Il-1ra blockers for treatment and prevention of sepsis

Abstract

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

Description

[0001] IL-1 Ra Blockers for Treatment and Prevention of Sepsis

[0002] This application claims the right of priority of European applications 23183633, filed 5 July 2023, and 23215259, filed 8 December 2023, both of which are incorporated herein by reference in their entirety.

[0003] Field

[0004] The present invention relates to the medical use of IL-1 Ra blockers in management of sepsis associated with systemic fungal infection.

[0005] Background

[0006] Sepsis is a severe, life-threatening condition triggered by microbial dissemination via the bloodstream and a maladapted systemic inflammatory response. Although fungal sepsis is less frequent, invasive fungal infections have particularly high mortality rates. Candida albicans represents the most common cause of fungal bloodstream infection; and despite adequate antifungal treatment, the overall mortality is 30-40%, exceeding 60% in critically ill patients. C. albicans is normally contained by epithelial barrier immunity and occurs as commensal in half of the population. Inborn errors of immunity highlight the importance of IL-17-mediated pathways to avert mucocutaneous colonization, whereas functional neutrophil responses are critical to prevent systemic infection (Puel A. (2020), Hum Genet. 139, 1011-1022; Desai JV, Lionakis MS. (2018), Curr Clin Microbiol Rep. 5, 181-189). Accordingly, individuals with immunosuppression due to hematologic malignancies, organ transplantation, AIDS, or prolonged intensive care hospitalization are highly susceptible to invasive candidiasis. However, preceding systemic viral infections, or medical interventions that compromise physiological barrier function, such as indwelling devices, parenteral nutrition, and abdominal surgery, may predispose otherwise immuno-competent hosts to disseminated Candida infection. Given the high mortality of invasive fungal infections, the emergence of drug resistant strains, and an increasing number of high-risk patients, there is great interest in defining underlying disease mechanisms in order to develop novel therapeutic strategies.

[0007] Inflammation is the physiological innate response to tissue damage aiming to eliminate the injuring agent and restore internal homeostasis. Both, the dynamics and the composition of its powerful effector functions must be precisely adapted to the characteristics of the invading pathogen in order to provide appropriate defense mechanisms that ensure pathogen clearance while avoiding extensive tissue damage. Therefore, interacting networks of pro- and anti-inflammatory cytokines orchestrate the differentiation and recruitment of functionally distinct immune cell populations. Moreover, there are negative feedback mechanisms, so-called immune checkpoints, which signal disproportionate immune activation and dampen the inflammatory process to prevent immunopathology. However, sepsis is characterized by a derailment of such pro- and antiinflammatory pathway homeostasis with concurrent hyper-inflammation and immune paralysis, resulting in a dysfunctional systemic inflammatory response, multi-organ damage, and failure to contain pathogen replication.

[0008] The prototypical pro-inflammatory cytokine interleukin-1 (IL-1 ) initiates and coordinates local and systemic inflammatory responses by activating the IL-1 receptor (IL-1 R) on immune and non- immune cells. Both IL-1 cytokines, IL-1 a and IL-10, are quintessential for antimicrobial immunity, including to C. albicans. Still, excessive IL-1 production is associated with severe acute and chronic inflammatory conditions, such as autoinflammatory syndromes, rheumatoid arthritis, sepsis, or metabolic disorders. Accordingly, IL-1oc and IL-10 are subject to highly complex regulatory mechanisms acting at the transcriptional and posttranslational level, which restrict the generation of the mature cytokines or influence their ability to activate the IL-1 R. The inflammatory effects of bioactive IL-1 a and IL-10 are controlled by the IL-1 R antagonist (IL-1 Ra), an endogenous inhibitor that competes for occupancy of the IL-1 R but does not trigger downstream signaling. One secreted and three intracellular isoforms of IL-1 Ra have been described and are derived from the same IL1RN gene. Adding to the complexity of IL-1 regulation, these individual isoforms are differentially expressed in various cell types. For example, intracellular IL-1 Ra is constitutively expressed in epithelial cells, whereas secreted and intracellular isoforms may be induced in diverse leukocyte subsets in response to pro-inflammatory cytokines, microbial products, or tissue injury. The potency of IL-1 Ra-mediated regulation is evident in the severe inflammatory syndrome of DIRA patients, and is exploited therapeutically for the management of IL-1 -mediated diseases. However, while the molecular pathways that govern the generation and secretion of mature IL-1 are well appreciated, much less is known about the regulatory mechanisms operating at the level of receptor binding, such as IL-1 Ra, particularly with respect to the cell type-specific regulation of IL-1-driven inflammation.

[0009] Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods to prevent death from sepsis associated with systemic fungal infection. This objective is attained by the subject-matter of the independent claims of the present specification, with further advantageous embodiments described in the dependent claims, examples, figures and general description of this specification.

