IL-1Ra blockers for the 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

[Technical Field]

[0001] This application claims priority to European Application No. 23183633, filed on 5 July 2023, and European Application No. 23215259, filed on 8 December 2023, both of which are incorporated herein by reference in their entirety.

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

[0003] Sepsis is a life-threatening illness caused by microbial dissemination through the bloodstream and a maladaptive systemic inflammatory response. While fungal sepsis is less common, invasive fungal infections, in particular, have a high mortality rate. Candida albicans is the most common cause of fungal bloodstream infections, with an overall mortality rate of 30–40% despite appropriate antifungal treatment, exceeding 60% in severely ill patients. C. albicans is usually suppressed by the epithelial barrier immune system and exists as a commensal organism in half of the population. In congenital immunodeficiency disorders, the importance of the IL-17-mediated pathway for avoiding mucocutaneous colony formation is emphasized, while the functional response of neutrophils is also important for preventing systemic infection (Puel A. (2020), Hum Genet. 139, 1011-1022; Desai JV, Lionakis MS. (2018), Curr Clin Microbiol Rep. 5, 181-189). Therefore, individuals in an immunosuppressed state due to hematological malignancies, organ transplants, AIDS, or prolonged intensive care hospitalization are highly susceptible to invasive candidiasis. However, preceding systemic viral infections, or medical interventions that impair physiological barrier function such as indwelling devices, parenteral nutrition, and abdominal surgery, may increase susceptibility to disseminated Candida infection even in otherwise immune-competent hosts. 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 elucidating the underlying mechanisms of these diseases in order to develop new therapeutic strategies.

[0004] Inflammation is a physiological, innate response to tissue damage aimed at removing damaging substances and restoring internal homeostasis. To provide adequate defense mechanisms that ensure pathogen clearance while avoiding widespread tissue damage, both the dynamics and composition of its powerful effector functions must be precisely adapted to the characteristics of the invading pathogen. Therefore, the interaction network of pro-inflammatory and anti-inflammatory cytokines orchestrates the differentiation and recruitment of functionally distinct immune cell populations. Furthermore, there are negative feedback mechanisms known as immune checkpoints, which signal unbalanced immune activation and suppress inflammatory processes to prevent immunopathology. However, sepsis is characterized by excessive inflammation and immunoparalysis, along with a breakdown of homeostasis in these pro-inflammatory and anti-inflammatory pathways. As a result, the systemic inflammatory response becomes dysfunctional, leading to multi-organ damage and an inability to suppress pathogen replication.

[0005] Interleukin-1 (IL-1), a typical pro-inflammatory cytokine, initiates and modulates local and systemic inflammatory responses by activating IL-1 receptors (IL-1Rs) on immune and non-immune cells. Both IL-1 cytokines, IL-1α and IL-1β, are typical of antimicrobial immunity, including C. albicans. Nevertheless, 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 the transcriptional and post-translational levels, which limit the production of mature cytokines or affect their ability to activate IL-1Rs. The inflammatory effects of physiologically active IL-1α and IL-1β are regulated by IL-1R antagonists (IL-1Ras), which are endogenous inhibitors that compete for IL-1R occupancy but do not trigger downstream signaling. One secreted isoform and three intracellular isoforms of IL-1Ra, derived from the same "IL1RN" gene, have been reported. Adding to the complexity of IL-1 regulation, these individual isoforms are expressed differently in various cell types. For example, intracellular IL-1Ra is constitutively expressed in epithelial cells, while the secreted and intracellular isoforms can be induced in diverse leukocyte subsets in response to pro-inflammatory cytokines, microbial products, or tissue injury. The effectiveness of IL-1Ra-mediated regulation is evident in severe inflammatory syndromes in DIRA (interleukin-1 receptor antagonist deficiency) patients and is used therapeutically for the management of IL-1-mediated diseases. However, while the molecular pathways governing the production and secretion of mature IL-1 are well-evaluated, regulatory mechanisms operating at the receptor binding level, such as IL-1Ra, particularly cell-type-specific regulation of IL-1-driven inflammation, remain largely unknown. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Puel A.(2020),Hum Genet.139,1011-1022 [Non-Patent Document 2] Desai JV, Lionakis MS. (2018), Curr Clin Microbiol Rep.5, 181-189 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] Based on the above-described state of the art, the object of the present invention is to provide means and methods for preventing death from sepsis associated with systemic fungal infection. This object is achieved by the subject matter of the independent claims herein and with further advantageous embodiments described herein by the dependent claims, examples, figures and general description. [Means for solving the problem]

[0008] Summary of the Invention The present invention provides 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.

[0009] Terms and Definitions General For the purposes of interpreting this Specified, the following definitions apply, wherever a term is used in the singular form, it also includes the plural form, and vice versa. In the event of any conflict between the following definitions and any document incorporated herein by reference, the definitions set forth herein shall prevail.

[0010] The terms “comprising,” “having,” “containing,” “including,” and other similar forms, as used herein, and their grammatical equivalents, are intended to be semantically equivalent and open-ended, meaning that the one or more items following any one of these words do not mean to exhaustively list the one or more items concerned, or to limit themselves to only the one or more items listed. For example, an item “comprising” components A, B, and C may consist of components A, B, and C (i.e., contain only components A, B, and C), or it may contain one or more other components in addition to components A, B, and C. Thus, “comprising” and its similar forms, and their grammatical equivalents, are intended and understood to include disclosures of embodiments of “consisting essentially of” or “consisting of.”

[0011] Where a range of values ​​is provided, unless the context clearly indicates otherwise, each intermediate value between the upper and lower limits of that range, up to one-tenth of the lower limit unit, and any other stated or intermediate values ​​within that range are understood to be included in this disclosure, subject to the specific exclusions of the stated range. Where the stated range includes one or both of the limit values, the range excluding one or both of those limit values ​​is also included in the disclosure.

[0012] In this specification, a “about” reference to a value or parameter includes (and describes) the variation directed toward the value or parameter itself. For example, a statement referring to “about X” also includes the statement “X”.

[0013] As used herein, including in the attached claims, the singular forms "a," "or," and "the" include plural references unless the context makes it clear otherwise.

[0014] As used herein, “and / or” is deemed to specifically describe each of the two specified features or components together with or without the other feature or component. Accordingly, the term “and / or” as used in expressions such as “A and / or B” is intended to include “A and B,” “A or B,” “A (alone),” and “B (alone).” Similarly, the term “and / or” as used in expressions such as “A, B, and / or C” is intended to include each of the following embodiments: 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).

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art (e.g., cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry, organic synthesis). Standard methods are used for molecular, genetic and biochemical methods (see generally below: Sambrook et al., Molecular Cloning: A Laboratory Manual, Part 4 (2012), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY and Ausubel et al., Short Protocols in Molecular Biology (2002), Part 5, John Wiley & Sons, Inc.) and chemical methods.

[0016] Binding; binders, ligands, antibodies: Unless more narrowly defined in the detailed description of the invention, references to binders and ligands include antibodies, antibody-like molecules, and aptamers as defined in the following paragraphs.

[0017] The term "specific binding" in the context of the present invention refers to the property of a ligand that binds to its target with a certain affinity and target specificity. The affinity of such a ligand is indicated by the dissociation constant of the ligand. A specifically reactive ligand has a dissociation constant of 10 -8 mol / L or less (particularly 10 -9 mol / L or less), but has at least a three-digit higher dissociation constant in the interaction with a molecule having substantially the same chemical composition as the target but a different three-dimensional structure.

[0018] The term "non-agonist ligand" refers to a ligand that can specifically bind to its target with a k -8 of 10 D mol / L or less, particularly 10 -9 mol / L or less, and further 10 -10 mol / L or less of k D , particularly a human or humanized monoclonal antibody. A "non-agonist" ligand interacts with its target without producing the biological effect of the target's physiological ligand. As an example, a non-agonist ligand for an interleukin receptor binds to the interleukin receptor (ILR, target) without producing the effect of the interleukin-ILR interaction and inhibits the binding of interleukin.

[0019] As used herein, the term "dissociation constant (K D )" is used in the meaning known in the technical fields of chemistry and physics: It means an equilibrium constant that measures the tendency of a complex composed of [usually two] different components to dissociate reversibly into its constituent components. This complex can be, for example, an antibody-antigen complex AbAg composed of an antibody Ab and an antigen Ag. K D is expressed in molar concentration [mol / l] and corresponds to the concentration of [Ab] at which half of the binding sites of [Ag] are occupied, or in other words, corresponds to the concentration of unbound [Ab] being equal to the concentration of the [AbAg] complex. The dissociation constant can be calculated by the following formula:

Number

[0020] In this specification, "off-rate (dissociation rate) (K) off ;[1 / sec]) and "On-rate (binding rate) (K on ;[L / (sec * The term "mol" is used in the sense known in the fields of chemistry and physics: these refer to the dissociation of an antibody with its target antigen (K off ) or bond (K on This refers to the rate constant used to measure (K). off and K on This can be determined experimentally using methods well established in the art. The K of the antibody off and K on Surface plasmon resonance is employed as a method for measuring this. This is the principle behind biosensor systems such as the Biacore® or ProteOn® systems. These also use the following equation to determine the dissociation constant K D It can be used to find:

number

[0021] On Rate K on The natural upper limit is 10 9 (This is L / sec * moles.)

[0022] The term "aptamer" refers to an oligonucleotide or peptide molecule that binds to a specific target molecule. Aptamers can be created by selecting them from a large pool of random sequences. Nucleic acid aptamers can be generated by repeated in vitro selection, or equivalently by SELEX (Systematic Evolution of Ligands by Exponential Enrichment), which binds to molecular targets such as small molecules, proteins, or nucleic acids via non-covalent interactions. Aptamers possess molecular recognition properties comparable to those of antibodies.

[0023] In the context of this specification, the term “antibody” refers to complete antibodies, including but not limited to immunoglobulin 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 constructs associated with or derived therefrom. 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 has a heavy chain variable region (V H ) and heavy chain constant region (C H ) is composed of. The heavy chain constant region of IgG is C H 1, C H 2, C H It consists of three domains. Each light chain has a light chain variable region (V in this specification). L (abbreviated as) and the light chain steady region (C L It is composed of ). The light chain constant region is a single domain called C L It consists of the following: The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody contains various cells of the immune system (e.g., effector cells) and the first component of the classical complement system, and can mediate the binding of immunoglobulins to host tissues or factors. Similarly, this term encompasses so-called nanobodies or single-domain antibodies, antibody fragments consisting of a single monomeric variable antibody domain.

[0024] In this specification, the term "humanized antibody" refers to an antibody originally produced by immune cells of a non-human species, whose protein sequence has been modified to increase its similarity to antibody variants naturally produced in humans. As used herein, the term "humanized antibody" includes antibodies in which a CDR sequence derived from the germline of another mammalian species, such as a mouse, has been transplanted onto a human framework sequence. Further modifications to the framework region may be made not only within the human framework sequence, but also within the CDR sequence derived from the germline of another mammalian species.

[0025] In this specification, the term "antibody-like molecule" refers to a molecule or target with high affinity / Kd ≤ 10 -7 mol / L (especially ≤10)-9 This refers to molecules that can specifically bind at a concentration of mol / L. Antibody-like molecules bind to their targets in a similar manner to the specific binding of antibodies. The term "antibody-like molecule" encompasses repeat proteins such as engineered ankyrin repeat proteins (Molecular Partners, Zurich) and engineered antibody-mimetic proteins that exhibit highly specific and high-affinity target protein binding (see U.S. Patent Publication Nos. 2012142611, 2016250341, 2016075767, and 2015368302). The term "antibody-like molecule" further encompasses, but is not limited to, polypeptides derived from armadillo repeat proteins, polypeptides derived from leucine-rich repeat proteins, and polypeptides derived from tetratricopeptide repeat proteins. The term "antibody-like molecule" further includes protein A domain, fibronectin domain FN3, consensus fibronectin domain, lipocalin (see Skerra, Biochim, Biophys, Acta 2000, 1482(1-2):337-50), polypeptides derived from zinc finger protein (see Kwan et al. Structure 2003, 11(7):803-813), Src homology domain 2 (SH2) or Src homology domain 3 (SH3), PDZ domain, gamma crystallin, ubiquitin, cysteine ​​notch polypeptide or nottin, cystatin, Sac7d, triple helix coiled-coil (also known as alpha), Knitz domain or Knitz-type protease inhibitors, and specific binding polypeptides derived from carbohydrate-binding module 32-2. The term "antibody-like molecule" further includes humanized camel antibodies. The term "antibody-like molecule" similarly includes scFv fragments.

[0026] The term "protein A domain-derived polypeptide" refers to a derivative of protein A that can specifically bind to the Fc and Fab regions of immunoglobulins.

[0027] The term "armadillo repeat protein" refers to a polypeptide containing at least one armadillo repeat, which is characterized by a pair of alpha helices that form a hairpin structure.

[0028] In this specification, the term "humanized camel antibody" refers to an antibody consisting solely of a heavy chain or a variable domain (VHH domain) of a heavy chain, whose amino acid sequence has been modified to increase its similarity to antibodies naturally produced in humans, resulting in reduced immunogenicity when administered to humans. General strategies for humanizing camel antibodies are described below: Vincke et al., "General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold," J Biol Chem. January 30, 2009; 284(5):3273-3284, and U.S. Patent Application Publication No. 2011165621 (A1).

