflammation in mice was characterized by absolute counts of leukocyte subsets (J) and cytokine concentrations (K) on day 2 post-infection. (n=6 mice / group).

[0117] Error bars represent the mean ± SEM.

[0118] Figure 3 Serum IL-1Ra is produced by CD169 + marginal zone macrophages produced

[0119] (A–B) Serum IL-1Ra expression in mice on day 3 post-infection (A) or 5 hours post-LPS injection (B). Scale bar, 200 µm. (n = 6–15 mice / group, combined into 3 experiments).

[0120] (C) Schematic diagram showing the EYFP reporter gene used in (F).

[0121] (D, E) On day 3 post-infection, serum IL-1Ra (D) and Candida titer (E) were analyzed in splenectomized (SE) mice and control (ctrl) mice.

[0122] (F, G) Histopathological evaluation of fungal dissemination (F) and inflammation (G) (n=4–6 mice / group, representative experiments).

[0123] (H, I) On day 3 post-infection, serum IL-1Ra (H) and renal CD101 levels were analyzed in clophosphamide-treated (CL) mice and control (CTRL) mice. + and pro-IL-1β + Cell (I).

[0124] Error bars represent the mean ± SEM.

[0125] Figure 4 During disseminated Candida infections, serum IL-1Ra mediates impaired pathogen control and dysfunctional hyperinflammatory processes.

[0126] (A, B) Analysis of IL-1Ra in serum (A) during Candida albicans infection and peritoneal lavage fluid (B) during yeast polysaccharide-induced peritonitis. Mafb IL-1Ra recombinant in mice. (A, n=7 mice / group; C, n=9 mice / group).

[0127] (C–E) Characterization of IL-1Ra reconstructed IL-1Ra by absolute count (C), fungicidal neutrophil activity (D), and phagocytosis (E). Mafb Peritoneal infiltration occurred in mice 18 hours after intraperitoneal injection of yeast polysaccharide. (n=6 mice / group, two pooling experiments).

[0128] (F) Western blot analysis of IL-1R signal transduction in neutrophils with or without prior IL-1Ra pulse treatment.

[0129] (G, H) Mice undergoing IL-1Ra remodeling were characterized by fungal titer (G) and cell infiltration (H) on day 3 post-infection. (n=7 mice / group, two pooling experiments).

[0130] (I) Histopathological quantification of fungal replication and inflammation.

[0131] (J–L) Prophylactic IL-1Ra neutralization (NT) was detected by serum IL-1Ra expression (J) and fungal kidney titer (K) on day 3 post-Candida infection. Scale bar, 1000 µm. (L) Histopathological quantification of Candida replication and inflammation (n=6–10 mice / group, three pooling experiments).

[0132] (M–O) Therapeutic IL-1Ra neutralization in wild-type mice infected with Candida albicans was assessed by serum IL-1Ra (M), renal Candida albicans titer (N), and survival (O). (n = 6–7 mice / group, two pooling experiments).

[0133] (P, Q) Inflammatory signature profile in the kidneys of the mice shown, determined by a cytokine array after IL-1Ra reconstruction (P) or neutralization (Q).

[0134] Error bars represent the mean ± SEM.

[0135] Figure 5 IL-1Ra from infiltrating macrophages limits the clearance of pathogens in infected tissues.

[0136] (A) mRNA expression of IL-1 family genes in the shown cell subset on day 2 post-infection.

[0137] (B, C) In vitro, IL-1Ra was produced from neutrophils and monocytes stimulated with Candida albicans in the presence or absence of IL-1β or IFN-β. Cells were purified from naïve mice (B) or on day 2 post-infection (C). (B, replicate culture, n=4 mice; C, n=4–7 mice).

[0138] (D) Western blot analysis of IL-1Ra isotypes in Candida-stimulated neutrophils and monocytes.

[0139] (E) The proportion of neutrophils and monocytes in renal leukocytes expressing IL-1R mRNA on day 2 post-infection. (n=7 mice / group, two pooling experiments).

[0140] (F) On day 2 post-infection, IL-1Ra fl / fl IL-1Ra Mafb and IL-1Ra CD11c The proportion of IL-1Ra mRNA-expressing cells in the indicated immune cell subsets of mice. (n=7 mice / group, two experiments).

[0141] (G–J) EYFP on day 2 (GI) and day 5 (J) post-infection. Mafb Mice and EYFP CD11c Analysis of Cre expression in mouse leukocytes expressing IL-1 RamRNA. EYFP Mafb (G) and EYFP CD11c (H) Expression of EYFP and IL-1Ra mRNA in mouse neutrophils or monocytes was detected and quantified by flow cytometry. (n=4-8 mice / group).

[0142] (I, J) On day 2 (I) and day 5 (J) post-infection, EYFP Mafb and EYFP CD11c Ly6G, which expresses IL-1 RamRNA in mice - CD11b + The proportion of cells expressing EYFP in the cell line. (n=4–8 mice / group).

[0143] (K, L) On day 3 post-infection, IFNAR was characterized by serum IL-1Ra expression (K) and renal fungal load (L). - / - Mice. (n=4–8 mice / group).

[0144] (M) EYFP purified by in vitro flow cytometry on day 5 post-infection. Mafb IL-1Ra secretion by macrophages under the in vitro stimulation conditions shown. (n=6 mice / group, two pooling experiments).

[0145] Error bars represent the mean ± SEM.