[0010] Summary of the Invention

[0011] The invention provides a pharmaceutical composition comprising an agent capable of inhibiting IL-1 Ra activity, for use in prevention or treatment of sepsis associated with systemic fungal infection. Terms and definitions

[0012] General

[0013] For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

[0014] The terms “comprising”, “having”, “containing”, and “including”, and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open-ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of or “consisting of.”

[0015] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0016] Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”

[0017] As used herein, including in the appended claims, the singular forms “a”, “or” and “the” include plural referents unless the context clearly dictates otherwise.

[0018] "And / or" where used herein is to be taken as specific recitation of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: 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).

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry, organic synthesis). Standard techniques are used for molecular, genetic, and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.

[0020] Binding; Binders, Ligands, Antibodies:

[0021] If not specified more narrowly in the Detailed Description of the Invention, reference to binders and ligands encompasses antibodies, antibody-like molecules and aptamers as defined in the following paragraphs.

[0022] The term specific binding in the context of the present invention refers to a property of ligands that bind to their 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 < 10'8mol / L (particularly < 10'9mol / L) when binding to its target, but a dissociation constant at least three orders of magnitude higher in its interaction with a molecule having a globally similar chemical composition as the target, but a different three-dimensional structure.

[0023] The term non-agonist ligand refers to a ligand, particularly a human or humanized monoclonal antibody, capable of specifically binding to its target, at a ko of 10'8mol / L or lower, particularly at a ko < (equal to or lower than) 10'9mol / L or even at a ko 10'10mol / L. 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 to an interleukin receptor binds to the interleukin receptor (ILR, the target) without producing the effect of the interleukin-ILR interaction, and inhibits binding of the interleukin.

[0024] In the context of the present specification, the term dissociation constant (KD) is used in its meaning known in the art of chemistry and physics; it refers to an equilibrium constant that measures the propensity of a complex composed of [in most cases, two] different components to dissociate reversibly into its constituent components. The complex can be e.g. an antibody-antigen complex AbAg composed of antibody Ab and antigen Ag. KD 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, in other words, the concentration of unbound [Ab] equals the concentration of the [AbAg] complex. The dissociation constant can be calculated according to the following formula:

[0025] [Ab]: concentration of antibody; [Ag]: concentration of antigen; [AbAg]: concentration of antibodyantigen complex

[0026] In the context of the present specification, the terms off-rate (KOff;[1 / sec]) and on-rate (Kon; [L / (sec*mol)]) are used in their meaning known in the art of chemistry and physics; they refer to a rate constant that measures the dissociation (KOff) or association (Kon) of an antibody with its target antigen. KOff and Koncan be experimentally determined using methods well established in the art. A method for determining the KOff and Konof an antibody employs surface plasmon resonance. This is the principle behind biosensor systems such as the Biacore® or the ProteOn® system. They can also be used to determine the dissociation constant KD by using the following formula:

[0027] The natural upper limit for the on-rate Kon is 109L / (sec*mol).

[0028] The term aptamer relates to an oligonucleotide or peptide molecule that binds to a specific target molecule. Aptamers can be created by selecting them from a large random sequence pool. Nucleic acid aptamers can be generated through repeated rounds of in-vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to molecular targets such as small molecules, proteins or nucleic acids through non-covalent interactions. Aptamers offer molecular recognition properties that rival that of antibodies.

[0029] In the context of the present specification, the term antibody refers to whole 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 chains thereof and related or derived constructs. A whole antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region of IgG is comprised of three domains, CH1 , CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system. Similarly, the term encompasses a so-called nanobody or single domain antibody, an antibody fragment consisting of a single monomeric variable antibody domain.

[0030] In the context of the present specification, the term humanized antibody refers to an antibody originally produced by immune cells of a non-human species, the protein sequences of which have been modified to increase their similarity to antibody variants produced naturally in humans. The term humanized antibody as used herein includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences as well as within the CDR sequences derived from the germline of another mammalian species.