[0029] In this specification, the term “fragment crystallizable (Fc) region” means, when applied to IgG, a C covalently bonded by disulfide bonds. H 2 and C H This refers to the portion of an antibody that contains two identical heavy chain fragments, each consisting of three domains.

[0030] In this specification, the term “single-chain variable fragment (scFv)” refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin, which yields high affinity for an antibody-like target from a single polypeptide chain. The VH and VL chains of the scFv are linked via a short linker peptide of 10 to approximately 25 amino acids [Huston et al. (1988). PNAS 85(16):5879-5883]. This linker can be configured either by linking the N-terminus of VH to the C-terminus of VL (VL-VH) or vice versa (VH-VL).

[0031] In this specification, the term "IL-1Ra inhibitor" refers to drugs that suppress the physiological response to the interleukin-1 receptor antagonist protein (IL-1Ra; Uniprot P18510).

[0032] General Molecular Biology: Nucleic Acid Sequences, Expression The terms “gene expression” or “expression,” or “gene product,” may refer to either or both the process of generating nucleic acids (RNA) or peptides or polypeptides, and their products, also known as transcription and translation, respectively, or to any intermediate process that modulates the processing of genetic information to produce polypeptide products. The term “gene expression” may also apply to the transcription and processing of RNA gene products, such as regulatory RNA or structural (e.g., ribosomal) RNA. When the expressed polynucleotides originate from genomic DNA, expression may include the splicing of mRNA in eukaryotic cells. Expression can be evaluated at both the transcription and translation levels, i.e., at the mRNA and / or protein product levels.

[0033] In this specification, the term “nucleotide” refers to building blocks of nucleic acids or nucleic acid analogs, whose oligomers can selectively hybridize with RNA oligomers or DNA oligomers based on base pairing. In this context, the term “nucleotide” includes the classical ribonucleotide building blocks adenosine, guanosine, uridine (and ribosylthymine), cytidine, and the classical deoxyribonucleotides deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. Furthermore, it includes nucleic acid analogs such as phosphothioates, 2'O-methylphosphothioates, peptide nucleic acids (PNA; N-(2-aminoethyl)-glycine units linked by peptide bonds with nucleic acid bases attached to the α-carbon of glycine), or loc nucleic acids (LNA; 2'O,4'C methylene-bridged RNA building blocks). Where “hybridized sequences” refer to in this specification, such hybridized sequences may consist of any of the above nucleotides or mixtures thereof.

[0034] As used herein, the term "phosphothioate" is synonymous with the terms phosphorothioate and thiophosphate.

[0035] In this specification, the term “can form a hybrid or hybridized sequence” refers to sequences that can selectively bind to their target sequences under conditions present in the cytosol of mammalian cells. Such hybridized sequences may be sequentially inversely complementary to the target sequence, or may contain gaps, mismatches, or further mismatched nucleotides. The minimum length of a sequence capable of hybridization depends on its composition (containment of C or G nucleotides that contribute more to binding energy than A or T / U nucleotides) and skeletal chemistry.

[0036] In this specification, the term “hybridize sequence” encompasses polynucleotide sequences comprising, or essentially comprising, RNA (ribonucleotides), DNA (deoxyribonucleotides), phosphothioate deoxyribonucleotides, 2'-O-methyl modified phosphothioate ribonucleotides, LNA and / or PNA nucleotide analogs. In certain embodiments, the hybridize sequence according to 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 certain embodiments, the hybridized sequence is at least 80% identical, and more preferably 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identical, to the reverse complementary sequence of NCBI reference sequence:XM_047444184.1, NCBI reference sequence:XM_047444185.1, or NCBI reference sequence:XM_011511121.2. In certain embodiments, the hybridized sequence comprises a deoxynucleotide, a phosphothioate deoxynucleotide, LNA and / or PNA nucleotide, or a mixture thereof.

[0037] In this specification, the term “antisense oligonucleotide” refers to an oligonucleotide having a sequence substantially complementary to RNA and capable of hybridizing with RNA. Such antisense action on RNA modulates, in particular inhibits, or represses the RNA’s biological activity. If the RNA is mRNA, the expression of the resulting gene product is inhibited or repressed. Antisense oligonucleotides may 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 theoretically optimal antisense sequences for a given target. Optimization can be performed from both the nucleic acid base sequence and the skeletal (ribo, deoxyribo, analog) composition. Numerous sources of actual physical oligonucleotides exist, and they are generally synthesized by solid-state synthesis.

[0038] The term "gapmer" refers to a short DNA antisense oligonucleotide structure that has RNA-like segments on both sides of its sequence, typically composed of a locating nucleic acid (LNA), 2'-OMe, or 2'-F modified base. Gapmers often contain nucleotides modified with phosphorothioate (PS) groups, particularly in their 5' and 3' terminal regions. Gapmers are designed to hybridize to a target region of RNA and silence the gene by inducing cleavage by RNase H. The binding of the gapmer to the target exhibits high affinity to the modified RNA facies and resistance to degradation by specific nucleases. Gapmers are being developed as therapeutic agents for various cancers, viruses, and other chronic genetic disorders.

[0039] In this specification, the term siRNA (small molecule / short interfering RNA) refers to RNA molecules that can interfere with (in other words, suppress or block) the expression of genes containing nucleic acid sequences complementary to or hybridizing with the siRNA sequence in a process called RNA interference. The term siRNA encompasses both single-stranded and double-stranded siRNA. siRNA is typically characterized by a length of 17–24 nucleotides. Double-stranded siRNA can be derived from longer double-stranded RNA molecules (dsRNA). It is widely believed that this longer double-stranded RNA is cleaved by an endo-ribonuclease (called Dicer) to form double-stranded siRNA. In the nucleoprotein complex (called RISC), the double-stranded siRNA is unwound to form single-stranded siRNA. Much of the RNA interference works by the binding of an siRNA molecule to an mRNA molecule with a complementary sequence, resulting in the degradation of the mRNA. RNA interference can also be achieved by siRNA molecules binding to the intron sequences of premRNA (immature, unspliced ​​mRNA) in the cell nucleus, thereby causing degradation of the premRNA.

[0040] In this specification, the term "shRNA (small hairpin RNA)" refers to an artificial RNA molecule having a tight hairpin turn that can be used to silence the expression of a target gene via RNA interference (RNAi).

[0041] Any patent document cited herein is deemed to be incorporated in its entirety by reference.

[0042] As used herein, the term “pharmaceutical composition” refers to a compound of the present invention or a pharmaceutically acceptable salt thereof, accompanied by at least one pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition according to the present invention is provided in a form suitable for topical, parenteral, or infusion administration.

[0043] As used herein, the term “pharmaceutically acceptable carrier” includes, as is known to those skilled in the art, any solvent, dispersion medium, coating, surfactant, antioxidant, preservative (e.g., antimicrobial, antifungal), isotonic, absorption retarder, salt, preservative, drug, drug stabilizer, binder, excipient, disintegrant, lubricant, sweetener, flavoring, coloring, etc., and combinations thereof (see, for example, Remington: The Science and Practice of Pharmacy, ISBN 0857110624). The present invention also encompasses nanoparticles, liposomes, or cell carriers in the sense of “pharmaceutically acceptable carrier.”

[0044] The term “inhibitor” as used herein refers to any pharmaceutically acceptable agent or compound that can be used for its biological activity, interaction, and specific interference with its designated target (in this case, IL-1Ra). Inhibitors include small molecule agents that meet the criteria summarized as Lipinski’s five rules (the agent satisfies at least three of the following rules: number of hydrogen bond donors ≤ 5, number of hydrogen bond acceptors ≤ 10, molecular weight ≤ 500 Da, and octanol / water partition coefficient ≤ 5). Specific examples of such inhibitors are described herein.

[0045] As used herein, the terms “to treat” or “to cure” any disease or disorder (e.g., sepsis) mean, in one embodiment, mitigating the disease or disorder (e.g., delaying, preventing, or reducing the onset of the disease or at least one of its clinical symptoms). In another embodiment, “to treat” or “to cure” means mitigating or improving at least one physical parameter, including one that may not be identifiable to the patient. In yet another embodiment, “to treat” or “to cure” means modulating the disease or disorder, either physically (e.g., stabilizing identifiable symptoms), physiologically (e.g., stabilizing physical parameters), or both. Methods for evaluating the treatment and / or prevention of diseases are generally known in the art unless specifically described below herein. [Modes for carrying out the invention]

[0046] Detailed description of the present invention 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.

[0047] The inventors suggest that the therapeutic method proposed herein is particularly useful in patients with acute sepsis, especially those with a confirmed primary or secondary fungal infection by Candida albicans, or in patients considered to be at particularly high risk of developing sepsis associated with Candida albicans or other fungal pathogens. However, inhibition of IL-1Ra may be contraindicated in patients with bacterial sepsis.

[0048] Ideally, patients suspected of having a systemic fungal infection should receive treatment at an early stage of symptom onset.

[0049] In certain embodiments, systemic fungal infection is an infection caused by Candida albicans.

[0050] In certain embodiments, the agents capable of inhibiting IL-1RA are ligands for IL-1Ra selected from monoclonal antibodies and antibody-like molecules.

[0051] Antibodies have the advantage of a long serum half-life and can provide long-term protection.

[0052] However, considering the fact that rapid clearance after bolus administration may be sufficient to disrupt the immune response caused by infection, the inventors believe that the data obtained suggest that protein-based binders such as ankyrin repeat proteins, or shorter-lived protein agents such as antibody-like molecules, e.g., nanobodies, variable fragments, or camelid antibodies, may also serve a useful purpose.

[0053] In certain embodiments, 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 this patent, the term “neutralizing antibody” includes both monoclonal and polyclonal antibodies, as well as any fragment, variant, or derivative thereof that retains the ability to neutralize a target.

[0054] It is also envisioned that polypeptide agents derived from soluble IL-1 receptors may be used. In these polypeptide agents, the epitopes that bind to IL-1 alpha and IL-1 beta are modified to prevent scavenging (removal) of these cytokines by the soluble receptors, while their binding to IL-Ra is maintained to function as an effective inhibitor of IL-1Ra.

[0055] In certain embodiments, the agent capable of inhibiting IL-1RA is an oligonucleotide agent capable of inhibiting IL-1RN gene expression.

[0056] In certain embodiments, oligonucleotide agents can hybridize to mRNA encoding IL-1Ra.

[0057] In certain embodiments, the oligonucleotide agent is selected from antisense oligonucleotides, gapmers, siRNAs, and shRNAs. Using siRNA transcribed from an expression vector under the control of a macrophage-specific promoter may allow for specific targeting of the IL-1Ra source (production source) discovered by the inventors.

[0058] Medical treatment Similarly, within the scope of the present invention, the treatment of sepsis associated with systemic fungal infection includes administering to a patient who requires such treatment an agent capable of inhibiting IL-1Ra activity in accordance with the above description, or a method thereof.

[0059] Pharmaceutical compositions, dosage forms and salts According to one embodiment of the compound of the present invention, the compound of the present invention is provided as a pharmaceutical composition, a pharmaceutical administration form, or a pharmaceutical dosage form, the pharmaceutical composition, pharmaceutical administration form, or pharmaceutical dosage form 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.

[0060] The present invention further encompasses pharmaceutical compositions comprising the compound of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In further embodiments, the composition comprises at least two pharmaceutically acceptable carriers as described herein.

[0061] Specific embodiments of the present invention relate to enteral administration forms such as nasal, oral, rectal, transdermal, or oral administration, or to inhalation forms or suppositories. Furthermore, the pharmaceutical compositions of the present invention may be in solid form (including, but not limited to, capsules, tablets, pills, granules, powders, or suppositories) or in liquid form (including, but not limited to, solutions, suspensions, or emulsions).

[0062] Specific embodiments of the present invention relate to dosage forms for parenteral administration, such as subcutaneous, intravenous, intrahepatic, or intramuscular injection. Optionally, pharmaceutically acceptable carriers and / or excipients may be present.

[0063] Specific embodiments of the present invention relate to dosage forms for topical administration. Those skilled in the art will be familiar with a wide range of possible formulations for providing topical preparations, as exemplified by the following: Benson and Watkinson (eds.), Topical and Transdermal Drug Delivery: Principles and Practice (Part 1, Wiley 2011, ISBN-13: 978-0470450291); and Guy and Handcraft: Transdermal Drug Delivery Systems: Revised and Expanded (Part 2, CRC Press 2002, ISBN-13: 978-0824708610); and Osborne and Amann (eds.), Topical Drug Delivery Formulations (Part 1, CRC Press 1989; ISBN-13: 978-0824781835). In embodiments of the present invention relating to the topical use of the compounds of the present invention, the pharmaceutical composition is formulated in a manner suitable for topical administration, such as an aqueous solution, suspension, ointment, cream, gel, or sprayable formulation, such as one for delivery by aerosol, and comprises the active ingredient together with one or more solubilizers, stabilizers, isotonic enhancers, buffers, and preservatives known to those skilled in the art.