[0146] Figure 6 Type I IFN amplifies macrophage IL-1Ra responses and exacerbates fungal sepsis.

[0147] (A, B) The induction effect of type I IFN on IL-1Ra in vivo was assessed by serum IL-1Ra (A) and renal mRNA expression (B) after injection of IFN-β or PIC, respectively. (n=7 mice / group, two pooling experiments).

[0148] (C, D) IL-1Ra levels were determined in serum (C, D) 5 hours after PIC injection (C) or 48 hours after another Candida infection (D). fl / fl and IL-1Ra Mafb PIC-induced IL-1Ra expression in mice. (C, n=4–7; B, n=3; E, n=3–6 mice / group).

[0149] (E) Inoculate Candida albicans and PIC IL-1Ra as shown. fl / fl Mice and IL-1Ra Mafb Survival rate of mice. (n=6 mice / group, two pooling experiments).

[0150] (F) Kidney fungal load (F) in mice treated according to group (E) as assessed on day 2 post-infection. (n=6 mice / group).

[0151] (G) Post-infection IL-1Ra fl / fl Mice and IL-1Ra Mafb Serum IL-1Ra levels in mice. The dashed line represents the baseline level in the untreated group. (n=3–7 mice / group, single experiment).

[0152] (H) shows the survival rate of infected mice in each group. (n=4 mice / group).

[0153] (I, J) On day 3 post-infection, co-infected mice were characterized by serum IL-1Ra expression (I) and renal Candida titer (J). (n=4 mice / group, representative experiment).

[0154] Error bars represent the mean ± SEM.

[0155] Figure 7 The exacerbation of virus-induced fungal transmission is heavily dependent on type I IFN and IL-1Ra produced by macrophages.

[0156] (A–D) On day 3 post-infection, IFNAR was analyzed in Candida infection (Ca) or LCMV co-infection with Candida (LCMV+Ca) by PAS staining and IL-1Ra staining of serum IL-1Ra (A), renal fungal titer (B), and mRNA expression of interferon-stimulated genes in spleen (C) and kidney (D). - / -Mice and wild-type mice. (n=5–7 mice / group, two pooling experiments). (E, F) IL-1Ra infection as described in (AE). fl / fl and IL-1Ra Mafb Serum IL-1Ra levels (E) and renal Candida titers (F) in mice. (n=4-5 mice / group, single experiment). Error bars represent mean ± SEM.

[0157] Figure 8 The efficiency and specificity of the Cre-driven vector used in this study. Figure 1 Related.

[0158] (A) In vitro evaluation of IL-1Ra LysM IL-1Ra Mafb IL-1Ra CD11c Mice and their respective IL-1Ra fl / fl Efficiency of cre-mediated IL-1Ra loss in the indicated cell subsets of the littermate control. Bone marrow-derived macrophages, bone marrow neutrophils, and spleen dendritic cells were stimulated with 10 ng / ml LPS for 24 h. IL-1Ra production was then assessed by ELISA in culture supernatant or by Western blotting in cell lysates. Representative Western blots are shown.

[0159] (B) Flow cytometry analysis of cells isolated from juvenile EYFP cells LysM EYFP Mafb and EYFP CD11c EYFP Cre reporter protein expression in designated immune cell subsets of bone marrow, spleen, and kidney in mice and their respective Cre-negative EYFPLL littermates.

[0160] (C) IL-1Ra in Candida albicans infection LysM IL-1Ra Mafb IL-1Ra CD11c and their respective IL-1Ra fl / fl Body weight of littermates. Body weight on days 3 and 6 post-infection, expressed as a percentage of initial body weight before infection (day 0). Dots represent individual mice. Figure A shows pooled data from three experiments, with at least six mice in each group. Figure B shows pooled data from one experiment, with two mice in each group. Bars indicate mean ± SEM.

[0161] Figure 9 Inflammatory response in conditionally IL-1Ra-deficient mice infected with Candida albicans. Figure 2 Related.

[0162] (A–B) Flow cytometry was used to identify IL-1Ra at 6 and 18 hours after intraperitoneal infusion of yeast polysaccharide A. fl / fl IL-1Ra LysM IL-1Ra Mafb and IL-1Ra CD11c Absolute counts of neutrophils and monocytes in the lavage fluid (A) and blood (B) of mice. Dots represent individual mice. Data representing two experiments are shown. Bars indicate mean ± SEM.

[0163] Figure 10 IL-1Ra in Candida albicans infection Mafb Early neutrophil recruitment and function in mice. Figure 2 Related.

[0164] (A) IL-1Ra on days 1, 2, and 3 after Candida albicans infection. fl / fl and IL-1Ra Mafb Kinetics of neutrophil recruitment in the spleen and kidney of mice. Representative data from two experimental groups, four mice in each group.

[0165] (B–C) Production of reactive oxygen species (ROS) in neutrophils as measured by luminol-enhanced chemiluminescence assay. (B) On day 2 post-Candida albicans infection, from IL-1 Rafl / fl and IL-1Ra Mafb Neutrophils isolated from mouse kidneys. (C) 18 hours after intraperitoneal injection of yeast polysaccharide A, from IL-1Ra fl / fl and IL-1Ra Mafb Neutrophils isolated from mouse lavage fluid.

[0166] (D) On day 2 after Candida albicans infection, IL-1Ra LysM IL-1Ra Mafb IL-1Ra CD11c Mice and their respective IL-1Ra fl / fl Splenic fungal titers in littermate control mice. Data were pooled from two experimental groups, eight mice per group. Bars represent mean ± SEM.