[0031] The term antibody-like molecule in the context of the present specification refers to a molecule capable of specific binding to another molecule or target with high affinity / a Kd < 10'7mol / L (particularly < 10'9mol / L). An antibody-like molecule binds to its target similarly to the specific binding of an antibody. The term antibody-like molecule encompasses a repeat protein, such as a designed ankyrin repeat protein (Molecular Partners, Zurich), an engineered antibody mimetic protein exhibiting highly specific and high-affinity target protein binding (see US2012142611 , US2016250341 , US2016075767 and US2015368302). The term antibody-like molecule further encompasses, but is not limited to, a polypeptide derived from armadillo repeat proteins, a polypeptide derived from leucine-rich repeat proteins and a polypeptide derived from tetratricopeptide repeat proteins. The term antibody-like molecule further encompasses a specifically binding polypeptide derived from a protein A domain, a fibronectin domain FN3, a consensus fibronectin domain, a lipocalin (see Skerra, Biochim. Biophys. Acta 2000, 1482(1- 2):337-50), a polypeptide derived from a Zinc finger protein (see Kwan et al. Structure 2003, 11 (7):803-813), a Src homology domain 2 (SH2) or Src homology domain 3 (SH3), a PDZ domain, a gamma-crystallin, ubiquitin, a cysteine knot polypeptide or a knottin, cystatin, Sac7d, a triple helix coiled coil (also known as alphabodies), a Kunitz domain or a Kunitz-type protease inhibitor and a carbohydrate binding module 32-2. The term antibody-like molecule further encompasses a humanized camelid antibody. The term antibody-like molecule similarly encompasses an scFv fragment.

[0032] The term protein A domains derived polypeptide refers to a molecule that is a derivative of protein A and is capable of specifically binding the Fc region and the Fab region of immunoglobulins.

[0033] The term armadillo repeat protein refers to a polypeptide comprising at least one armadillo repeat, wherein an armadillo repeat is characterized by a pair of alpha helices that form a hairpin structure.

[0034] The term humanized camelid antibody in the context of the present specification refers to an antibody consisting of only the heavy chain or the variable domain of the heavy chain (VHH domain) and whose amino acid sequence has been modified to increase their similarity to antibodies naturally produced in humans and, thus show a reduced immunogenicity when administered to a human being. A general strategy to humanize camelid antibodies is shown in Vincke et al. “General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold”, J Biol Chem. 2009 Jan 30;284(5):3273-3284, and US2011165621 A1 .

[0035] In the context of the present specification, the term fragment crystallizable (Fc) region refers to a fraction of an antibody comprising, if applied to IgG, two identical heavy chain fragments consisting of a CH2 and a CH3 domain, covalently linked by disulfide bonds.

[0036] In the context of the present specification, the term single-chain variable fragment (scFv) relates to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin, resulting in an antibody-like, high affinity to the target from a single polypeptide chain. The VH and VL chains of the scFv are connected through a short linker peptide of ten to about 25 amino acids [Huston et al. (1988). PNAS 85 (16): 5879-5883] The linker can either connect the N-terminus of the VH with the C-terminus of the VL (VL-VH), or adopt the reverse configuration (VH-VL). The term IL-1Ra inhibitor in the context of the present specification relates to a pharmaceutical drug that suppresses the physiologic response to interleukin-1 receptor antagonist protein (IL- 1 Ra; Uniprot P18510).

[0037] General Molecular Biology: Nucleic Acid Sequences, Expression

[0038] The terms gene expression or expression, or alternatively the term gene product, may refer to either of, or both of, the processes - and products thereof - of generation of nucleic acids (RNA) or the generation of a peptide or polypeptide, also referred to transcription and translation, respectively, or any of the intermediate processes that regulate the processing of genetic information to yield polypeptide products. The term gene expression may also be applied to the transcription and processing of a RNA gene product, for example a regulatory RNA or a structural (e.g. ribosomal) RNA. If an expressed polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. Expression may be assayed both on the level of transcription and translation, in other words mRNA and / or protein product.

[0039] The term Nucleotides in the context of the present specification relates to nucleic acid or nucleic acid analogue building blocks, oligomers of which are capable of forming selective hybrids with RNA or DNA oligomers on the basis of base pairing. The term nucleotides in this context includes the classic ribonucleotide building blocks adenosine, guanosine, uridine (and ribosylthymine), cytidine, the classic deoxyribonucleotides deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine. It further includes analogues of nucleic acids such as phosphothioates, 2’0-methylphosphothioates, peptide nucleic acids (PNA; N-(2-aminoethyl)- glycine units linked by peptide linkage, with the nucleobase attached to the alpha-carbon of the glycine) or locked nucleic acids (LNA; 2’0, 4’C methylene bridged RNA building blocks). Wherever reference is made herein to a hybridizing sequence, such hybridizing sequence may be composed of any of the above nucleotides, or mixtures thereof.

[0040] The term phosphothioate as used herein is synonymous with the terms phosphorothioate and thiophosphate.

[0041] The terms capable of forming a hybrid or hybridizing sequence in the context of the present specification relate to sequences that under the conditions existing within the cytosol of a mammalian cell, are able to bind selectively to their target sequence. Such hybridizing sequences may be contiguously reverse-complimentary to the target sequence, or may comprise gaps, mismatches or additional non-matching nucleotides. The minimal length for a sequence to be capable of forming a hybrid depends on its composition, with C or G nucleotides contributing more to the energy of binding than A or T / U nucleotides, and on the backbone chemistry.