[0064] The administration regimen of the compounds of the present invention varies depending on known factors such as the pharmacodynamic properties of the particular drug and its mode and route of administration; the recipient's species, age, sex, health status, medical condition, and weight; the nature and severity of symptoms; the type of concurrent treatment; the frequency of treatment; the route of administration, the patient's renal and hepatic function, and the desired effect. In certain embodiments, the compounds of the present invention may be administered once daily, or the total daily dose may be divided into two, three, or four doses per day.

[0065] Manufacturing method and treatment method according to the present invention In a further aspect, the present invention also includes the use of agents capable of inhibiting IL-1Ra activity as identified herein for use in a method for producing a medicament for the treatment or prevention of sepsis associated with systemic fungal infection.

[0066] Similarly, the present invention encompasses a method for treating a patient diagnosed with sepsis associated with a systemic fungal infection. This method involves administering to the patient an effective amount of a drug capable of inhibiting IL-1Ra activity, as detailed herein.

[0067] Where, in this specification, alternative forms of a single separable feature are described as “embodiments,” it should be understood that such alternative forms can be freely combined to form separate embodiments of the invention disclosed herein.

[0068] The present invention can be further illustrated by the following embodiments and figures, from which further embodiments and advantages can be derived. These embodiments are for illustrative purposes only and do not limit the scope of the present invention. [Examples]

[0069] The inventors investigated IL-1Ra expression in various myeloid cell subsets in a mouse model of invasive candidiasis and tested the effects of IL-1Ra produced by specific cell types on antifungal immunity. They demonstrated that IL-1Ra secreted by macrophages acts as an innate immune checkpoint that hinders efficient pathogen clearance, and that targeted removal of this checkpoint provides protection against lethal C. albicans sepsis. Furthermore, they found that IL-1Ra secreted by macrophages is positively regulated by type I interferon (IFN), reflecting a correlation between secondary invasive candidiasis and preceding viral infection. Taken together, these results provide a mechanistic explanation for high disease susceptibility to Candida bloodstream infection and suggest that IL-1Ra-targeted therapy could be a novel therapeutic approach.

[0070] result IL-1Ra is rapidly expressed in response to C. albicans bloodstream infection. To establish the relevance of IL-1 signaling during invasive fungal infections, 2.5 × 10⁻⁶ 5The expression of IL-1 family cytokines in the kidneys was examined after intravenous infection of CFUs with Candida albicans. IL-1β and IL-1Ra proteins were strongly expressed throughout the infection site on post-infection day 3, but IL-1α-positive cells were hardly observed at the periphery of the lesion. IL-1β transcripts were already present in the kidneys of naive animals, but IL-1R1 and IL-1Ra could only be detected at very low levels. However, fungal infection rapidly increased the gene expression of IL-1β and IL-1Ra in the kidneys, while IL-1R1 mRNA expression remained unchanged compared to naive mice (Figure 1A). Furthermore, significant levels of IL-1Ra protein were present in the serum of infected mice, peaking on post-infection day 2 (Figure 1B). Meanwhile, mature IL-1β cytokines remained undetectable in peripheral blood, suggesting they were likely produced locally in the infected tissue. IL-1Ra protein expression in the kidney was consistent with leukocyte recruitment and limited to inflammatory infiltration, suggesting that immune cells are the primary producers of IL-1Ra. Indeed, primary neutrophils, monocytes, dendritic cells, and bone marrow (BM)-derived macrophages (BMDMs) secreted IL-1Ra in vitro in response to both Candida morphotypes, with the highest IL-1Ra levels induced by monocytes exposed to Candida hyphae (Figure 1C). These data confirm that the IL-1β / IL-1R1 axis is a crucial component of early antifungal immune defense and suggest that myeloid cells are involved in its regulation through the production of IL-1Ra.

[0071] Removal of IL-1Ra produced by macrophages acts protectively against invasive fungal infections. Therefore, the inventors attempted to clarify the effect of IL-1Ra produced by specific leukocyte subsets on the immune response to C. albicans by selectively using conditional deletion of IL-1Ra in neutrophils, macrophages, or dendritic cells. fl / flMice were crossed with each Cre driver strain to produce neutrophils and macrophages (LysM-Cre, IL-1Ra LysM (named as), macrophages (Mafb-Cre, IL-1Ra) Mafb (named as), CD11c-expressing cells (CD11c-Cre, IL-1Ra CD11c IL-1Ra was deleted in (named as) (Figures 1D, 8A, and 8B). Macrophage-specific removal of IL-1Ra fl / fl Compared to control littermates, IL-1Ra LysM and IL-1Ra Mafb In mice, the initial immune control of C. albicans infection was dramatically improved (Figures 1E and 1H). Both strains already showed a decrease in renal fungal titer by day 3 post-infection, and C. albicans was controlled to below the detection limit in most animals within 7 days. IL-1Ra LysM Mice lack IL-1Ra in both macrophages and neutrophils, but their phenotype is one in which IL-1Ra is selectively lacking in macrophages. Mafb It was almost identical to a mouse. Furthermore, IL-1Ra LysM Mouse macrophages and neutrophils showed residual IL-1Ra expression in both in vivo and in vitro, but IL-1Ra Mafb In mouse macrophages, IL-1Ra deletion was highly specific and efficient (Figures 8A and 8B). This suggests that neutrophil-expressed IL-1Ra is unlikely to play a significant role, and that macrophage-produced IL-1Ra is a crucial negative regulator of IL-1R signaling during systemic Candida infection. However, removal of IL-1Ra in CD11c-expressing cells provided only weaker, transient protection. CD11c In mouse kidneys, IL-1Ra was present on day 3 after infection. fl / fl Although the amount of fungus was kept lower than in littermates, this difference disappeared by day 7 (Figure 1G). Thus, IL-1Ra produced by CD11c-expressing cells affected early antifungal defense, but did not affect long-term control.

[0072] These observations were supported by histopathological examination of infected kidneys. IL-1Ra LysM Mouse and IL-1Ra Mafb Mice were found to have a superior ability to suppress fungal replication early on (Figures 1E and 1F), exhibiting very limited distribution of C. albicans in the kidneys and minimal inflammation 3 days post-infection (Figures 1H-1I). Furthermore, IL-1Ra LysM Mouse and IL-1Ra Mafb The mice experienced less weight loss compared to the control group, which indicated a general reduction in morbidity (Figure 8C). In contrast, IL-1Ra CD11c Mice exhibited an intermediate phenotype on day 3, with reduced Candida dissemination and inflammation, but unchanged weight loss (Figure 1G, 1J, and 8C). Consistent with improved immunoregulation in C. albicans, kidney sections of all three IL-1Ra-deficient lines showed significantly fewer infection foci and reduced IL-1Ra-positive areas (Figure 1H-K). The beneficial effects of IL-1Ra removal in macrophages were demonstrated by decreased creatinine and serum urea nitrogen levels (Figure 1L), and reduced expression of renal impairment markers Lcn2 and Kim-1 (Figure 1M). Mafb This effect was most pronounced in mice. Overall, these results demonstrate that removing IL-1Ra produced by macrophages significantly improves the ability to control invasive Candida infections, resulting in rapid pathogen clearance, reduced disease severity, and better maintenance of organ function.

[0073] IL-1Ra controls neutrophil recruitment and bactericidal activity. To investigate whether systemic candidiasis can be efficiently suppressed by removing IL-1Ra produced by macrophages, the antifungal immune response characteristics of each IL-1Ra-deficient strain were evaluated. Macrophage-specific IL-1Ra deletion was indicated by a higher percentage of mature CD101+Ly-6G high neutrophils in the blood two days post-infection (Figure 2A-B), indicating that IL-1Ra Mafb Mouse and IL-1Ra LysMIt increases the early mobilization and maturation of neutrophils in mice. This effect is due to IL-1Ra Mafb The effect was most pronounced in mice, and IL-1RaMafb mice also showed a reduction in the proportion of circulating immature neutrophils and inflammatory monocytes (Figure 2B).

[0074] Deleting IL-1Ra produced by macrophages also promoted neutrophil recruitment to the site of inflammation. To rule out feedback mechanisms caused by differences in microbial control, the inflammatory response to non-replicating fungal cell wall components was examined. Zymosan-primed IL-1Ra Mafb Mouse and IL-1Ra LysM The peritoneal lavage fluid from the mice contained 3 times and 2 times the amount of neutrophils, respectively (Figures 2C, 9A, and 9B). IL-1Ra fl / fl Compared to the control, IL-1Ra Mafb Mouse and IL-1Ra LysM Mouse tissue-infiltrating neutrophils showed significantly increased bactericidal activity, secreted large amounts of IL-1β, and were shown to be highly activated (Figures 2D and 2E). However, deletion of IL-1Ra in CD11c-expressing cells was shown to be related to IL-1Ra CD11c In mice, it did not affect the number of neutrophils in the blood and only slightly increased their recruitment to the peritoneum (Figures 2B and 2C); however, IL-1Ra Mafb Mouse and IL-1Ra LysM Similar to what was observed in mice, enhanced bactericidal activity was conferred to tissue-infiltrating neutrophils (Figure 2D).

[0075] Characterization of neutrophil phenotypes in blood, spleen, and kidneys revealed the presence of infected IL-1Ra Mafb In mouse kidneys, IL-1Ra fl / fl Compared to the control group, CD101+ neutrophils producing high levels of reactive oxygen species were concentrated, and the frequency of neutrophils expressing IL-1β and MPO was higher (Figure 2F-I). LysM Mouse and IL-1Ra CD11cNeutrophils in mouse kidneys exhibited an intermediate phenotype (Figures 2F-I). CD63 expression correlated with Candida exposure in the infected organ but was not affected by IL-1-Ra (Figure 2H). Removal of IL-1Ra did not affect the phenotype or fungal titer of splenic neutrophils (Figures 3F-I and 10D). Thus, the deficiency of macrophage-produced IL-1Ra promoted the rapid recruitment of highly bactericidal neutrophils to the infected kidney.

[0076] IL-1Ra Mafb Mouse, IL-1Ra LysM Mice, and to a lesser extent, IL-1Ra CD11c In mice, fungal replication in the kidneys was limited by post-infection (pi) day 3 (Figure 1E-1G). As a result, IL-1R afl / fl No dysfunctional excessive inflammatory response was observed in the control group (Figure 1H-1J), and the expression levels of pro-inflammatory cytokines were low (Figure 2K). Furthermore, IL-1Ra and IL-1Ra LysM In the mouse kidneys, the number of neutrophils and inflammatory monocytes present was reduced at this point, which may reflect a less severe fungal load and accelerated resolution of inflammation in these mice (Figures 2J, 4C, and 10A). Bulk tissue RNA sequencing and gene set enrichment analysis (GSEA) of infected kidneys showed IL-1Ra Mafb We associated the absence of IL-1Ra expression in macrophages in mice with the suppression of inflammatory pathways, including IL-6 / Jak / Stat3 signaling, IFN-α response, and TNF-α signaling. Mafb In addition to Il1rn, the reduced transcripts in mouse kidneys included the major chemotaxis Ccl2, Cxcl2, and Cxcl1; the adhesion molecule Icam1; the NFκB signaling regulator Nfkbia; and the antifungal effector Cybb (not shown). The inventors concluded that macrophage-specific IL-1Ra deletion promotes rapid neutrophil-mediated elimination of pathogens and limits harmful hyperinflammation caused by persistent fungal growth.

[0077] Serum IL-1Ra is produced by CD169+ macrophages in the splenic marginal zone. Since residual Cre activity in neutrophils and monocytes was minimal, we investigated the possibility of an intrinsic function of IL-1Ra. However, partial removal of IL-1Ra in mixed BMCs equally affected the recruitment and effector function of cells arising from both IL-1Ra-expressing BM (bone marrow) and IL-1Ra gene-modified BM (Figures 11A-11G). This suggests an extracellular (cell-extrinsic) effect, likely mediated by IL-1Ra secreted from macrophages. Considering these results, the important role of IL-1Ra secreted from macrophages is highlighted, and since we detected significant levels of serum IL-1Ra circulation in infected mice (Figure 1B), we then aimed to evaluate the effects of IL-1Ra secreted into the bloodstream and identify macrophage subsets contributing to serum IL-1Ra production.

[0078] Bloodstream infection with C. albicans stimulated the release of serum IL-1Ra in wild-type mice, but this response was due to IL-1Ra LysM , IL-1Ra Mafb , and IL-1Ra CD11c This was not observed in mice (Figure 3A). Similarly, infusion of lipopolysaccharide (LPS) increased serum IL-1Ra production, which was significantly reduced in all conditional IL-1Ra-deficient lines tested (Figure 3B). Intravenous infusion of either Candida or LPS induced IL-1Ra protein expression in the splenic marginal zone. Splenic IL-1Ra production corresponds to the presence of IL-1Ra in serum, and IL-1Ra primed with Candida or LPS LysM , IL-1Ra Mafb and IL-1Ra CD11c In mice, it was strongly reduced (this is not shown). EYFP expression leads to EYFP LysM Mouse, EYFP Mafb Mouse, and EYFP CD11cThe history of Cre activity in cells present in the mouse marginal zone was confirmed. Mafb-Cre activity was most extensively observed in EYFP-labeled cells in the splenic marginal zone and also affected macrophages in the red pulp (Figure 3C). This is related to IL-1Ra Mafb This effectively demonstrated the deletion of IL-1Ra in mouse spleens and serum, and their robust protection against invasive candidiasis. Immunofluorescence co-staining identified CD169+ macrophages in the splenic marginal zone as the primary IL-1Ra producers, while MARCO+ macrophages were found to be IL-1Ra-negative.