[0167] Figure 11 The inhibitory effect of IL-1Ra is extracellular and mediated by IL-1Ra secreted by macrophages in the splenic marginal zone. Figure 3 Related.

[0168] (A) 18 hours after intraperitoneal infusion of yeast polysaccharide A, EYFP LSL EYFP LysM EYFP Mafb、 and EYFP CD11cEYFP in mouse blood and peritoneal lavage fluid + The proportion of cells in the shown cell subpopulation.

[0169] (B) Protocol for generating mixed bone marrow chimeras (BMC). Using an equal proportion of CD45.1 + IL-1RA + / + Wild-type bone marrow (as an internal reference present in all chimeras) and bone marrow derived from IL-1Ra + / + IL-1Ra Mafb IL-1Ra LysM Or compare with IL-1Ra fl / fl The corresponding CD45.2 in mice + Experimental bone marrow reconstitution of allogeneic CD45.1 + IL-1Ra + / + recipient.

[0170] (C–E) Immune cell subsets in the mixed BMC were analyzed on day 3 post-Candida infection. (C) Total CD45 in the kidney of the illustrated mixed BMC, as determined by flow cytometry. + Absolute counts of neutrophils, inflammatory monocytes, and renal resident macrophages. (D) CD45.2 / 2 in neutrophils and monocytes in the kidneys of the mixed BMC mice shown on day 3 post-infection. + Cells (solid column) and CD45.1 / 2 + The proportion of cells (hollow column). CD45.2 / 2 + The cell ratio was normalized to CD45.1 / 2 in the bone marrow. + B220 + The average proportion of cells was determined to normalize potential differences in bone marrow remodeling efficiency among different recipient mice. (E) In vitro determination of IL-1Ra from infected mice after CD11b, Ly6G, CD45.1, and CD45.2 staining and flow cytometry analysis. fl / fl BMC, IL-1Ra LysM BMC and IL-1Ra Mafb The phagocytic activity of neutrophils separated from BMC by the kidney, namely the uptake of pHrodo Green yeast glycan particles.

[0171] (F) Generation of chimeric mice: from a donor strain (IL-1Ra) fl / fl or IL-1Ra Mafb The isolated bone marrow cells were transplanted into irradiated recipient wild-type mice (C57BL / 6), left to stand for at least 8 weeks, and then infected with Candida albicans.

[0172] (G) Kidney fungal load IL-1Ra on days 3 and 7 after Candida infectionfl / fl >B6 and IL-1Ra Mafb B6BMC mice.

[0173] (H) A protocol was used to selectively remove macrophages residing in the peripheral regions of C57BL / 6 mice using clophosphonate liposomes (CL), followed by Candida albicans infection. Mice were sacrificed 3 days after infection.

[0174] (I–L) Absolute counts of neutrophils and monocytes in the kidneys (I) and spleen (J) of mice in the control and CL treatment groups on day 3 post-infection with Candida albicans. (K, L) Absolute numbers of CD101-positive mature neutrophils (K) and activated pro-IL-1β-positive neutrophils (L) in the spleen were assessed by flow cytometry. Figure A shows pooled data from at least three experiments, with at least eight mice in each group. Figures CE show pooled data from two experiments, with five mice in each group. Figure G shows pooled data from two experiments, with four mice in each group. Data in Figures I–L are pooled from two experiments, with twelve mice in each group. Bars indicate mean ± SEM.

[0175] Figure 12 The neutralizing effect of serum IL-1Ra produced by macrophages improves early pathogen control and prevents excessive inflammation of the kidneys. Figure 4 and Figure 5 Related.

[0176] (A) Experimental protocol for rIL-1 Ra reconstruction.

[0177] (B) Experimental design for preventive IL-1Ra neutralization.

[0178] (C) Experimental design for therapeutic IL-1Ra neutralization. Monoclonal IL-1Ra antibodies were administered intravenously from day 2 to day 6 post-infection. Mice used for survival assessment received a dose of 3.5 × 10⁻⁶. 5 CFU was used, while the injection dose for mice used in the fungal titer assays on days 5 and 7 was 2 × 10⁻⁶. 5 CFU.

[0179] (D) IL-1Ra from Candida albicans attack Mafb Mice and IL-1Ra fl / fl Kaplan-Meier survival plot for control. Data were pooled from two experiments with at least seven mice in each group.

[0180] (E) Using 1×10 5 WT and IFNAR-deficient mice were infected with CFU (Candida albicans) for 7 days. Fungal load in the kidneys was assessed using CFU. Data describe the results of one experiment involving four mice in each group.