[0042] In the context of the present specification, the term hybridizing sequence encompasses a polynucleotide sequence comprising or essentially consisting of RNA (ribonucleotides), DNA (deoxyribonucleotides), phosphothioate deoxyribonucleotides, 2’-O-methyl-modified phosphothioate ribonucleotides, LNA and / or PNA nucleotide analogues. In certain embodiments, a hybridizing sequence according to the 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 hybridizing sequence is at least 80% identical, more preferred 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to the reverse complimentary 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 hybridizing sequence comprises deoxynucleotides, phosphothioate deoxynucleotides, LNA and / or PNA nucleotides or mixtures thereof.

[0043] The term antisense oligonucleotide in the context of the present specification relates to an oligonucleotide having a sequence substantially complimentary to, and capable of hybridizing to, an RNA. Antisense action on such RNA will lead to modulation, particular inhibition or suppression of the RNA’s biological effect. If the RNA is an mRNA, expression of the resulting gene product is inhibited or suppressed. Antisense oligonucleotides can consist of DNA, RNA, nucleotide analogues and / or mixtures thereof. The skilled person is aware of a variety of commercial and noncommercial sources for computation of a theoretically optimal antisense sequence to a given target. Optimization can be performed both in terms of nucleobase sequence and in terms of backbone (ribo, deoxyribo, analogue) composition. Many sources exist for delivery of the actual physical oligonucleotide, which generally is synthesized by solid state synthesis.

[0044] The term gapmer refers to a short DNA antisense oligonucleotide structure with RNA-like segments on both sides of the sequence, which are typically composed of locked nucleic acids (LNA), 2'- OMe, or 2'-F modified bases. Gapmers often comprise nucleotides modified with phosphoroth ioate (PS) groups, particularly in their 5’ and 3’ terminal regions. Gapmers are designed to hybridize to a target piece of RNA and silence the gene through the induction of RNase H cleavage. Binding of the gapmer to the target has a higher affinity due to the modified RNA flanking regions, as well as resistance to degradation by certain nucleases. Gapmers are being developed as therapeutics for a variety of cancers, viruses, and other chronic genetic disorders.

[0045] The term siRNA (small / short interfering RNA) in the context of the present specification relates to an RNA molecule capable of interfering with the expression (in other words: inhibiting or preventing the expression) of a gene comprising a nucleic acid sequence complementary or hybridizing to the sequence of the siRNA in a process termed RNA interference. The term siRNA is meant to encompass both single stranded siRNA and double stranded siRNA. siRNA is usually characterized by a length of 17-24 nucleotides. Double stranded siRNA can be derived from longer double stranded RNA molecules (dsRNA). According to prevailing theory, the longer dsRNA is cleaved by an endo-ribonuclease (called Dicer) to form double stranded siRNA. In a nucleoprotein complex (called RISC), the double stranded siRNA is unwound to form single stranded siRNA. RNA interference often works via binding of an siRNA molecule to the mRNA molecule having a complementary sequence, resulting in degradation of the mRNA. RNA interference is also possible by binding of an siRNA molecule to an intronic sequence of a pre-mRNA (an immature, non-spliced mRNA) within the nucleus of a cell, resulting in degradation of the pre-mRNA.

[0046] The term shRNA (small hairpin RNA) in the context of the present specification relates to an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi).

[0047] Any patent document cited herein shall be deemed incorporated by reference herein in its entirety.

[0048] As used herein, the term pharmaceutical composition refers to a compound of the invention, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition according to the invention is provided in a form suitable for topical, parenteral or injectable administration.

[0049] As used herein, the term pharmaceutically acceptable carrier includes any solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (for example, antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington: the Science and Practice of Pharmacy, ISBN 0857110624). The invention also encompasses nanoparticles, liposomes, or cellular carriers within the meaning of pharmaceutically acceptable carrier.

[0050] The term inhibitor in the context of the present specification relates to any pharmaceutically acceptable agent or compound that may be used to interact with and specifically interfere with the biological activity of its designated target (IL-1 Ra in the case of the present specification). Inhibitors include small molecule drugs that fulfil the criteria summarized as Lipinski’s Rules of five (the drug fulfils at least three of the following rules: number of H-bond donors is < 5; number of H-bond acceptors is < 10; molecular mass is <500Da; octanol / water partition coefficient < 5). Specific examples of such inhibitors are mentioned herein.

[0051] As used herein, the term treating or treatment of any disease or disorder (e.g. sepsis) refers in one embodiment to ameliorating the disease or disorder (e.g. slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment "treating" or "treatment" refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. Methods for assessing treatment and / or prevention of disease are generally known in the art, unless specifically described hereinbelow. Detailed Description of the Invention

[0052] The invention relates to a pharmaceutical composition comprising an agent capable of inhibiting IL-1 Ra activity, for use in prevention or treatment of sepsis associated with systemic fungal infection.