[0079] The inventors directly investigated the relationship between splenic IL-1Ra production and serum IL-1Ra response and antifungal immune defense in splenectomized wild-type mice infected with C. albicans. Surgical removal of the spleen beforehand reduced Candida-induced serum IL-1Ra levels compared to simulated Candida-infected controls (Figure 3D). Furthermore, splenectomy increased resistance to Candida replication, as indicated by reduced fungal load (Figure 3E), limited Candida dissemination, and decreased inflammation in the kidneys. To confirm that serum IL-1Ra is actually secreted from splenic CD169+ macrophages, the inventors employed a clodronate depletion protocol. This protocol selectively targets marginal zone resident macrophages by utilizing the dynamics of different macrophage regrowth (Figure 11H). Depletion of splenic marginal zone macrophages and marginal metallophilic macrophages reduced serum IL-1Ra levels to those of uninfected mice (Figure 3H), and also reduced CD101+ neutrophils and IL-1β + This was associated with increased neutrophil recruitment to the kidneys (Figure 3I, and 11I-11L). Taken together, these findings demonstrate a direct correlation between IL-1Ra expression in CD169+ macrophages in the splenic marginal zone and serum IL-1Ra production after hematogenous dissemination of C. albicans.

[0080] Serum IL-1Ra is a natural immune checkpoint that mediates pathogen control disorders and dysfunctional over-inflammation during invasive fungal infections. Previous findings have revealed that IL-1Ra produced by macrophages inhibits the efficient early containment of disseminated candidiasis by restricting neutrophil recruitment to tissues and antifungal ability. Furthermore, these have shown a significant contribution of serum IL-1Ra released from splenic CD169+ macrophages. To examine the effect of IL-1Ra circulation on the immune response against Candida, the inventors next Mafb intravenously injected recombinant IL-1Ra protein into mice to reconstitute the IL-1Ra pool in the serum. IL-1Ra Mafb mice were thought to be able to selectively examine the contribution of serum IL-1Ra by this method because both circulating serum IL-1Ra and IL-1Ra produced by macrophages in infected tissues were deficient. When recombinant IL-1Ra was bolus-injected, the serum IL-1Ra level of Candida-infected IL-1Ra Mafb mice initially exceeded that of IL-1Ra-competent mice but maintained wild-type levels until 18 hours (Figures 4A and 12A). Therefore, the inventors Mafb performed injections twice a day during the experiment to completely replenish the serum IL-1Ra of IL-1Ra Mafb mice. Since no IL-1Ra was observed in the splenic marginal zone after Candida infection or in the peritoneal exudate during zymosan peritonitis (Figure 4B), it was confirmed that this regimen exclusively restored only serum IL-1Ra and did not restore locally secreted cytokines. When serum IL-1Ra was reconstituted, the protective phenotype of IL-1Ra fl / fl mice almost completely returned to the protective phenotype of IL-1Ra Mafb mice controls. For example, the enhanced neutrophil recruitment to zymosan priming observed in IL-1Ra MafbThe bactericidal and phagocytic activities of neutrophils from the mice were restricted to the levels of IL-1Ra-competent wild-type mice. Furthermore, the IL-1Ra Mafb mice, rather than the IL-1Ra Mafb ex vivo effector functions of neutrophils from the mice were amplified by in vitro IL-1β stimulation, suggesting that the lack of serum IL-1Ra in vivo increased the sensitivity of neutrophils to local IL-1β stimulation (Figures 4D and 4E). Indeed, pre-stimulating primary neutrophils with IL-1Ra in vitro resulted in the disappearance of IL-1R signaling upon subsequent exposure to IL-1β even after removing unbound extracellular IL-1Ra (Figure 4F). As expected from these results, the IL-1Ra Mafb mice no longer efficiently controlled Candida replication in the kidney as well as the IL-1Ra Mafb mice. Instead, they had a higher fungal burden, showed increased Candida seeding, and stronger kidney inflammation (Figures 4G - 4I), and exhibited a phenotype similar to that of the IL-1Ra fl / fl controls, indicating that the marked immediate resistance of IL-1Ra Mafb mice to Candida was mainly due to the lack of serum IL-1Ra in these mice, and highlighting the impact of serum IL-1Ra on the initial immune control of C. albicans. Furthermore, targeted removal of IL-1Ra was suggested to offer a potentially effective strategy for exploiting the endogenous antifungal response during disseminated candidiasis.

[0081] Therefore, the inventors tested whether neutralizing IL-1Ra in vivo before infection would enhance the ability of wild-type mice to suppress fungal infections (Figure 12B). Daily injection of a neutralizing antibody against mouse IL-1Ra efficiently depleted serum IL-1Ra but did not affect the expression of IL-1Ra protein in the splenic marginal zone (Figure 4J). Furthermore, prophylactic IL-1Ra neutralization significantly improved the immune control of Candida infection, as indicated by a decrease in fungal titer, limitation of Candida dissemination, and reduced tissue inflammation in infected kidneys (Figures 4K and 4L). Serum concentrations of IL-1Ra peaked on post-infection day 2 and returned to naive levels on post-infection day 7 (Figures 1C and 4M). Therefore, the inventors investigated whether therapeutic IL-1Ra neutralization would provide protection against lethal C. albicans infection (Figure 12C). In fact, neutralization of IL-1Ra, initiated on day 2 post-infection, reduced fungal titer in wild-type mice during high-dose C. albicans challenges and significantly improved their survival rate (Figures 4M, 4N, and 4O). LysM Mouse, IL-1Ra Mafb Mouse, IL-1Ra CD11c Based on the mouse phenotype, early suppression of fungal replication leads to the development of wild-type IL-1Ra fl / fl It has already been shown that it prevents the dysfunctional inflammatory response observed in controls (Figure 2K). These observations were confirmed after serum IL-1Ra depletion and reconstitution. Mafb While the mice showed very low levels of pro-inflammatory cytokines in the kidney, conversely, reconstitution of serum IL-1Ra in these mice not only inhibited their ability to limit Candida proliferation (Figure 4G), but also inhibited IL-1Ra fl / flIt also induced hyperinflammation of the kidneys, similar to that seen in mice (Figure 4L). Conversely, neutralization of IL-1Ra promoted rapid immune regulation against Candida in wild-type mice and was associated with a significant reduction in the inflammatory profile in the kidneys (Figures 4K and 4M). These data suggest that macrophage-produced IL-1Ra is directly linked to ineffective immune regulation of invasive fungal infections and the resulting dysfunctional inflammatory response, thereby suggesting that serum IL-1Ra is a biomarker and potential therapeutic target for disseminated candidiasis.

[0082] IL-1Ra secreted by infiltrating macrophages limits the elimination of pathogens in infected tissue. Our findings demonstrate that serum IL-1Ra has a significant impact on immune defense against Candida. Nevertheless, IL-1Ra CD11cThe transient protection observed in mice indicated that the later stages of the response were influenced by IL-1Ra secreted from a second population of macrophages sensitive to gene deletions mediated by Mafb-Cre rather than CD11c-Cre. Therefore, we investigated the relevance of locally produced IL-1Ra in infected tissue and characterized the IL-1Ra response in renal infiltrating leukocytes. Neutrophils, inflammatory monocytes, and renal resident macrophages constituted the main leukocyte populations present in the kidney three days post-infection (Figure 2J). Analysis of cells purified by FACS from infected mice revealed that IL-1Ra mRNA expression was highest in neutrophils, but IL-1β and IL-1R1 were expressed at similar levels in all three populations (Figure 5A). Nevertheless, when naive primary cells were exposed to Candida in vitro, IL-1Ra protein secretion from monocytes was significantly stronger than that from neutrophils. Simultaneous stimulation with the IL-1β cytokine did not affect IL-1Ra secretion in either monocytes or neutrophils (Figure 5B). However, type I IFN significantly increased Candida-induced IL-1Ra secretion in monocytes but did not affect IL-1Ra release from neutrophils (Figure 5B). Similarly, neutrophils isolated from infected kidneys secreted low levels of IL-1Ra independently of ex vivo exposure to Candida or type I IFN (Figure 5C). In contrast, inflammatory monocytes isolated from the same animals released significant amounts of IL-1Ra in response to Candida, which was further enhanced by additional IFNβ stimulation (Figure 5C). Western blot analysis revealed that neutrophils primarily express the 16kDa intracellular isoform of IL-1Ra. This may explain the discrepancy between mRNA expression detected in these cells in response to Candida and cytokine secretion (Figure 5D). In summary, these data identified inflammatory monocytes as the primary early IL-1Ra-producing cells in infected tissue, suggesting that their response is positively regulated by type I interferon (IFN).

[0083] Intracellular mRNA staining by prime flow analysis confirmed that inflammatory monocytes accounted for over 50% of all IL-1Ra mRNA-positive leukocytes on day 2 post-infection (Figure 5E). However, IL-1Ra Mafb Mouse or IL-1Ra CD11c The contribution of IL-1Ra secreted by monocytes to the enhanced protection observed in mice seemed unlikely. This was because both strains included a proportion of wild-type IL-1Ra mRNA-positive monocytes (Figure 5F), and EYFP Mafb Mouse and EYFP CD11c This was because Cre activity in renal infiltrating monocytes was negligible in reporter mice (Figures 5G and 5H). Nevertheless, the emergence of a second Ly-6Clo MHC II+F4 / 80+IL-1Ra producing population (presumably monocyte-derived macrophages) was observed, which comprised 40% of IL-1Ra-producing cells by day 5 post-infection (Figures 5I and 5J). This subset was identified as EYFP. CD11c EYFP is better than reporter mouse. Mafb Since it showed higher Cre activity in IL-1Ra Mafb In mice, it was suggested that IL-1Ra would be more efficiently deleted in these cells (Figure 5J). Assuming that type I IFN increases IL-1Ra secretion from monocytes, we investigated its effect on the IL-1Ra response produced by macrophages. IFNAR signaling-deficient mice (IFNAR - / - The group showed wild-type IL-1Ra expression in the spleen and serum (Figure 5K) and controlled fungal replication in the kidney to a similar degree as the control group (Figures 5L and 12E), indicating that type I IFN was not necessary for the induction of serum IL-1Ra in response to Candida infection. Type I IFN preferentially increased IL-1Ra secretion in a subset of MafbCre-labeled monocyte-derived macrophages, which the inventors identified as the major IL-1Ra-producing cells. In particular, EYFP showed increased secretion on day 5 post-infection. MafbAmong all CD11c+ macrophages isolated from infected mouse kidneys, only the EYFP+MafbCre-labeled population showed substantial IL-1Ra secretion in response to Candida, which was simultaneously amplified by IFN-β stimulation (Figure 5M). These results reveal the influence of IL-1Ra secreted from monocyte-derived macrophages in infected tissue. Furthermore, IL-1Ra Mafb In mice, particularly efficient gene targeting of this type I IFN-sensitive population suggests that it contributes to sustained long-term protection against Candida.

[0084] Type I IFN amplifies the macrophage IL-1Ra response and exacerbates fungal sepsis. Hospitalized patients may be susceptible to secondary invasive fungal infections due to preceding viremia. Our data demonstrate that type I IFN signaling enhances Candida-induced IL-1Ra secretion by macrophages. Therefore, we evaluated whether a type I IFN response induced in the context of viral infection can induce IL-1Ra as a permissive factor for subsequent invasive candidiasis. Intravenous infusion of IFN-β confirmed its ability to directly induce a serum IL-1Ra response (Figure 6A). Furthermore, intravenous administration of the synthetic TLR3 agonist polyinosinate-polycytidylic acid (PIC) (a well-established method for inducing potent IFN-I production in mice) rapidly induced IL-1Ra mRNA and protein expression in the spleen and induced significant levels of IL-1Ra circulation in the serum (Figures 6B and 6C). However, IL-1Ra Mafb In mice, PIC did not induce such IL-1Ra production, suggesting that fungal infection and IFN-I stimulated the same macrophage population to release IL-1Ra (Figure 6C). Furthermore, PIC treatment significantly amplified the Candida-induced IL-1Ra response in the spleen and serum (Figure 6D), resulting in mice becoming highly susceptible to fungal infections. In particular, PIC-treated mice showed a 2.5 × 10⁶ increase in IL-1Ra. 5Infection of CFUs with Candida significantly increased morbidity and required early exclusion from the experiment; however, in the absence of PIC-induced IFN-I, this fungal load was tolerable (Figure 6E). In contrast, PIC-induced IFN-I failed to induce detectable IL-1Ra production, and Candida infection IL-1Ra Mafb It did not worsen the disease severity in mice (Figure 6E). This confirmed that IFN-driven amplification of IL-1Ra produced by macrophages is the underlying mechanism for enhanced susceptibility to Candida infection after PIC injection in wild-type mice. Co-injection of PIC also enhances IL-1Ra + / + In mice, lower Candida intake worsened disease severity during infection (Figure 6F). The inventors of 10 5 In mice administered CFU-containing Candida alone, minimal levels of serum IL-1Ra were detected. However, co-induction of IFN-I strongly enhanced the serum IL-1Ra response, interfered with the initial immune regulation of C. albicans, and increased renal fungal titers by two orders of magnitude (Figure 6F). Supporting these observations, infection with microbial pathogens known to stimulate type I IFN (IFN-I) production in vivo, such as lymphocytic choriomeningitis virus (LCMV), varicella stomatitis virus (VSV), vaccinia virus (VV), or Listeria monocytogenes, induced IL-1Ra expression in the spleen to varying degrees (data not shown). In particular, LCMV and VSV, which are potent IFN-I inducers, strongly induced serum IL-1Ra production from MafbCre-labeled macrophages (Figure 6G).