[0181] Experimental model

[0182] animal

[0183] Mice were bred and housed in the specific pathogen-free (SPF) animal facility of the Institute of Pathology, University of Bern. All experimental procedures were conducted in accordance with ethical guidelines and with the animal testing permits approved by the State of Bern (BE3 / 18 and BE31 / 2021). Mice were kept in 12-hour light-dark cycles with regulated temperature and humidity and unrestricted access to food and water. C57BL / 6J, CD45.1 (B6.SJL-PtprcaPepcb / BoyJ), Mafb-cre (Mafbtm1.1(cre)Kmm / J), and R26R-EYFP (B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos / J) mice were purchased from Jackson Laboratory and housed in-house. IL-1Ra LysM Mice are IL-1Ra fl / fl (Il1rntm1.1Cga) mice were bred by crossing LysM-Cre (Lyz2tm1(cre)Ifo / J) mice and were kindly provided by Marc Donath (Department of Biomedical Sciences, University Hospital, Basel). This was done to obtain IL-1Ra... Mafb and IL-1Ra CD11c In mice, we will use IL-1Ra fl / fl Mice were crossed with either Mafb-cre or CD11c-cre (B6.Cg-Tg(Itgax-cre)1-1Reiz) mice. The CD11c-cre mice were a gift from Manfred Kopf (Institute of Molecular Health Sciences, ETH Zurich). Typically, IL-1Ra... fl / fl Cre-negative littermate mice were used as controls; for some experiments, IL-1Ra... wt / wt Cre-positive mice were used as controls to produce wild-type results. R26R-EYFP mice (here referred to as EYFP) were used as controls. LSL EYFP-cre reporter strains were generated by crossing LysM-cre, Mafb-cre, or CD11c-cre mice with LysM-cre, Mafb-cre, or CD11c-cre mice. These are referred to here as EYFP-cre. LysM EYFP Mafb or EYFP CD11c All mouse strains were backcrossed onto C57BL / 6 for more than 10 generations or bred on a C57BL / 6 background. Age- and sex-matched animals were randomly assigned to experimental groups. To generate bone marrow (BM) chimeras (BMCs), 5 × 10⁻⁶ mice were injected intravenously. 6Twenty-four hours before receiving donor BM cells, six-week-old recipient mice were lethally irradiated with gamma rays using a GammaCell X40 irradiator. The mice received sulfamethoxazole and trimethoprim via drinking water for two weeks and were kept at rest for at least eight weeks prior to the experiment to allow for bone marrow remodeling.

[0184] Candida albicans and inflammation model

[0185] Candida albicans (SC5413) was grown in YPD medium (BD Difco™ YPD Broth, BD Sciences) at 30°C for 18 hours. Candida albicans yeast cells were washed twice in sterile PBS and counted using a hemocytometer. To obtain Candida albicans hyphae, the washed Candida albicans yeast was cultured in RPMI 1640 (10% FBS) at 37°C for 4 hours. Heat-inactivated yeast and hyphae were prepared by culturing at 72°C for 1 hour and then incubated on YPD agar plates at 30°C for 48 hours to confirm complete inactivation. For in vivo experiments, 0.5–2.5 × 10⁻⁶ cells were cultured. 5 Colony-forming units (CFU) of Candida albicans were infected via the lateral tail vein in mice aged eight to ten weeks. To determine fungal organ titers, mice were euthanized, organs were weighed, and homogenized in 0.5% NP-40 water with TissueLyser II (Qiagen) at 25 Hz for 2 × 3 minutes. Serial dilutions of the tissue homogenate in PBS were then plated onto YPD agar and incubated for 24–48 hours. Fungal load was calculated as CFU of Candida albicans per gram of tissue. To evaluate the effects of type I IFN on in vivo IL-1Ra production and Candida albicans infection, mice were injected alone with 2 µg of recombinant mouse IFN-β (Biolegen), and alone or 5 hours before Candida albicans infection with the synthetic TLR3 ligand polyinosinic acid-polycytidylic acid (PIC, InvivoGen). Similarly, alone or one day before Candida albicans infection, 1 × 10⁻⁶ IFN-β was injected. 4 Or 2×10 6 Mice were infected with the lymphocytic choroid plexus meningitis virus (LCMV) strain WE containing plaque-forming units (pfu). 10 6 PFU vesicular stomatitis virus (Indiana strain, VSV Indiana), 2×10 6On day 1 post-infection with pfu vaccinia virus (VV) or 3000 CFU Listeria monocytogenes (strain 10403S), IL-1Ra expression levels in spleen and serum were also detected. Pathogens were diluted from frozen viral stock solutions grown in MDCK (VV), BHK21 (LCMV WE), and Vero (VSV) cells, or freshly prepared as overnight cultures in brain-heart infusion broth (Listeria). To evaluate early cell recruitment induced by the fungal component, mice were intraperitoneally injected with 1 mg of fresh yeast glycan A from Saccharomyces cerevisiae (Sigma) dissolved in sterile PBS. Blood and peritoneal exudates were collected at 6 and 18 hours post-injection. Serum and peritoneal lavage fluid were stored at -80°C before IL-1Ra measurement by ELISA. Leukocytes in blood and peritoneal lavage fluid were characterized by flow cytometry. Prior to evaluating the killing and phagocytic effects of Candida albicans in vitro, peritoneal neutrophils were purified from the lavage fluid using magnetic bead-based separation. To assess LPS-induced IL-1Ra expression, mice were intraperitoneally injected with ultrapure LPS O111:B4 (150 µg / kg, Sigma) and D-galactosamine (800 mg / kg, Carbosynth Ltd.), and serum and tissues were collected 5 hours later.

[0186] Method details

[0187] In vivo operation of spleen macrophages

[0188] Splenectomy was performed under isoflurane anesthesia in a laminar flow hood. The abdominal skin and peritoneum were opened through two small incisions to expose the spleen. The spleen was removed by cauterizing the splenic artery and vein at the splenic hilum. The peritoneum was closed with absorbable sutures, and the skin incisions were closed with wound clamps. Postoperatively, mice were allowed to recover under a heat lamp and remained rested for eight weeks prior to Candida albicans infection. To selectively deplete macrophages in the splenic marginal zone, mice were intravenously injected with 1 mg of commercially available clophosphamide liposomes (Liposma) per animal, while control mice received an equal volume of sterile PBS. Ten days later, all mice were treated with 2.5 × 10⁻⁶ splenic phosphamides. 5 CFU (Candida albicans) infection. Serum and organs were collected on day 3 post-infection for analysis.