[0053] The inventors propose that the results suggest that the treatment proposed herein is of particular use in acute sepsis, for patients in whom a primary or secondary fungal infection, particularly by Candida albicans, has already been confirmed, or who are deemed to be of particularly high risk of developing Candida albicans or other fungal pathogen-related sepsis. Of note, inhibition of IL-1 Ra is likely contraindicated for patients with bacterial sepsis.

[0054] Ideally, a patient suspected of suffering from systemic fungal infection is treated at the earliest onset of symptoms.

[0055] In certain embodiments, the systemic fungal infection is infection by Candida albicans.

[0056] In certain embodiments, the agent capable of inhibiting IL-1 RA is a ligand to IL-1 Ra selected from a monoclonal antibody and an antibody-like molecule.

[0057] Antibodies have an advantage in serum half-life, and may therefore provide long lasting protection.

[0058] The inventors believe however that the data obtained suggest that also a shorter-lived protein agent, as may be a protein-based binder such as a designed ankyrin repeat protein or an antibody like molecule, for example a nanobody, a variable fragment, or a camelid antibody, may serve a useful purpose given the fact that rapid clearance after a bolus administration might be sufficient to break the immune reaction triggered by the infection.

[0059] In particular embodiments, the ligand to IL-1 Ra is a neutralizing antibody or neutralizing antibodylike molecule. The term “neutralizing antibody or neutralizing antibody-like molecule” refers to an antibody or an antibody fragment that can specifically bind to a the target IL-1 RA in a manner that inhibits the biological activity of the target. In the context of this patent, the term "neutralizing antibody" encompasses both monoclonal and polyclonal antibodies, as well as any fragments, variants, or derivatives thereof that retain the ability to neutralize the target.

[0060] It is also conceivable that a polypeptide agent derived from a soluble IL-1 receptor is employed, in which the epitopes that bind to IL-1 alpha and IL-1 beta have been altered in a way to preclude scavenging of these cytokines by the soluble receptor, whereas binding to IL-Ra is maintained to function as an effective inhibitor of IL-1 Ra.

[0061] In certain embodiments, the agent capable of inhibiting IL-1 RA is an oligonucleotide agent capable of inhibiting IL-1 RN gene expression.

[0062] In certain embodiments, the oligonucleotide agent is capable of hybridizing to a mRNA encoding IL-1 Ra. In certain embodiments, the oligonucleotide agent is selected from an antisense oligonucleotide, a gapmer, an siRNA and a shRNA. Using siRNA transcribed from an expression vector under control of a macrophage-specific promoter might allow to specifically target the origin of the IL-1 Ra as discovered by the inventors.

[0063] Medical treatment

[0064] Similarly, within the scope of the present invention is a method or treating sepsis associated with systemic fungal infection in a patient in need thereof, comprising administering to the patient an agent capable of inhibiting IL-1 Ra activity according to the above description.

[0065] Pharmaceutical Compositions, Administration / Dosage Forms and Salts

[0066] According to one aspect of the compound according to the invention, the compound according to the invention is provided as a pharmaceutical composition, pharmaceutical administration form, or pharmaceutical dosage form, said pharmaceutical composition, pharmaceutical administration form, or pharmaceutical dosage form comprising at least one of the compounds of the present invention or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier, diluent or excipient.

[0067] The invention further encompasses a pharmaceutical composition comprising a 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, such as those described herein.

[0068] Certain embodiments of the invention relate to a dosage form for enteral administration, such as nasal, buccal, rectal, transdermal or oral administration, or as an inhalation form or suppository. In addition, the pharmaceutical compositions of the present invention can be made up in a solid form (including without limitation capsules, tablets, pills, granules, powders or suppositories), or in a liquid form (including without limitation solutions, suspensions or emulsions).

[0069] Certain embodiments of the invention relate to a dosage form for parenteral administration, such as subcutaneous, intravenous, intrahepatic or intramuscular injection forms. Optionally, a pharmaceutically acceptable carrier and / or excipient may be present.