[0085] LCMV is a well-characterized experimental model that replicates 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 dissemination in LCMV-WE infection. Similar to our findings with PIC-induced IFN-I, high-dose LCMV co-infection significantly worsened morbidity in Candida-infected mice; and the tolerable fungal load in control mice without LCMV co-infection could no longer be suppressed, and morbidity worsened (Figure 6H). Low-dose LCMV-infected mice remained responsive to small amounts of Candida inoculation (10) up to day 3 post-infection. 5 Although control was achieved against CFU, serum IL-1Ra levels remained very high (Figure 6I), indicating that fungal dissemination in the kidneys was not controlled (Figure 6J). In co-infected IFNAR-deficient mice, the increase in splenic and serum IL-1Ra responses was completely absent, indicating that the exacerbating effect of viral infection was strictly type I IFN-dependent; and their fungal titers were comparable to those of mice infected with C. albicans alone (Figures 7A and 7B). Therefore, the kidneys of co-infected IFNAR-deficient mice contained isolated infection foci and were thus similar to the kidneys of controls infected with C. albicans alone, while the kidneys of co-infected wild-type mice showed unrestricted fungal dissemination. Transcripts of IFN regulatory genes Ifit1, Isg15, and Mx1 revealed that C. albicans infection itself induces significant IFNAR signaling in the spleen and kidneys, which is further enhanced by virus-induced IFN-I (Figures 7C and 7D). Nevertheless, while Il1rn expression in the context of C. albicans infection appeared to be IFN-independent, its viral amplification was partially IFNAR-dependent (Figures 7C and 7D). MafbIn mice, co-infection confirmed that LCMV-induced IFNAR signaling induced serum IL-1Ra production from MafbCre-labeled macrophages (Figure 7E). Furthermore, while virus-induced IFN-I partially exacerbated Candida infection by amplifying the IL-1Ra response produced by macrophages, it was also revealed that Candida infection was exacerbated by an IFNAR-dependent mechanism unrelated to IL-1Ra produced by macrophages (Figure 7F). In summary, these observations demonstrate that type I IFN induced during viral infection strongly increases susceptibility to Candida bloodstream infection, leading to detrimental consequences for host survival, and that IL-1Ra produced by macrophages is involved in this process.

[0086] Discussion Invasive fungal infections remain an urgent and under-addressed medical problem due to their high disease-related mortality and limited treatment options. Our research elucidates the high disease susceptibility of disseminated candidiasis and the disease mechanisms contributing to known amplification due to viral co-infection. We identified serum IL-1Ra produced by macrophages as a disease-promoting innate immune checkpoint and found that inhibiting this macrophage-produced serum IL-1Ra provides protection against lethal C. albicans sepsis in a mouse model. These findings are immediately important for understanding the pathogenesis of invasive fungal infections and may open new avenues for their treatment.

[0087] Our results highlight the crucial role of serum IL-1Ra in invasive candidiasis. Serum IL-1Ra produced by hepatocytes has been reported in various inflammatory conditions. In contrast, we identified splenic CD169+ macrophages as the primary cells producing serum IL-1Ra during fungal infections. While serum IL-1Ra may be beneficial in bacterial sepsis by preventing excessive IL-1 signaling, it was found to be detrimental in invasive candidiasis. Genetic ablation of macrophage-produced IL-1Ra and liposome-mediated depletion of marginal macrophages suppressed serum IL-1Ra levels and enhanced the protective neutrophil response. This highlighted the influence of CD169+ macrophage-derived IL-1Ra. Marginal zone macrophages sense bloodborne pathogens such as Candida and direct the induction of innate and adaptive immunity. G-CSF released by CD169+ macrophages colonizing the spleen promotes neutrophil dysfunction in fungal sepsis. While no effect on splenic neutrophils was detected in IL-1Ra-deficient mice (Figures 2F, 2G, 2H, 2I, and 10A), reconstitution or neutralization of IL-1Ra regulated neutrophil function and excessive renal inflammation. This suggests that IL-1Ra derived from CD169+ macrophages inhibits antifungal immunity by limiting the maturation and rapid recruitment of IL-1-driven, highly bactericidal neutrophils to tissues (Figures 2 and 10A). We have identified a method to detect a second wave of IL-1Ra produced from macrophages in infected tissues and efficiently target this population. MafbThis may contribute to the excellent resistance of mice to Candida. Besides macrophages, neutrophils and monocytes infiltrating the kidney showed considerable IL-1Ra expression, while dendritic cells or renal macrophages showed only minimal expression. Neutrophils primarily contained intracellular IL-1Ra isoforms and secreted limited amounts of IL-1Ra in vitro. This suggests that IL-1Ra expressed by neutrophils is primarily biologically activated after being released upon neutrophil death. Unlike neutrophils, monocytes secreted considerable amounts of IL-1Ra, which was further enhanced by IFN-I stimulation. The Cre driver used here did not delete IL-1Ra from monocytes (Figure 5); and we are unable to conclude to what extent IL-1Ra secreted from monocytes affects immunodefence against disseminated Candida. However, the fact that a strong protective effect was observed when macrophages removed IL-1Ra, even in the presence of IL-1Ra expressed by monocytes, suggests that the role of IL-1Ra secreted by macrophages is dominant.

[0088] Our research identified neutrophils as a major defense mechanism regulated by IL-1Ra secreted from macrophages, and demonstrated that neutrophil maturation, recruitment to tissues, and enhanced functionality mediate the protective effects of IL-1Ra removal. Neutrophil effector pathways, including phagocytosis, reactive oxygen species production, and NET formation, are essential for systemic antifungal immunity. Our data suggest that exposure of neutrophils to IL-1Ra in the blood determines their IL-1β responsiveness after recruitment to inflammatory tissues (Figure 4). IL-1 programs neutrophil clustering behavior and the execution of defense mechanisms during antifungal responses. Therefore, the increased sensitivity to IL-1β stimulation (Figure 4) and increased IL-1β production (Figure 2) observed in neutrophils in the absence of IL-1Ra may enhance such IL-1 neutrophil-targeting activity. MafbThe superior neutrophil response in mice is likely also due to increased IL-1 signaling in other cell types, including endothelial cells. Enhanced IL-1 signaling may also promote granulocyte formation and act protectively against C. albicans through neutrophil-independent mechanisms.

[0089] The inventors found that IL-1Ra secreted from macrophages is positively regulated by IFN-I. Type I IFN signaling was associated with host defense against Candida in human patients. However, its role in protective immunity remains controversial, and both positive and negative disease outcomes have been reported in IFNAR-deficient mice. The inventors did not observe a significant effect of IFNAR deficiency on Candida monoinfection, which may suggest differences in genetic background or microbiome composition between these studies. Candida stimulates IFN-I secretion from conventional dendritic cells, which enhances bactericidal capacity and licenses monocytes to promote protective NK cell and neutrophil responses. Conversely, type I IFN may exacerbate the severity of Candida infection by inhibiting inflammasome activation and the production of bioactive IL-1, and by promoting hyperinflammatory kidney damage. Furthermore, the IFN-inducible factor IFIT2 limits leukocyte reactive oxygen species production and bactericidal activity. The inventors demonstrated that IFN-I inhibits protective IL-1 signaling by increasing IL-1Ra secretion from macrophages (Figures 6 and 7). This suggests that IL-1Ra is involved in the crosstalk between IFN-driven and IL-1-driven inflammation in invasive candidiasis, as proposed in bacterial infections. Candida induced IFNAR-independent IL-1Ra production, but the Candida-induced IL-1Ra response was significantly amplified by additional IFN-I signaling. IFN-I is induced by numerous pathogens, and prior viremia constitutes a risk factor for invasive candidiasis. The inventors showed that viral co-infection dramatically worsens disease mortality via IFN-dependent enhancement of IL-1Ra production in Mafb-Cre-labeled macrophages, providing a potential disease mechanism for secondary candidiasis or polybacterial sepsis.In this type of IFN-I / IL-1 crosstalk, targeting IL-1Ra may prove advantageous because it enhances protective IL-1R signaling by increasing the efficacy of physiologically secreted endogenous IL-1 without inhibiting beneficial defense mechanisms induced by type I IFN.

[0090] As the fact that defects in the entire IL-1 pathway dramatically increase disease mortality clearly demonstrates, IL-1-mediated inflammation is absolutely essential for controlling Candida bloodstream infection. However, when the physiological balance of IL-1 by IL-1Ra is disrupted due to genetic deficiencies or neutralization of anti-IL1Ra autoantibodies, IL-1 signaling becomes unregulated, and multi-organ inflammatory syndromes occur. Disruptions in the IL-1 / IL-1Ra balance are associated with hyperinflammatory states, and anakinra is a target for consideration in bacterial sepsis. In contrast, IL-1Ra deficiency during Candida infection... MafbIn mice, neither after IL-1Ra neutralization nor after other mechanisms of inflammation were observed. Instead, removal of IL-1Ra not only accelerated pathogen clearance, but surprisingly, the pathogenic hyperinflammation observed in mice expressing functional IL-1Ra rapidly and paradoxically decreased. Therefore, the dysfunctional inflammatory response in Candida sepsis is not due to excessive IL-1 signaling, but rather to a failure to eliminate the pathogen. Therapeutic neutralization significantly improved the survival rate of wild-type mice. This will likely prompt the development of more efficient IL-1Ra targeting approaches. Enhancing IL-1-driven mechanisms by neutralizing IL-1Ra may be beneficial in patients with active fungal replication, but may not be suitable for inflammatory states caused by residual fungal antigens. For example, IL-1α-induced neutrophilic inflammation is necessary for the clearance of pulmonary Aspergillus infection, but worsens disease outcomes in Aspergillus-induced asthma. Therefore, increased neutrophil recruitment after IL-1Ra neutralization may promote resistance to invasive fungal infections, but it may also persist pathogenic inflammation against non-replicating fungal components. Whether such mechanisms contribute to Immune Reconstitution Inflammatory Syndrome (IRIS) in chronic disseminated candidiasis has not yet been investigated.

[0091] In conclusion, our research enabled us to both enhance the effects of the physiological IL-1 response by removing the endogenous IL-1 inhibitor IL-1Ra in place of the IL-1 cytokine or its receptor, and to gain valuable insights into its regulation via IL-1Ra expressed by various subsets of immune cells. This approach confirmed the beneficial role of IL-1 in antifungal defense, while also revealing that IL-1Ra secreted from macrophages has detrimental effects on the ability to suppress bloodstream Candida infection. Furthermore, it revealed that the increased inflammation observed during invasive candidiasis does not reflect excessive signaling via IL-1Ra, but rather a failure to eliminate the fungal pathogen. Taken together, these observations suggest that serum IL-1Ra could be a further biomarker and therapeutic target for invasive candidiasis. [Brief explanation of the drawing]