[0189] In vivo reconstruction and neutralization of IL-1Ra

[0190] Recombinant human IL-1Ra (anaretin) was kindly provided by Marianne Böni-Schnetzler (Department of Biomedical Sciences, University Hospital of Basel). IL-1Ra was observed on days 0, 1, and 2 following Candida albicans infection. MafbMice received intraperitoneal administration of recombinant IL-1Ra (500 µg per mouse) twice daily. In these experiments, non-recombinant IL-1Ra... fl / fl and IL-1Ra Mafb Mice were injected with an equal volume of sterile PBS. Hybridomas producing neutralizing anti-mouse IL-1Ra antibodies were kindly provided by Naofumi Mukoda (University of Kanazawa, Japan) and Russell Vance (University of California, Berkeley, USA). Monoclonal antibodies were prepared and purified in-house using protein G resin (GenScript). For prophylactic IL-1Ra neutralization experiments in vivo, mice were intraperitoneally injected with either anti-IL-1Ra neutralizing antibodies or the control Ultra-LEAF purified Armenian hamster IgG isotype antibody (BioLegend) one day before infection and on days 1 and 2 post-infection. For therapeutic IL-1Ra neutralization experiments, mice were intravenously injected with either anti-IL-1Ra antibodies or the control antibody on days 2 to 6 post-Candida albicans infection.

[0191] Isolation of white blood cell population

[0192] Leukocytes were isolated from the kidney using a modified protocol by Swamydas et al. The kidney was cut into 1 mm sections using a scalpel. 3Small pellets were placed in 6 mL of serum-free RPMI 1640 medium, and 0.2 mg / mL Liberase™ (Roche) and 0.2 mg / mL DNase I (Sigma) were added. The mixture was digested at 37°C for 45 minutes. At the end of the incubation, an equal volume of complete RPMI 1640 medium was added; the cell suspension was filtered through a 40 µm cell filter and washed twice with PBS. The pellet was then resuspended in 40% Percoll (Sigma), gently spread onto 70% Percoll, and centrifuged at 880 g for 30 minutes at room temperature. Spleen single-cell suspensions were obtained by enzymatic digestion with 2 mg / mL type IV collagenase (Worthington) and 0.2 mg / mL DNase I (Sigma) at 37°C for 45 minutes. Neutrophils were purified using the EasySep™ Mouse Neutrophil Enrichment Kit (StemCell Technologies) supplemented with the following biotinylated antibodies (all BioLegend): anti-CD3ε (3.45 µg / ml), anti-B220 (2.5 µg / ml), anti-TER119 (0.25 µg / ml), anti-F4 / 80 (3.45 µg / ml), anti-CD11c (3.45 µg / ml), anti-CD19 (3.45 µg / ml), anti-NK1.1 (3.45 µg / ml), and anti-CD317 (2.5 µg / ml). Mononuclear cells were isolated using the EasySep™ Mouse Mononuclear Cell Isolation Kit (StemCell Technologies) with biotinylated antibodies (all BioLegend) at the following final concentrations: anti-CD3ε (3.45 µg / ml), anti-B220 (2.5 µg / ml), anti-TER119 (0.25 µg / ml), anti-F4 / 80 (3.45 µg / ml), anti-CD19 (3.45 µg / ml), anti-NK1.1 (3.45 µg / ml), and anti-CD317 (2.5 µg / ml). To analyze gene expression or the production of single leukocyte subsets by IL-1Ra in vitro, single-cell suspensions were first prepared from infected organs as described above. After staining the cell surface to identify leukocyte subsets, single mouse cells were purified by FACS using a MoFlo Astrios EQ sorter (Beckman Coulter) with the flow cytometry core equipment at the Department of Biomedical Research, University of Bern. Cells were then sorted into preheated complete culture medium. Bone marrow cells were collected by rinsing the femur and tibia from both hind legs with sterile PBS. Red blood cells were lysed with ACK lysis buffer, and primary bone marrow neutrophils and bone marrow mononuclear cells were isolated using the EasySep™ Mouse Neutrophil Enrichment Kit and the EasySep™ Mouse Monocyte Isolation Kit, respectively, following the methods described above.To analyze LPS-induced IL-1Ra production in vitro, splenic macrophages and splenic dendritic cells were sorted from a single-cell suspension using anti-CD11b and CD11c immunomagnetic beads (Miltenyi Biotec) according to the manufacturer's instructions. Bone marrow-derived macrophages were prepared by culturing bone marrow cells in complete RPMI 1640 medium supplemented with 10% L929 cell supernatant (as a source of M-CSF). The medium was changed every 3 days, and bone marrow-derived macrophages were harvested for experiments on day 7. Peritoneal macrophages were harvested 4 days after intraperitoneal instillation of 1 ml of 3.8% thioglycolate (Becton Dickinson AG). Perfused cells were washed with PBS and cultured overnight with or without 10 ng / ml LPS (InvivoGen). IL-1Ra production was then assessed by ELISA in the culture supernatant or by Western blotting in cell lysates.