[0070] Certain embodiments of the invention relate to a dosage form for topical administration. The skilled artisan is aware of a broad range of possible recipes for providing topical formulations, as exemplified by the content of Benson and Watkinson (Eds.), Topical and Transdermal Drug Delivery: Principles and Practice (1st Edition, Wiley 2011 , ISBN-13: 978-0470450291 ); and Guy and Handcraft: Transdermal Drug Delivery Systems: Revised and Expanded (2ndEd., CRC Press 2002, ISBN-13: 978-0824708610); Osborne and Amann (Eds.): Topical Drug Delivery Formulations (1stEd. CRC Press 1989; ISBN-13: 978-0824781835). In embodiments of the invention relating to topical uses of the compounds of the invention, the pharmaceutical composition is formulated in a way that is suitable for topical administration such as aqueous solutions, suspensions, ointments, creams, gels or sprayable formulations, e.g., for delivery by aerosol or the like, comprising the active ingredient together with one or more of solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives that are known to those skilled in the art.

[0071] The dosage regimen for the compounds of the present invention will vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, health, medical condition, and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment; the frequency of treatment; the route of administration, the renal and hepatic function of the patient, and the effect desired. In certain embodiments, the compounds of the invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three, or four times daily.

[0072] Method of Manufacture and Method of Treatment according to the invention

[0073] The invention further encompasses, as an additional aspect, the use of an agent capable of inhibiting IL-1 Ra activity as identified herein, for use in a method of manufacture of a medicament for the treatment or prevention of sepsis associated with systemic fungal infection.

[0074] Similarly, the invention encompasses methods of treatment of a patient having been diagnosed with sepsis associated with systemic fungal infection. This method entails administering to the patient an effective amount of an agent capable of inhibiting IL-1 Ra activity, as specified in detail herein.

[0075] Wherever alternatives for single separable features are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein.

[0076] The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.

[0077] Examples

[0078] Here, we investigated the expression of IL-1 Ra across different myeloid cell subsets in a murine model of invasive candidiasis and examined the impact of IL-1 Ra produced by specific cell types on antifungal immunity. We showed that macrophage-secreted IL-1 Ra acted as an innate immune checkpoint that prevented efficient pathogen clearance; and its targeted removal protected against lethal C. albicans sepsis. Moreover, we found that macrophage-secreted IL-1 Ra was positively regulated by type I interferon (IFN), reflecting the association between secondary invasive Candidiasis and preceding viral infections. Together, these results provide a mechanistic explanation for the high disease susceptibility to Candida bloodstream infection and suggest IL- 1 Ra-targeted therapies as a novel treatment approach.

[0079] Results

[0080] IL-1 Ra is rapidly expressed upon bloodstream infection with C. albicans. To establish the relevance of IL-1 signaling during invasive fungal infection we examined IL-1 family cytokine expression in the kidney following intravenous infection with 2.5 x 105 CFU Candida albicans. IL-1 £ and IL-1 Ra protein were strongly expressed throughout infectious foci at day 3 post infection (p.i.), whereas few IL-1oc positive cells were situated at the outer margin of lesions. Transcripts for IL-1 £ were already present in kidneys of naive animals, yet IL-1 R1 and IL-1 Ra could only be detected at very low levels. However, fungal infection rapidly triggered increased gene expression of both IL-1 £ and IL-1 Ra in kidney, whilst IL-1 R1 mRNA expression stayed unchanged as compared to naive mice (Figure 1A). Furthermore, substantial levels of IL-1 Ra protein were present in serum of infected mice and peaked at day 2 p.i. (Figure 1 B). In contrast, mature IL-1 £ cytokine remained undetectable in peripheral blood and was likely produced locally in infected tissues. Expression of IL-1 Ra protein in kidneys coincided with leukocyte recruitment and was limited to inflammatory infiltrates, suggesting immune cells as main IL-1 Ra producers. Indeed, primary neutrophils, monocytes, dendritic cells and bone marrow (BM)-derived macrophages secreted IL-1 Ra in response to both Candida morphotypes in vitro with highest IL-1 Ra levels elicited from monocytes exposed to Candida hyphae (Figure 1 C). These data confirm the IL-1 £ / IL-1 R1 axis as an important component of the early antifungal immune defense, and implicate IL-1 Ra produced by myeloid cells in its regulation.