[0092] [Figure 1-1] Macrophage-mediated ablation of IL-1Ra provides protection against invasive fungal infections. (A) mRNA expression of IL-1β, IL-1Ra, and IL-1R in the kidney at indicated days post-infection (pi). (n=4-8 mice / time point, 2 pooled experiments). (B) Quantification of serum IL-1Ra at indicated days post-infection. (n=12-20 mice / time point). (C) IL-1Ra production by indicated myeloid cell subsets upon stimulation with LPS, heat-killed (HK) C. albicans yeast, or mycelium in vitro. (D) Creation of conditional IL-1Ra-deficient lines. (E-G) Candida titers in the kidney of IL-1RaLysM, IL-1RaMafb, IL-1RaCD11c, and wild-type IL-1Rafl / fl mice at 3 and 7 days post-infection. (n≧7 mice / group, 3 pooled experiments). [Figure 1-2](E~G) Candida titers in the kidneys of IL-1RaLysM, IL-1RaMafb, IL-1RaCD11c, and wild-type IL-1Rafl / fl mice at 3 and 7 days post-infection. (n≧7 mice / group, 3 pooled experiments). (H~K) Histopathological analysis (H~J) and quantification of IL-1Ra-positive areas (K). (L) Plasma creatinine and BUN (blood urea nitrogen) concentrations of the indicated strains at 2 days post-infection. The dashed line indicates the baseline state of uninfected mice (naive). (n=7~13 mice / group, 2 pooled experiments). [Figure 1-3] (M) Quantification of renal Kim-1 and Lcn2 mRNA expression. (n=4-9 mice / group, 2 pooled experiments). Error bars represent mean ± SEM. [Figure 2-1] IL-1Ra produced by macrophages hinders rapid neutrophil recruitment and the resolution of inflammation. (A-B) Analysis of blood leukocytes of the indicated strains for Ly-6G fluorescence intensity of neutrophils (A) and frequency of mature neutrophils (Matneut), immature neutrophils (Immatneut), and monocytes (Mono) (B) on day 2 post-infection. (n=6-8 mice / group, 2 pooled experiments). (C) Absolute number of neutrophils (Neut) and monocytes (Mono) in peritoneal lavage fluid of indicated mice 18 hours after zymosan infusion. [Figure 2-2] (D, E) Bactericidal activity (D) and IL-1β secretion (E) of purified renal neutrophils from mice shown on day 2 post-infection. (n=6 mice / group). (F~I) Frequency of CD101+ROShi (F), pro-IL-1β+ (G), CD63+ (H), and MPO+ (I) neutrophils (Neut) in the blood, spleen, and kidney of the shown strains on day 2 post-infection. (n=4 mice / group, 2 representative mice). [Figure 2-3] (J) Characterization of renal inflammation in mice 2 days post-infection based on absolute number of leukocyte subsets (J) and cytokine concentration (K). (n=6 mice / group). Error bars represent mean ± SEM. [Figure 3-1]Serum IL-1Ra is produced by CD169+ marginal zone macrophages. (A-B) IL-1Ra expression in serum (A,B) of shown mice 3 days post-infection (A) or 5 hours after LPS infusion (B). Scale bar is 200 μm. (n=6-15 mice / group, 3 experiments pooled). (C) Diagram showing the EYFP reporter used in (F). (D, E) Analysis of serum IL-1Ra (D) and Candida titer (E) 3 days post-infection in splenectomized (SE) mice and control (ctrl) mice. (F, G) Histopathological evaluation of fungal dissemination (F) and inflammation (G) (n=4-6 mice / group, representative experiment). [Figure 3-2] (H, I) Analysis of serum IL-1Ra(H), kidney CD101+, and pro-IL-1β+(I) in clodronate liposome-treated mice (CL) and control mice (ctrl) 3 days post-infection. Error bars represent mean ± SEM. [Figure 4-1] Serum IL-1Ra mediates impaired pathogen control and dysfunctional hyperinflammation during disseminated Candida infection. (A, B) Analysis of IL-1Ra rearrangement in serum (A) during Candida infection and in peritoneal lavage fluid (B) during zymosan-induced peritonitis in IL-1RaMafb mice. (A, n=7 mice / group; C, n=9 mice / group). (C~E) Characterization of peritoneal infiltration in IL-1Ra-rearranged IL-1RaMafb mice 18 hours after zymosan infection by absolute number (C), bactericidal neutrophil activity (D), and phagocytic activity (E). (n=6 mice / group, 2 pooled experiments). [Figure 4-2](C~E) Characterization of peritoneal infiltration in IL-1Ra-converted IL-1RaMafb mice 18 hours after zymosan infection by absolute number (C), bactericidal neutrophil activity (D), and phagocytic activity (E). (n=6 mice / group, 2 pooled experiments). (F) Western blot analysis of IL-1R signaling in neutrophils with or without IL-1Ra pulse treatment. (G, H) Characterization of fungal titer (G) and cell infiltration (H) at 3 days post-infection in IL-1Ra-converted mice (n=7 mice / group, 2 pooled experiments). (I) Histopathological quantification of fungal replication and inflammation. (J~L) Prophylactic IL-1Ra neutralization (NT) at 3 days post-infection of Candida infection was tested by serum IL-1Ra expression (J) and fungal renal titer (K). Scale bar is 1000 μm. (L) Pathological and histological quantification of Candida replication and inflammation (n=6-10 mice / group, 3 pooled experiments). [Figure 4-3] (J~L) Prophylactic IL-1Ra neutralization (NT) on day 3 after Candida infection was assessed by serum IL-1Ra expression (J) and fungal renal titer (K). Scale bar is 1000 μm. (L) Histopathological quantification of Candida replication and inflammation (n=6~10 mice / group, 3 pooled experiments). (M~O) Therapeutic IL-1Ra neutralization in wild-type mice infected with Candida was evaluated by serum IL-1Ra (M), renal Candida titer (N), and survival rate (O). (n=6~7 mice / group, 2 pooled experiments). [Figure 4-4] (P, Q) Inflammatory profiles in the kidneys of shown mice after IL-1Ra reconstitution (P) or neutralization (Q) were determined by cytokine arrays (n=5-7 mice / group). Error bars represent mean ± SEM. [Figure 5-1] IL-1Ra from infiltrating macrophages limits the elimination of pathogens in infected tissue. (A) mRNA expression of IL-1 family genes in a shown cell subset on post-infection day 2. [Figure 5-2](B, C) IL-1Ra production by neutrophils (Neut) and monocytes (Mono) stimulated with Candida (Ca) in vitro, in the presence or absence of IL-1β or IFN-β. Cells were purified from naive mice (B) or 2 days post-infection (C). (B, dual culture, n=4 mice; C (control), n=4-7 mice). (D) Western blot analysis of IL-1Ra isoforms in Candida-stimulated neutrophils and monocytes. [Figure 5-3] (E) Frequency of neutrophils and monocytes in renal leukocytes expressing IL-1Ra mRNA on day 2 post-infection. (n=7 mice / group, 2 pooled experiments). (F) Frequency of IL-1Ra mRNA-expressing cells in the indicated immune cell subsets on day 2 post-infection of IL-1Rafl / fl mice, IL-1RaMafb mice, and IL-1RaCD11c mice. (n=7 mice / group, 2 experiments). (G~J) Analysis of Cre expression in IL-1Ra mRNA-expressing leukocytes of EYFPMafb mice and EYFPCD11c mice on day 2 (G~I) and day 5 (J) post-infection. Expression of EYFP and IL-1Ra mRNA in neutrophils or monocytes of EYFPMafb mice (G) and EYFPCD11c mice (H) was evaluated and quantified by flow cytometry. (n=4~8 mice / group). (I, J) Frequency of EYFP expression in Ly6G-CD11b+ cells expressing IL-1Ra mRNA from EYFPMafb mice and EYFPCD11c mice on day 2 (I) and day 5 (J) post-infection. (n=4-8 mice / group). [Figure 5-4] (K, L) Characterization of IFNAR- / - mice by serum IL-1Ra expression (K) and renal fungal load (L) on day 3 post-infection. (n=4-8 mice / group). (M) IL-1Ra production by EYFPMafb macrophages, purified ex vivo via FACS and stimulated in vitro as shown, on day 5 post-infection. (n=6 mice / group, 2 pooled experiments). Error bars represent mean ± SEM. [Figure 6-1]Type I IFN amplifies the macrophage IL-1Ra response and exacerbates fungal sepsis. (A, B) In vivo IL-1Ra induction by type I IFN, evaluated by serum IL-1Ra (A) and kidney mRNA expression (B) after IFN-β or PIC injection, respectively. (n=7 mice / group, 2 pooled experiments). (C, D) Serum (C, D) and PIC-induced IL-1Ra expression in IL-1Rafl / fl mice and IL-1RaMafb mice were measured 5 hours after PIC injection (C) or 48 hours after additional Candida infection (D). (C, n=4~7; B, n=3; E, n=3~6 mice / group). (E) Survival probability of IL-1Rafl / fl mice and IL-1RaMafb mice injected with Candida and PIC, as shown. (n=6 mice / group, 2 pooled experiments). [Figure 6-2] (F) Fungal load in the kidneys of mice treated in the same manner as (E) as evaluated on day 2 post-infection (n=6 mice / group). (G) Serum IL-1Ra levels in IL-1Rafl / fl and IL-1RaMafb infected mice. The dashed line shows the naive (uninfected) baseline. (n=3-7 mice / group, single experiment). (H) Survival probability of the indicated group of infected mice. (n=4 mice / group). (I, J) Characterization of co-infected mice on day 3 post-infection by serum IL-1Ra expression (I) and kidney Candida titer (J) (n=4 mice / group, representative experiment). Error bars represent mean ± SEM. [Figure 7-1] Exacerbation of virus-induced fungal dissemination is critically dependent on type I IFN and IL-1Ra produced by macrophages. (A-D) IFNAR- / - mice infected with Candida (Ca) or co-infected with LCMV and Candida (LCMV+Ca) and wild-type mice were analyzed at 3 days post-infection by PAS staining and IL-1Ra staining of serum IL-1Ra (A), fungal renal titer (B), and mRNA expression of IFN-stimulated genes in the spleen (C) and kidney (D). (n=5-7 mice / group, 2 pooled experiments). (E, F) Serum IL-1Ra levels (E) and renal Candida titer (F) of IL-1Rafl / fl mice and IL-1RaMafb mice infected in the same manner as (A-E). (n=4-5 mice / group, single experiment). Error bars represent mean ± SEM. [Figure 7-2] (A-D) IFNAR- / - mice infected with Candida (Ca) or co-infected with LCMV and Candida (LCMV+Ca) and wild-type mice were analyzed on day 3 post-infection by PAS staining and IL-1Ra staining of serum IL-1Ra (A), fungal renal titer (B), and mRNA expression of IFN-stimulated genes in the spleen (C) and kidney (D). (n=5-7 mice / group, 2 pooled experiments). (E, F) Serum IL-1Ra levels (E) and renal Candida titer (F) of IL-1Rafl / fl mice and IL-1RaMafb mice infected in the same manner as (A-E). (n=4-5 mice / group, single experiment). Error bars represent mean ± SEM. [Figure 8-1] The efficiency and specificity of the Cre driver used in this study (related to Figure 1). (A) The efficiency of Cre-mediated IL-1Ra deletion was evaluated in vitro in specified cell subsets of IL-1RaLysM mice, IL-1RaMafb mice, IL-1RaCD11c mice, and their respective IL-1Rafl / fl littermate controls. BM (bone marrow)-derived macrophages (BMDM), BM neutrophils, and spleen DCs were stimulated with 10 ng / ml LPS for 24 hours. IL-1Ra production in the culture supernatant was evaluated by ELISA, or IL-1Ra production in the whole cell lysate was evaluated by Western blot analysis. Representative Western blots are shown. [Figure 8-2](B) Flow cytometry analysis of EYFP Cre-reporter expression in indicated immunocell subsets isolated from the BM, spleen, and kidney of naive EYFPLysM mice, EYFPMafb mice, and EYFPCD11c mice, and their respective Cre-negative EYFPLSL littermates. (C) Body weight of IL-1RaLysM, IL-1RaMafb, IL-1RaCD11c mice, and their respective IL-1Rafl / fl littermates infected with C. albicans. Body weight at 3 and 6 days post-infection is expressed as a percentage of the initial body weight before infection (day 0). A dot represents one mouse. Panel A shows pooled data from three experiments using at least 6 mice per group. Panel B shows pooled data obtained from one experiment using 2 mice per group. Bars represent mean ± SEM. [Figure 9] Inflammatory response of conditional IL-1Ra-deficient mice infected with C. albicans (related to Figure 2). (A-B) The absolute numbers of neutrophils and monocytes in the lavage solution (A) and blood (B) of IL-1Rafl / fl, IL-1RaLysM, IL-1RaMafb, and IL-1RaCD11c mice were determined by flow cytometry at 6 and 18 hours after post-infection administration of zymosan A. Representative data from two experiments are shown. Bars indicate mean ± SEM. [Figure 10-1] (A) Early neutrophil recruitment and functionality in IL-1RaMafb mice infected with C. albicans (related to Figure 2). (A) Neutrophil recruitment dynamics in the spleen and kidneys of IL-1Rafl / fl mice and IL-1RaMafb mice 1, 2, and 3 days after C. albicans infection. Representative data obtained from two experiments using 4 mice per group. [Figure 10-2] (B-C) Reactive oxygen species (ROS) production by neutrophils as measured by luminol-enhanced chemiluminescence assay. (B)C. Neutrophils isolated from the kidneys of IL-1Rafl / fl and IL-1RaMafb mice 2 days after albicans infection. (C) Neutrophils isolated from the lavage fluid of IL-1Rafl / fl and IL-1RaMafb mice 18 hours after zymosan A injection following infection. [Figure 10-3] (D) Fungal titers in the spleen of IL-1RaLysM, IL-1RaMafb, IL-1RaCD11c, and their respective IL-1Rafl / fl littermates 2 days after C. albicans infection. Data were pooled from two experiments using 8 mice per group. Bars indicate mean ± SEM. [Figure 11-1] The inhibitory effect of IL-1Ra is extracellular and mediated by IL-1Ra secreted from splenic marginal zone macrophages (see Figure 3). (A) Frequency of EYFP+ cells in the indicated cell subsets in the blood and peritoneal lavage fluid of EYFPLSL, EYFPLysM, EYFPMafb, and EYFPCD11c mice 18 hours after post-infection administration of zymosan A. (B) Scheme for the creation of mixed BM (myelocyte) chimeras (BMCs). Congenic CD45.1+IL-1Ra+ / + recipients were reconstituted in equal proportions with CD45.1+IL-1RA+ / + wild-type BM (as an internal reference present in all chimeras) and CD45.2+ experimental BM from IL-1RaMafb, IL-1RaLysM, or control IL-1Rafl / fl mice. [Figure 11-2] (C~E) Analysis of immune cell subsets in mixed BMCs 3 days post-Candida infection. (C) Absolute number of total CD45+ neutrophils, inflammatory monocytes, and renal resident macrophages in the kidney of the shown mixed BMC, determined by flow cytometry. (D) Frequency of CD45.2 / 2+ cells (black bars) and CD45.1 / 2+ cells (white bars) among neutrophils and monocytes in the infected kidney of the shown mixed BMC mouse 3 days post-infection. The frequency of CD45.2 / 2+ cells was normalized by the mean frequency of CD45.1 / 2+B220+ cells in BM to normalize potential differences in BM reconstitution efficiency in different recipient mice. (E) The phagocytic activity of neutrophils isolated from the kidneys of infected IL-1Rafl / flBMCs, IL-1RaLysMBMCs, and IL-1RaMafbBMCs was measured in vitro as the uptake of pHrodo green zymosan particles after staining for CD11b, Ly6G, CD45.1, and CD45.2 and flow cytometry analysis. [Figure 11-3] (F) Creation of chimeric mice: BM cells isolated from one donor strain (IL-1Rafl / fl or IL-1RaMafb) were transplanted into irradiated recipient wild-type mice (C57BL / 6), rested for at least 8 weeks, and then infected with C. albicans. (G) Renal fungal load IL-1Rafl / fl>B6 and IL-1RaMafb>B6 BMC mice at 3 and 7 days post-C. albicans infection. (H) Scheme for selective depletion of marginal zone resident macrophages in C57BL / 6 mice using clodronate liposomes (CL) after C. albicans infection. Mice were euthanized 3 days post-infection. [Figure 11-4] (I-L) Absolute numbers of neutrophils and monocytes in the infected kidney (I) and spleen (J) of control mice and CL-treated mice 3 days after albicans infection. (K, L) Absolute numbers of CD101-positive mature neutrophils (K) and activated pro-IL-1β-positive neutrophils (L) in the spleen were evaluated by flow cytometry. Panel A consists of pooled data from at least 3 experiments using at least 8 mice per group. Panels C-E consist of pooled data from 2 experiments using 5 mice per group. Panel G consists of pooled data from 2 experiments using 4 mice per group. The data in Figures I-L are pooled from 2 experiments using 12 mice per group. Bars represent mean ± SEM. [Figure 12-1] Neutralizing serum IL-1Ra produced by macrophages improves early pathogen control and prevents excessive renal inflammation (see Figures 4 and 5). (A) Experimental scheme for rIL-1Ra rearrangement. (B) Experimental design for prophylactic IL-1Ra neutralization. [Figure 12-2](C) Experimental design for therapeutic IL-1Ra neutralization. Monoclonal IL-1Ra antibody was administered intravenously from day 2 to day 6 post-infection. Mice were injected with 3.5 × 10⁵ CFU to assess survival rate, and with 2 × 10⁵ CFU on days 5 and 7 to examine fungal titer. (D) Kaplan-Meier survival plots of IL-1RaMafb mice and IL-1Rafl / fl controls challenged with C. albicans. Data were pooled from two experiments using at least 7 mice per group. (E) WT (wild-type) and IFNAR-deficient mice were infected with 1 × 10⁵ CFU of C. albicans for 7 days. Renal fungal load was assessed in CFU. Data show the results of one experiment using 4 mice per group.