[0193] Flow cytometry

[0194] Single-cell suspensions were prepared as described above and stained at 4°C for 15 minutes with an inactivated / deactivated dye (Invitrogen) and an anti-mouse CD16 / 32 antibody (Biolegend). Immune cell subsets were characterized using antibodies against CD90, CD19, CD49b, CD45, CD11b, CD11C, F4 / 80, Ly-6C, Ly-6G, IA / IE, and CD115 (all BioLegend), as well as antibodies against CD101 (eBioscience™) and SiglecF (Miltenyi Biotec). Neutrophils, resident macrophages, Ly-6... hi Monocytes, Ly-6C lo Monocytes and dendritic cells (DCs) are defined as CD45. + CD11b + Ly-6G hi CD45 + CD11b + F4 / 80 + CD45 + CD11b + F4 / 80 - MHCII - Ly6C hi CD45 + CD11b + F4 / 80 - MHCII - Ly6C - CD11c + and CD45 + CD11b+ F4 / 80 - MHC + CD11c + Mature neutrophils, immature neutrophils, and monocytes in the blood were identified as CD11b. + CD115 - Ly6G + CD101 + CD11b + CD115 - Ly6G + CD101 - and CD11b + CD115 + Ly6C + To characterize the phenotype of neutrophils, cells were stained with antibodies against CD45.2, CD11b, Ly-6G, CD63 (all BioLegend), CD101, and pro-IL-1β (all from eBioscience™) and MPO (Hycult Biotech). Cell populations positive for CD90, CD19, CD49b, CD11c, F4 / 80, IA / IE, and Siglec-F were excluded from flow cytometry analysis. Cells were fixed in 4% paraformaldehyde (PFA) for 5 minutes before obtaining cells. Intracellular IL-1Ra transcripts in renal infiltrating leukocytes were revealed using PrimeFlow™ RNA analysis according to the manufacturer's instructions. Cells were fixed and permeabilized after staining the cell surface against CD45.2, CD11b, CD11C, MHC II, F4 / 80, Ly-6C, and Ly-6G, followed by hybridization with the Il1rn probe, signal amplification, and fluorescent labeling. All samples were acquired on an LSRII cytometer (BD Biosciences) and analyzed using FlowJo (BD Biosciences).

[0195] Reactive oxygen species measurement

[0196] Freshly isolated neutrophils (1 × 10⁶ cells per well) 6Cells were equilibrated at 37°C for 60 min and then stained with 5 µM 2′,7′-dichlorofluorescein (DCF) in serum-free RPMI medium for 30 min, followed by exposure to heat-inactivated Candida albicans (MOI 3) in the presence or absence of IL-1β (20 ng / ml) for 30 min. Cells were then stained with both live and dead fluorescent dyes and labeled with antibodies against CD45.2, CD11b, Ly-6G, and Ly6C. ROS production was quantified in leukocytes isolated from the blood, spleen, and kidney of Candida albicans-infected mice. Cells were incubated with dihydrorhodamine 123 (Sigma Aldrich) at 37°C for 30 min before staining with live and dead fluorescent dyes and antibodies against CD45.2, CD11b, Ly-6G, Ly-6C, and CD101. The proportion of ROS-producing neutrophils was analyzed using FlowJo (BD Biosciences).

[0197] Candida albicans kill test

[0198] To test the antifungal ability, 5×10 4 2 × 10 neutrophils 4 Incubate with *Candida albicans* for 3 hours. Then lyse the cells with 1% NP-40 water, and spread serially diluted lysates onto YPD agar. Incubate at 30°C for 24 hours, then count the colonies. Fungicidal activity is expressed as the percentage of *Candida albicans* inoculum killed in the presence of neutrophils detected in control wells without leukocytes.

[0199] Phagocytic activity

[0200] Phagocytic activity was measured using pHrodo™ Green yeast glycan bioparticles (Thermo Fisher) according to the manufacturer's instructions. Neutrophils were purified from mouse peritoneal cavity induced by yeast glycan A, at 10 5Cells / well were plated in 100 µL of Opti-MEM® (Gibco) in black clear-bottomed 96-well plates (Corning) and equilibrated at 37°C for 1 h. The medium was then replaced with 100 µL of uptake buffer containing 0.5 mg / mL pHrodo™ Green yeast polysaccharide bioparticles, and incubated for another 2 h at 37°C with or without IL-1β (20 ng / mL). Bioparticles in pH 5.0 buffer served as a positive control. Wells without bioparticles and wells containing bioparticles in uptake buffer served as background and negative controls, respectively. Fluorescence intensity was evaluated using an Infinite M200 pro Tecan microplate reader at 490 nm excitation and 535 nm emission. Net phagocytosis was calculated by correcting for the fluorescence intensity of the background and negative controls. Phagocytosis was expressed as a percentage of net fluorescence intensity in the experimental wells compared to the net positive control.

[0201] Blood sampling

[0202] Collect samples from the distal end of the vena cava into syringes containing heparin (for creatinine and blood urea nitrogen) or EDTA (for cytokine analysis) as anticoagulants. All samples were kept on ice during processing. Transfer blood to a blood collection tube (BD Microtainer) for serum separation or a 1.5 ml reaction tube for plasma collection and centrifuge at 2000 g for 10 minutes at 4°C. Transfer 100 µl of the resulting supernatant to a clean polypropylene tube and process directly or store at -80°C until analysis. Plasma creatinine and blood urea nitrogen concentrations were determined by the Laboratory Medicine Center at the University Hospital of Bern.