[0081] Ablation of macrophage-produced IL-1 Ra protects against invasive fungal infection. We therefore sought to dissect the impact of IL-1 Ra produced by specific leukocyte subsets on the immune response to C. albicans using conditional deletion of IL-1 Ra selectively in neutrophils, macrophages or dendritic cells. We crossed IL-1 Rafl / flmice with respective Cre-driver strains to delete IL-1 Ra in neutrophils and macrophages (LysM-Cre, termed IL-1 RaLysM), in macrophages (Mafb-Cre, termed IL-1 RaMafb) and in CD1 1 c-expressing cells (CD1 1 c-Cre, termed IL-1 RaCD11c) (Figures 1 D, 8A, and 8B). Macrophage-specific ablation of IL-1 Ra drastically improved the early immune control of C. albicans infection in IL-1 RaLysMand IL-1 RaMafbmice as compared to IL-1 Rafl / fllittermate controls (Figures 1 E and 1 H). Both strains reduced fungal titers in kidneys already at day 3 p.i. and controlled C. albicans below the limit of detection in the majority of animals within 7 days. Although IL-1 RaLysMmice lacked IL-1 Ra in both macrophages and neutrophils, their phenotype was almost identical to that of IL-1 RaMafbmice lacking IL-1 Ra selectively in macrophages. In addition, macrophages and neutrophils of IL-1 RaLysMmice exhibited residual IL-1 Ra expression in vivo and in vitro, whereas IL-1 Ra deletion was highly specific and efficient in macrophages of IL-1 RaMafbmice (Figures 8A and 8B). This rendered major contributions of neutrophil-expressed IL-1 Ra unlikely and identified macrophage-produced IL-1 Ra as an important negative regulator of IL-1 R signaling during systemic Candida infection. However, ablation of IL-1 Ra in CD1 1 c-expressing cells provided weaker and only transient protection. Although kidneys of IL-1 RaCD11cmice contained lower fungal loads than IL-1 Rafl / fllittermates at 3 day p.i. , this difference had disappeared by day 7 (Figure 1 G). Thus, IL-1 Ra produced by CD11 c-expressing cells influenced the initial antifungal defense, but did not impact long-term control.

[0082] These observations were corroborated by the histopathological examination of infected kidneys. Confirming their superior ability to contain fungal replication early on (Figures 1 E and 1 F), IL- 1 RaLysMand IL-1 RaMafbmice showed very restricted distribution of C. albicans and less inflammation in kidneys 3 days p.i. (Figures 1 H-1 I). Moreover, IL-1 RaLysMand IL-1 RaMafbmice lost less weight as compared to controls, which was indicative for their reduced general morbidity (Figure 8C). In comparison, IL-1 RaCD11cmice exhibited an intermediate phenotype with reduced Candida dissemination and inflammation at day 3, but unaltered weight loss (Figures 1 G, 1 J, and 8C). Consistent with their improved immune control of C. albicans, we found markedly fewer infectious foci and reduced IL-1 Ra-positive areas in kidney sections of all three IL-1 Ra-deficient strains (Figures 1 H-K). The beneficial effect of IL-1 Ra ablation in macrophages was most pronounced in IL-1 RaMafbmice, as illustrated by their reduced creatinine and blood urea nitrogen levels (Figure 1 L), and lower expression of kidney injury markers Lcn2 and Kim-1 (Figure 1 M). Altogether, our results establish that ablation of macrophage-produced IL-1 Ra strongly improves the ability to control invasive Candida infection, resulting in rapid pathogen clearance, reduced disease severity and a better preservation of organ function.

[0083] IL-1 Ra controls the recruitment and fungicidal capacity of neutrophils. To investigate how removal of macrophage-produced IL-1 Ra enabled the efficient containment of systemic candidiasis, we characterized antifungal immune responses in the respective IL-1 Ra-deficient strains. Macrophage-specific deletion of IL-1 Ra increased the early mobilization and maturation of neutrophils in IL-1 RaMafband IL-1 RaLysMmice, as indicated by greater proportions of mature CD101 + Ly-6G high neutrophils in blood on day 2 p.i. (Figures 2A-B). This effect was strongest in IL-1 RaMafbmice, which also showed reduced proportions of circulating immature neutrophils and inflammatory monocytes (Figure 2B).

[0084] Absence of macrophage-produced IL-1 Ra also accelerated the recruitment of neutrophils to inflammatory sites. To exclude feedback mechanisms triggered by differential microbial control, we examined inflammatory responses to non-replicating fungal cell wall components. The peritoneal lavages of zymosan-primed IL-1 RaMafband IL-1 RaLysMmice contained three- and twofold more neutrophils, respectively (Figures 2C, 9A, and 9B). Relative to IL-1 Rafl / flcontrols, tissue-infiltrating neutrophils in IL-1 RaMafband IL-1 RaLysMmice exhibited substantially augmented fungicidal activity and secreted higher amounts of IL-1 p, indicating their increased activation (Figures 2D and 2E). Still, deletion of IL-1 Ra in CD11 c-expressing cells did not affect neutrophil numbers in blood and only slightly enhanced their peritoneal recruitment in IL-1 RaCD11cmice (Figures 2B and 2C); yet it endowed tissue-infiltrating neutrophils with enhanced fungicidal capacity, similar to that observed in IL-1 RaMafband IL-1 RaLysMmice (Figure 2D). Characterization of neutrophil phenotypes in blood, spleen and kidney revealed an enrichment of CD 101 + neutrophils producing high levels of ROS and higher frequencies of neutrophils expressing IL-1 p and MPO in kidneys of infected IL-1 RaMafbmice as compared to IL-1 Rafl / flcontrols (Figures 2F-I). Neutrophils in kidneys of IL-1 RaLysMand IL-1 RaCD11cmice displayed intermediate phenotypes (Figures 2F-I). CD63 expression correlated with exposure to Candida in infected organs, but was not influenced by IL1-Ra (Figure 2H). Removal of IL-1 Ra affected neither neutrophil phenotypes nor fungal titers in spleen (Figures 3F-I, and 10D). Thus, absence of macrophage-produced IL-1 Ra promoted the rapid recruitment of highly fungicidal neutrophils to infected kidneys.