[0093] Experimental model animal Mice were housed and managed in a specific-pathogen-free (SPF) facility at the Bern University Institute of Pathology. All procedures were carried out in accordance with the Bern Cantonal Ethical Guidelines and approved animal use permit protocols (BE3 / 18 and BE31 / 2021). Mice were housed in a 12-hour light-dark cycle, with controlled 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 The Jackson Laboratory and bred in-house. IL-1Ra LysM The mouse is IL-1Ra fl / fl This mouse was created by crossing (II1rntm1.1Cga) mice with LysM-Cre(Lyz2tm1(cre)Ifo / J) mice and was kindly provided by Marc Donath (Department of Biomedical Sciences, University Hospital Basel). IL-1Ra Mafb Mouse and IL-1Ra CD11c To obtain a mouse, IL-1Ra fl / flMice were crossed with either Mafb-Cre mice or CD11c-Cre mice (B6.Cg-Tg(Itgax-cre)1-1Reiz). The CD11c-Cre mice were donated by Manfred Kopf (Institute of Molecular Health Sciences, ETH Zurich). Generally, IL-1Ra fl / fl Cre-negative littermates served as controls, and in some experiments, IL-1Ra wt / wt Cre-positive mice were used as controls, and wild-type results were obtained. R26R-EYFP mice (EYFP as used herein) LSL (referred to as EYFP) was crossed with LysM-Cre mice, Mafb-Cre mice, or CD11c-Cre mice to create EYFP-Cre reporter lines for each (EYFP is referred to as EYFP in this specification). LysM EYFP Mafb , or EYFP CD11c (This is referred to as [the C57BL / 6 strain]). All mouse strains were either backcrossed to C57BL / 6 for more than 10 generations or created from a C57BL / 6 background (genetic background). Age and sex-matched animals were randomly assigned to the experimental group. To create bone marrow (BM) chimeras (BMCs), 6-week-old recipient mice were given 5 × 10⁶ doses. 6 Individual donor BM cells were lethally irradiated with gamma rays using a GammaCellX40 irradiation device 24 hours prior to intravenous infusion. Mice were administered sulfamethoxazole and trimethoprim via drinking water for two weeks, rested for at least eight weeks before the experiment, and then BM was reconstituted.

[0094] Candida albicans and inflammation models C. albicans (SC5413) was cultured in YPD medium (BD Difco® YPD Broth, BD Sciences) at 30°C for 18 hours. C. albicans yeast cells were washed twice with sterile PBS and counted using a hemocytometer. To obtain C. albicans hyphae, the washed C. albicans yeast was cultured in RPMI1640 (10% FBS) at 37°C for 4 hours. Heat-dead yeast and hyphae were prepared by incubation at 72°C for 1 hour, and complete inactivation was confirmed by plating on YPD agar plates at 30°C for 48 hours. In in vivo experiments, 0.5–2.5 × 10⁶ mice aged 8–10 weeks were given 10⁶ cells. 5 Colony-forming units (CFUs) of C. albicans were intravenously infected via the lateral tail vein. To measure the fungal organ titer, mice were euthanized, their organs were weighed, and homogenized in 0.5% NP-40 water at 25 Hz for 2 x 3 minutes using TissueLyser II (Qiagen). Subsequently, the tissue homogenates were serially diluted with PBS, plated on YPD agar, incubated for 24–48 hours, and the fungal load was calculated as C. albicans CFUs / g tissue. To evaluate the effect of type I IFN on IL-1Ra production and C. albicans infection in vivo, mice were injected with 2 μg of recombinant mouse IFN-β (Biolegend) alone, and with the synthetic TLR3 ligand polyinosinate-polycytidylic acid (PIC, InvivoGen) alone or 5 hours prior to C. albicans infection. Similarly, 1 × 10⁶ 4 or 2 × 10 6 Mice were infected with the plaque-forming unit (pfu) lymphocytic choriolarinitis virus (LCMV) strain WE either alone or one day prior to C. albicans infection. Spleen and serum IL-1Ra production was also investigated. 6 pfu vesicular stomatitis virus (VSV Indiana), 2 × 10 6Evaluators were assessed on day 1 post-infection using pfu vaccinia virus (VV) or 3000 CFU Listeria monocytogenes (10403S strain). Pathogens were prepared by dilution from frozen virus stocks grown in MDCK cells (VV), BHK21 cells (LCMV WE), or Vero cells (VSV), or by overnight culture in cerebral myocardial infarction broth (Listeria). To evaluate the initial cell recruitment induced by fungal components, 1 mg of Zymosan A from Saccharomyces cerevisiae (Sigma), freshly lysed in sterile PBS, was intraperitoneally injected into mice. Blood and peritoneal exudate were collected 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. Peritoneal neutrophils were purified from the lavage solution using a magnetic bead separation method before evaluating their killing and phagocytic activity against C. albicans in vitro. To evaluate LPS-induced IL-1Ra expression, mice were intraperitoneally injected with ultra-high purity LPS O111:B4 (150 μg / kg, Sigma) and D-galactosamine (800 mg / kg, Carbosynth Ltd.), and serum and organs were collected 5 hours later.

[0095] Method details In vivo manipulation of spleen macrophages Splenectomy was performed under isoflurane anesthesia in a laminar flow bench. The spleen was exposed by opening the abdominal skin and peritoneum through two small incisions. 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 incision was closed with wound clips. Postoperatively, the mice were allowed to recover under a heating lamp and rested for 8 weeks before C. albicans infection. To selectively reduce macrophages in the splenic marginal zone, the mice were intravenously injected with 1 mg of commercially available clodronate liposomes (Liposoma) per mouse, while control mice were administered the same amount of sterile PBS. After 10 days, all mice received 2.5 × 10⁶ 5CFUs were infected with C. albicans. Serum and organs were collected and analyzed three days after infection.

[0096] Reconstitution and neutralization of IL-1Ra in vivo The genetically modified human IL-1Ra (anakinra) was donated by Marianne Boni-Schnetzler (Department of Biomedical Sciences, University Hospital of Basel). Mafb Mice were administered recombinant IL-1Ra (500 μg per mouse) intraperitoneally twice daily on days 0, 1, and 2 after C. albicans infection. In these experiments, IL-1Ra fl / fl and IL-1Ra Mafb Equal volumes of sterile PBS were injected into mice that had not been reconstituted. Hybridomas producing neutralizing anti-mouse IL-1Ra antibodies were provided by Naofumi Mukaida (Kanazawa University, Japan) and Russell Vance (University of California, Berkeley, USA). Monoclonal antibodies were manufactured and purified in-house using protein G resin (GenScript). For prophylactic IL-1Ra neutralization in vivo, mice were intraperitoneally injected with either the neutralizing anti-IL-1Ra antibody or a control of Ultra-LEAF purified Armenian hamster IgG isotype antibody (BioLegend) on days 1-1, 1, and 2 after infection. For therapeutic IL-1Ra neutralization, mice were intravenously administered anti-IL-1Ra antibody or control antibody 2-6 days after C. albicans infection.

[0097] Isolation of a white blood cell population Leukocytes were isolated from the kidney using a modified protocol by Swamydas et al. The kidney was cut using a scalpel to a size of 1 mm. 3The cells were excised and digested in 6 ml of serum-free RPMI1640 medium containing 0.2 mg / ml Liberase® (Roche) and 0.2 mg / ml DNase I (Sigma) at 37°C for 45 minutes. At the end of incubation, the same volume of complete RPMI1640 medium was added, the cell suspension was filtered through a 40 μm cell strainer, and washed twice with PBS. This pellet was suspended in 40% Percoll (Sigma), gently placed on top of 70% Percoll, and centrifuged at 880 g at room temperature for 30 minutes. Spleen single-cell suspension was 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). Monocytes were isolated using the EasySep® Mouse Monocyte 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 in vitro IL-1Ra production in individual leukocyte subsets, single-cell suspensions were first prepared from infected organs as described above. After cell surface staining to identify leukocyte subsets, individual mouse cells were purified using FACS on a MoFlo Astrios EQ sorter (Beckman Coulter) at the Flow Cytometry Core Facility of the Biomedical Research Department, University of Bern.Cells were sorted and placed in pre-warmed complete culture medium. BM cells were collected by washing the femurs and tibias of both hind limbs with sterile PBS. After lysing the erythrocytes with ACK lysis buffer, primary BM neutrophils and BM monocytes were isolated using the EasySep® Mouse Neutrophil Enrichment Kit and EasySep® Mouse Monocyte Isolation Kit as described above. To analyze LPS-induced IL-1Ra production in vitro, spleen macrophages and spleen dendritic cells were purified from single-cell suspensions using magnetic anti-CD11b microbeads and CD11c microbeads (Miltenyi Biotec) according to the manufacturer's instructions. BM-derived macrophages were prepared by culturing BM cells in complete RPMI1640 medium supplemented with 10% L929 cell supernatant as a source of M-CSF. The medium was changed every 3 days, and BM-derived macrophages were collected for experimentation on day 7. Peritoneal macrophages were collected four days after intraperitoneal administration of 1 ml of 3.8% thioglycolate (Becton Dickinson AG). Cells in the lavage solution were washed with PBS and cultured overnight in or without 10 ng / ml of LPS (InvivoGen). Subsequently, IL-1Ra production was evaluated in the culture supernatant by ELISA or in the cell lysate by Western blotting.