[0203] Analyze cytokine responses

[0204] The concentrations of cytokines in serum, peritoneal lavage fluid, or cell culture supernatant were determined by ELISA. Following the manufacturer's guidelines, the production of IL-1Ra and G-CSF in serum and culture supernatant was measured using the Mouse IL-1Ra / IL-1F3 DuoSet Kit and the Mouse G-CSF DuoSet Kit (RnDSystems). Mouse IL-1β and IL-6 were quantified using a sandwich ELISA with anti-IL-1β / biotinylated anti-IL-1β or anti-IL-6 / biotinylated anti-IL-6 antibody pairs (both from eBioscience), followed by detection with streptoacid-alkaline phosphatase (Southern Biotech) and p-nitrophenyl phosphate. Density readings were taken on an Infinite M200 proTecan microplate reader, and concentrations were calculated using recombinant standards for mouse IL-1β (RnDSystems) and IL-6 (eBioscience), respectively. Cytokine expression profiles in infected kidneys were analyzed using a mouse cytokine / chemokine 31-in-1 detection array (Eve Technologies). In short, infected kidneys were rapidly frozen, homogenized in modified RIPA buffer, and the tissue homogenate was stored at -80°C until analysis.

[0205] Western blot analysis

[0206] Purified leukocyte subsets were lysed in modified RIPA buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% Triton X100, 1 mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS, and 10 mM NaF) supplemented with protease inhibitors (Roche), benzyl sulfonyl fluoride, and phosphatase inhibitor mixtures 2 and 3 (Sigma Aldrich). Protein extracts were normalized using a Pierce® Protein Assay Kit (Thermo Fisher); and 25 µg of protein was assessed by reducing SDS-PAGE (10–12%). After transferring proteins to polyvinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories), immunoblotting was performed using primary antibodies against IL-1Ra (Thermo Fisher), p38, phosphorylated p38 (all CellSignaling), and β-actin (Santa Cruz Biotechnology), along with their corresponding horseradish peroxidase (HRP)-labeled secondary antibodies (Santa Cruz). Imaging was then performed using SuperSignal™ West Pico chemiluminescent substrates. Staining was visualized using a ChemiDoc™ MP imaging system (Bio-Rad Laboratories).

[0207] Immunohistochemistry and immunofluorescence

[0208] Dissected organs were fixed in 4% paraformaldehyde for 6 to 8 hours, followed by routine paraffin embedding. Tissue sections (2.5 µm) were stained on Immunostainer Leica Bond RX (Leica Biosystems) with periodic acid Schiff (PAS), hexamethylenetetramine silver (Grocott), or with antibodies against IL-1Ra, IL-1α, IL-1β, IL-1R1 (all from R&D Systems), F4 / 80 (Bio-Rad Laboratories), CD68, Ly-6G, CCR2, Iba1, histone H3 (citrulline R2+R8+R17) (all from Abcam), anti-MPO (Agilent), or anti-GFP (Novus). Embedding, sectioning, and staining were performed by the Translational Research Platform of the Institute of Pathology, University of Bern. Images were acquired using a Pannoracmi 250 scanner (3DHistech) prior to analysis. Histological grading of PAS-stained sections was performed by trained pathologists unfamiliar with the characteristics of the samples. Inflammation and Candida albicans dissemination were evaluated separately for the tubulointerstitial compartments and glomeruli. Scores ranging from 0 to 3 were defined for each category and criterion, as described below. The final score for each section represents the sum of the scores for the tubulointerstitial compartments and glomeruli.

[0209]

[0210] Spleen tissue was frozen in OCT embedding medium and cut (5 µm) using a Leica CM1950 cryostat. Acetone-fixed frozen sections were incubated for 1 hour in PBS containing 10% goat serum and 0.1% Triton X-100 (Sigma-Aldrich) to block nonspecific binding, followed by overnight staining with anti-mouse MARCO (Bio-Rad Laboratories) and anti-mouse CD169 (BioLegend) antibodies. Rinsed sections were stained with DAPI (Sigma-Aldrich) for 20 minutes and then mounted with Dako fluorescent mounting medium. Fluorescence images were obtained using a Pannoracamic 250 Flash II slide scanner (3DHistech). IL-1Ra positive areas in kidney sections were quantitatively stained using ImageScope (v12.4.0.5043). To observe the co-localization of macrophages and IL-1Ra in the marginal zone, spleen tissue was frozen in OCT embedding medium and cut (10 µm) using a Leica CM1950 cryostat. PFA-fixed (4%) frozen sections treated with multi-stage buffer (Lunaphore) were stained using LabSat technology (Lunaphore). In the first cycle, anti-mouse CD169 (Biolegen), anti-mouse IL1RA (R & D Systems), rabbit anti-goat Alexa Fluor 546 secondary antibody (Invitrogen), and DAPI were stained and imaged. In the second cycle, after using quenching buffer (Lunaphore), MARCO (Bio-Rad Laboratories), corresponding secondary antibodies (PE goat anti-rat Ig, SouthernBiotech), and DAPI were stained and imaged.

[0211] Quantitative reverse transcription PCR

[0212] Kidney destruction was performed for two 2-minute cycles at 25 Hz using 0.5 to 1 ml of TRIzol reagent (Ambion Life Technologies) with stainless steel beads (5 mm; Qiagen) in TissueLyser II (Qiagen). Total mRNA was isolated according to the manufacturer's instructions. Contaminated DNA was digested with RNase-free DNase, and mRNA concentration was measured using a NanoDrop™ One spectrophotometer (Thermo Fisher). Reverse transcription was performed on 1 µg mRNA / reactant using GoScript™ reverse transcriptase (Promega) in the presence of an RNase inhibitor (BioLabs). Quantitative PCR was performed on a StepOnePlus™ Real-Time PCR System (Thermo Fisher) using the KAPA SYBR® FAST qPCR Master Mix (2X) kit (Sigma), and expression was normalized to G6pdx or Actb expression. For FACS-sorted cells, RNA was purified using the ReliaPrep™ RNA Mini-Preparation System (Promega) according to the manufacturer's instructions.

[0213] RNA sequencing and bioinformatics analysis

[0214] On day 2 post-infection, kidneys from infected mice were harvested, flash-frozen in liquid nitrogen, and then placed in BashingBeat™ lysis tubes containing TRI Reagent® lysis buffer (Zymo Research). Homogenization was performed using TissueLyser II (Qiagen) at 30 Hz for three 1-minute cycles. Total RNA was extracted using the Direct-ZolhZxf93 RNA Miniprep Plus kit (Zymo Research) and stored at -80°C until use. The quantity and quality of purified total RNA were assessed using a Qubit 4.0 fluorometer with the Qubit RNA BR assay kit (Thermo Fisher) and an Advanced Fragment Analyzer system using the Fragment Analyzer RNA kit (Agilent). RiboCop for HMR+ Globin Removal Kit (Lexogen) was used to remove 200 ng of ribosomal RNA and globin mRNA from the input RNA according to the manufacturer's instructions. Subsequently, following a long insert size protocol, cDNA libraries were prepared using the CORALL Total RNA-SeqV2 Library Construction Kit (Lexogen) with UDIs 12nt A1 group. The quantity and length of the cDNA libraries were investigated using a Qubit 4.0 fluorometer and the Advanced Fragment Analyzer system described above. Library quantification was also determined using the JetSeq Library Quantification Lo-ROX Kit (Bioline) according to the manufacturer's instructions. On an Illumina NovaSeq 6000 instrument (Illumina), paired-end sequencing of the equimolar pooled cDNA libraries was performed using the Illumina NovaSeq 6000 S1 Kit v1.5 (300 cycles). The run produced 50-60 million reads / sample. The quality of the sequencing run was evaluated using the Illumina Sequencing Analysis Observer (Illumina, version 2.4.7), and all base call files were multiplexed and converted to FASTQ files using Illumina bcl2fastq conversion software v2.20. Quality control assessment, library generation, and sequencing were performed at the University of Bern's next-generation sequencing platform. From the raw sequencing reads, adaptors and poly(A) sequences were removed using cutAppt (v3.4). Subsequently, 12 bp UMI sequences were extracted using the UMI tool (v1.2.2) according to the manufacturer's recommendations for the library preparation kit, and high-error-rate sequences were removed. The reads were aligned to the whole mouse genome (GRCm38.p6) using hisat2 (v2.2.1). The alignment was repeated using the UMI tool (v1.2.2).Based on Ensembl mouse gene annotation v102, featureCounts (v2.0.1) was used to count fragments aligned to gene features. DESeq2 was used to calculate differential gene expression from the raw count matrix. Gene set enrichment analysis (GSEA) was performed using the R package clusterProfiler based on gene ordination, using test statistics obtained from the differential gene expression results from DESeq. Gene ontology overrepresentation analysis was performed on genes differentially expressed with adjusted p-values ​​less than 0.05 using the R package topGO. Heatmap visualizations were generated using the R package ComplexHeatmap. All downstream analyses in R were performed using R version 4.1.0.

[0215] Quantitative and statistical analysis

[0216] Unless data for a single mouse are presented, data are expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism software. Two-tailed Student's t-test, one-way ANOVA with Bartlett's multiple comparisons, or two-way ANOVA with Tukey's multiple comparisons were used to compare two or more groups, as specified in the figure descriptions. Statistical significance was considered to be p < 0.05. An asterisk indicates statistical significance (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

Claims

1. A pharmaceutical composition for use in the prevention or treatment of sepsis associated with systemic fungal infection, comprising an agent capable of inhibiting IL-1 Ra activity.

2. The pharmaceutical composition according to claim 1, wherein the systemic fungal infection is a Candida albicans infection.

3. The pharmaceutical composition according to claim 1 or 2, wherein the agent capable of inhibiting IL-1 RA is a ligand for IL-1 RA selected from the group consisting of monoclonal antibodies and antibody-like molecules.

4. The pharmaceutical composition according to claim 3, wherein the agent capable of inhibiting IL-1 RA is an IL-1 RA neutralizing antibody or neutralizing antibody-like molecule.

5. The pharmaceutical composition according to claim 1 or 2, wherein the agent capable of inhibiting IL-1 RA is an oligonucleotide agent capable of inhibiting IL-1 RN gene expression.

6. The pharmaceutical composition according to claim 5, wherein the oligonucleotide agent is capable of hybridizing to mRNA encoding IL-1 Rα.

7. The pharmaceutical composition according to claim 5 or 6, wherein the oligonucleotide agent is selected from the group consisting of antisense oligonucleotides, gapmers, siRNAs and shRNAs.

Images