[0098] Flow cytometry analysis Single-cell suspensions were prepared as described above and stained with bio / dead dye (Invitrogen) and anti-mouse CD16 / 32 antibody (BioLegend) at 4°C for 15 minutes. Immunocyte 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 Siglec F (Miltenyi Biotec). Neutrophils, resident macrophages, Ly-6 hi Single ball, Ly-6C lo The monocyte and DC are each 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 + This was defined as follows: Mature neutrophils, immature neutrophils, and monocytes in the blood were each classified as CD11b + CD115 - Ly6G + CD101 + CD11b + CD115 - Ly6G + CD101 - , and CD11b + CD115 + Ly6C +This was identified. To characterize the neutrophil phenotype, cells were stained with antibodies against CD45.2, CD11b, Ly-6G, CD63 (all BioLegend), CD101, and pro-IL-1 beta (both 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 with 4% paraformaldehyde (PFA) for 5 minutes before acquisition. Intracellular IL-1Ra transcripts from renal infiltrating leukocytes were elucidated using the PrimeFlow® RNA assay (Thermo Fisher) according to the manufacturer's instructions. After staining the cell surface with CD45.2, CD11b, CD11c, MHC II, F4 / 80, Ly-6C, and Ly-6G, the cells were fixed and permeabilized. Hybridization, signal amplification, and fluorescent labeling were then performed using an Il1rn probe. All samples were acquired using an LSRII cytometer (BD Biosciences) and analyzed using FlowJo (BD Biosciences).

[0099] Measurement of reactive oxygen species Newly purified neutrophils (10 per well) 6 After equilibrating cells at 37°C for 60 minutes, they were stained with 5 μM 2',7'-dichlorofluorescein (DCF) in serum-free RPMI for 30 minutes, followed by further exposure to heat-killed C. albicans (MOI 3) in or without IL-1β (20 ng / ml) for another 30 minutes. Subsequently, the cells were stained with live / dead fluorescent dyes and antibodies against CD45.2, CD11b, Ly-6G, and Ly6C. Reactive oxygen species production was quantified in leukocytes isolated from the blood, spleen, and kidneys of C. albicans-infected mice. Cells were incubated with dihydrorhodamine 123 (Sigma Aldrich) for 30 minutes at 37°C before staining with live / dead fluorescent dyes and antibodies against CD45.2, CD11b, Ly-6G, Ly-6C, and CD101. The frequency of reactive oxygen species-producing neutrophils was analyzed using FlowJo (BD Biosciences).

[0100] C. albicans sterilization assay To test the sterilization ability, 5 × 10 4 Each neutrophil 2 × 10 4 Cells were incubated with C. albicans for 3 hours. The cells were then lysed with 1% NP-40 water, and serial dilutions of the lysate were plated onto YPD agar. Colonies were measured after 24 hours at 30°C. Bactericidal activity was expressed as the percentage of C. albicans inoculations detected in a control well without leukocytes that died in the presence of neutrophils.

[0101] Phagocytosis assay Phagocytic activity was measured using pHrodo® Green Zymosan Bioparticles (Thermo Fisher) according to the manufacturer's instructions. Neutrophils were purified from the peritoneal cavity of mice primed with Zymosan A and 100 μl were added to 100 μl of Opti-MEM® (Gibco). 5 Cells were plated in black transparent-bottom 96-well plates (Corning) at a concentration of cells / well and equilibrated at 37°C for 1 hour. The culture medium was then replaced with 0.5 mg / ml pHrodo® green zymosan bioparticles in 100 μl of uptake buffer, and incubation was continued at 37°C for 2 hours, either in the presence or absence of 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. Fluorescence intensity was evaluated using an Infinite M200 pro Tecan microplate reader at an excitation wavelength of 490 nm and an emission wavelength of 535 nm. Net phagocytosis was calculated by correcting for the fluorescence intensity of the background and negative controls. Phagocytosis was expressed as a percentage of the net fluorescence intensity of the experimental well compared to the net positive control.

[0102] Blood sampling Blood samples were collected from the vena cava using syringes containing heparin (for creatinine and blood urea nitrogen) or EDTA (for cytokine analysis) as an anticoagulant. All samples were kept on ice during handling. Blood was transferred to serum collection tubes (BD microtainers) or 1.5 ml plasma collection tubes and centrifuged at 2000 g at 4°C for 10 minutes. 100 μl of the supernatant was transferred to a clean polypropylene tube and either processed immediately or stored at -80°C until analysis. Plasma creatinine and blood urea nitrogen concentrations were measured by the Laboratory Medicine Center at Bern University Hospital.

[0103] Analysis of cytokine response Cytokine concentrations in serum, peritoneal lavage fluid, or cell culture supernatant were measured by ELISA. IL-1Ra and G-CSF production in serum and culture supernatant was measured using the mouse IL-1ra / IL-1F3 DuoSet kit and the mouse G-CSF DuoSet kit (RnD Systems) according to the manufacturer's guidelines. Mouse IL-1β and IL-6 were quantified using sandwich ELISA with anti-IL-1β / biotinylated anti-IL-1β or anti-IL-6 / biotinylated anti-IL-6 antibody pairs (all from eBioscience), and subsequently detected with streptavidin-alkaline phosphatase (Southern Biotech) and para-nitrophenyl phosphate. Optical density was read using an Infinite M200 pro Tecan microplate reader, and concentrations were calculated using recombinant standards for mouse IL-1β (RnD Systems) and IL-6 (eBioscience), respectively. Cytokine profiles in infected kidneys were analyzed using the Mouse Cytokine / Chemokine 31-Plex Discovery Assay® Array (Eve Technologies). Briefly, infected kidneys were rapidly frozen, homogenized in modified RIPA buffer, and the tissue homogenates were stored at -80°C until analysis.

[0104] Western blot analysis Purified leukocyte subsets were dissolved 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 a protease inhibitor (Roche), phenylmethylsulfonyl fluoride, and phosphatase inhibitor cocktails 2 and 3 (Sigma Aldrich). Protein extracts were standardized using the Pierce® BCA Protein Assay Kit (Thermo Fisher), and 25 μg of protein was evaluated by reduced SDS-PAGE (10-12%). After transfer to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories), immunoblotting was performed using primary antibodies against IL-1Ra (Thermo Fisher), anti-p38, anti-phospho-p38 (all from Cell Signaling), and anti-β-actin (Santa Cruz Biotechnology), along with their corresponding HRP-labeled secondary antibodies (Santa Cruz). The samples were then developed with SuperSignal® West Pico chemiluminescent substrate. Staining was visualized using the ChemiDoc® MP Imaging System (Bio-Rad Laboratories).

[0105] Immunohistochemistry and immunofluorescence Organs were dissected, fixed with 4% paraformaldehyde for 6-8 hours, and then embedded in standard paraffin. Tissue sections (2.5 μm) were stained using a Leica Bond RX immunohistochemical system (Leica Biosystems) with periodic-acid Schiff (PAS), methenamine silver (Grocott), or 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, and anti-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 Unit at the Institute of Pathology, University of Bern. Images were acquired using a Panoramic250 scanner (3DHistech) before analysis. Histological grading was performed by trained pathologists blinded to specimen identity, using periodate-Schiff (PAS) stained sections. Inflammation and C. albicans dissemination were assessed separately for the tubulointerstitial compartment and glomeruli. A score from 0 to 3 was defined for each category and criterion, as follows. The final score for each section represents the sum of the scores for the tubulointerstitial compartment and glomeruli. [Table 1]

[0106] Spleen tissue was frozen in OCT medium and sectioned using a Leica CM1950 cryostat (5 μm). 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, and then stained overnight with anti-mouse MARCO (Bio-Rad Laboratories) antibody and anti-mouse CD169 (BioLegend) antibody. Rinsed sections were stained with DAPI (Sigma-Aldrich) for 20 minutes and mounted using Dako Fluorescence Mounting Medium (Dako). Fluorescence images were acquired using a Panoramic 250 flash II slide scanner (3DHistech). IL-1Ra-positive regions of stained kidney sections were quantified using ImageScope (v12.4.0.5043). To visualize the co-localization of marginal zone macrophages and IL-1Ra, spleen tissue was frozen in OCT medium and cut using a Leica CM1950 cryostat (10 μm). PFA-fixed (4%) frozen sections were treated with multi-staining buffer (Lunaphore) and stained using LabSat technology (Lunaphore). In the first cycle, rabbit anti-goat Alexa Fluor 546 antibody (Invitrogen) was used as the secondary antibody to stain and image with anti-mouse IL1RA (R&D Systems), anti-mouse CD169 (Biolegend), and DAPI. After applying quenching buffer (Lunaphore), in the second cycle, the corresponding secondary antibody (PE goat anti-rat Ig, SouthernBiotech) was used to stain and image with MARCO (Bio-Rad Laboratories) and DAPI.

[0107] Quantitative reverse transcription PCR In TissueLyser II (Qiagen), 0.5–1 ml of TRIzol reagent (Ambion Life Technologies) was mixed with stainless steel beads (5 mm; Qiagen), and kidneys were lysed at 25 Hz for 2 minutes for 2 cycles. Total mRNA was isolated according to the manufacturer's instructions. Contaminated DNA was digested with RNase-free DNase (Life Technologies), and mRNA concentration was measured using a NanoDrop® One spectrophotometer (Thermo Fisher). 1 μg mRNA / reaction was reverse transcribed using GoScript® Reverse Transcriptase (Promega) in the presence of an RNase inhibitor (BioLabs). Quantitative PCR was performed using KAPA SYBR® FAST qPCRMaster Mix (2X) Kit (Sigma) on a StepOnePlus® Real-Time PCR system (Thermo Fisher), and expression levels were normalized to the expression levels of G6pdx or Actb. For cells sorted by FACS, RNA was purified using ReliaPrep® RNA Miniprep Systems (Promega) according to the manufacturer's instructions.

[0108] RNA sequencing and bioinformatics analysis Kidneys from infected mice were collected two days post-infection, rapidly frozen in liquid nitrogen, and then homogenized using TissueLyser II (Qiagen) with TRI Reagent (registered trademark) (Zymo Research) in BushingBeat (trademark) lysis tubes (Zymo Research) for three cycles at 30 Hz for 1 minute each. Total RNA was extracted using the Direct-zol (trademark) RNA miniprep Plus kit (Zymo Research) and stored at -80°C until use. The yield and quality of the purified total RNA were evaluated using a Qubit 4.0 fluorometer with the Qubit RNA BR Assay Kit (Thermo Fisher) and an Advanced Analytical Fragment Analyzer System with the Fragment Analyzer RNA Kit (Agilent). First, 200 ng of input RNA was decontaminated with ribosomal RNA and globin mRNA using the RiboCop for HMR+Globin Depletion Kit (Lexogen) according to the manufacturer's instructions. Subsequently, cDNA libraries were prepared using the CORALL Total RNA-Seq V2 library Prep.kit and UDI 12nt set A1 (Lexogen) according to the protocol for long insert sizes. The volume and length of the cDNA libraries were determined using the Qubit 4.0 fluorometer and Advanced Analytical Fragment Analyzer System, as described above. Library quantification was also determined using the JetSeq library Quantification Lo-ROX kit (Bioline) according to the manufacturer's instructions. Equimolar pooled cDNA libraries were sequenced pair-end using the illumina NovaSeq 6000 instrument (illumina) with the illumina NovaSeq 6000 S1 Reagent Kit v1.5 (300 cycles). This run yielded 50 to 60 million reads / sample.The quality of the sequencing execution was evaluated using Illumina's Sequencing Analysis Viewer (Illumina, version 2.4.7). All base call files were demultiplexed using Illumina's bcl2fastq conversion software v2.20 and converted to FASTQ files. Quality control evaluation, library preparation, and sequencing were performed on the next-generation sequencing platform at the University of Bern. Adapter sequences and poly(A) sequences were removed from the raw sequencing reads using cutadapt (v3.4). Subsequently, 12bp UMI sequences were extracted, and sequences with high error rates were removed using UMI-tools (v1.2.2) according to the library preparation kit manufacturer's suggestions. Reads were aligned to the ensemble mouse genome (GRCm38.p6) using hisat2 (v2.2.1). Read alignment was deduplication using UMI-tools (v1.2.2). Fragment counts aligned to genetic features were performed using featureCounts(v2.0.1) based on ensembl mouse gene annotation v102. 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 ordering according to the test statistics obtained from the differential gene expression results of DESeq. Overexpression analysis of gene ontology was performed using the R package topGO for differentially expressed genes with corrected p-values ​​less than 0.05. Heatmap visualization was created using the R package ComplexHeatmap. All downstream analyses in R were performed using R version 4.1.0.

[0109] Quantitative and statistical analysis Unless otherwise specified, data represent the mean ± SEM. Statistical analysis was performed using GraphPad Prism software. For comparisons between two or multiple groups, one-way ANOVA with two-tailed Student's t-test, Bartlett's multiple comparison test, or two-way ANOVA with Tukey's multiple comparison test was applied, as specified in the figure caption. Statistical significance was defined as p<0.05. Asterisks indicate statistical significance (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001).

Claims

1. 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.

2. The pharmaceutical composition for use according to claim 1, wherein the systemic fungal infection is an infection caused by Candida albicans.

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

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

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

6. The pharmaceutical composition for use according to claim 5, wherein the oligonucleotide agent can hybridize to mRNA encoding IL-1Ra.

7. The oligonucleotide agent is selected from antisense oligonucleotides, gapmers, siRNA and shRNA, in the pharmaceutical composition for use according to claim 5 or 6.

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