[0085] IL-1 RaMafb, IL-1 RaLysMand to a lesser extent also IL-1 RaCD11cmice, restricted fungal replication in kidneys by day 3 p.i. (Figures 1 E-1 G). As a result, they did not exhibit the dysfunctional hyper- inflammatory response detected in IL-1 Rafl / flcontrols (Figures 1 H-1 J), and expressed lower levels of pro-inflammatory cytokines (Figures 2K). In addition, kidneys of IL- 1 Ra and IL-1 RaLysMmice contained less neutrophils and inflammatory monocytes at this time, which may reflect the lower fungal burden and accelerated inflammation resolution in these mice (Figures 2J, 4C, and 10A). Bulk tissue RNAseq of infected kidneys and gene set enrichment analysis (GSEA) associated the absence of macrophage-expressed IL-1 Ra in IL-1 RaMafbmice with a suppression of inflammatory pathways including IL-6 / Jak / Stat3 signaling, IFN-oc response and TNF-oc signaling. Besides Il1rn, transcripts reduced in kidneys of IL-1 RaMafbmice included key chemoattractants Cc / 2, Cxcl2, Cxcl1 ; adhesion molecule Icarnl the regulator of NFkB signaling, Nfkbia and antifungal effector Cybb (not shown). We conclude that deletion of macrophage-specific IL-1 Ra promotes rapid neutrophil- mediated pathogen elimination, which limits the detrimental hyper-inflammation triggered by sustained fungal proliferation.

[0086] Serum IL-1 Ra is produced by CD169+ macrophages of the splenic marginal zone. Minimal residual Cre activity in neutrophils and monocytes prompted us to address a potential cell-intrinsic function of IL-1 Ra. However, partial removal of IL-1 Ra in mixed BMC equally affected the recruitment and effector functions of cells originating from both, IL-1 Ra-expressing and IL-1 Ra gene-modified BM (Figure 1 1A-1 1 G). This suggested a cell-extrinsic effect, likely mediated by IL-1 Ra secreted from macrophages. Given that these results emphasized an essential role of macrophage-secreted IL- 1 Ra and provided that we detected substantial levels of circulating serum IL-1 Ra in infected mice (Figure 1 B), we next sought to assess the impact of IL-1 Ra secreted into the blood circulation and to identify the macrophage subset contributing to serum IL-1 Ra production.

[0087] Bloodstream infection with C. albicans stimulated the release of serum IL-1 Ra in wild type mice; yet this response was absent from IL-1 RaLysM, IL-1 RaMafband IL-1 RaCD11cmice (Figure 3A). Likewise, lipopolysaccharide (LPS) infusion elicited vigorous serum IL-1 Ra production, which was considerably reduced in all conditional IL-1 Ra-deficient strains tested (Figure 3B). Intravenous injection of either Candida or LPS induced expression of IL-1 Ra protein in the splenic marginal zone. Splenic IL-1 Ra production corresponded to the presence of IL-1 Ra in serum, and was strongly reduced in IL-1 RaLysM, IL-1 RaMafband IL-1 RaCD11cmice primed with Candida or LPS (not shown). EYFP-expression confirmed a history of Cre-activity in cells present in the marginal zone of EYFPLysM, EYFPMafband EYFPCD11cmice. Mafb-Cre-activity appeared to EYFP-mark cells in the splenic marginal zone most extensively and also affected macrophages in the red pulp (Figure 3C). This corresponded well to the efficient IL-1 Ra deletion in spleens and sera of IL-1 RaMafbmice, as well as their robust protection against invasive candidiasis. Immunofluorescence co-staining identified CD169+ macrophages of the splenic marginal zone as main producers of IL-1 Ra, whereas MARCO+ macrophages appeared IL-1 Ra-negative.

[0088] We directly examined the relevance of splenic IL-1 Ra production for the serum IL-1 Ra response and antifungal immune defense in splenectomized wild type mice infected with C. albicans. Prior surgical removal of the spleen reduced the levels of Cand / da-induced serum IL-1 Ra as compared to mock-treated, Cand / da-infected controls (Figure 3D). Moreover, splenectomy conferred increased resistance against Candida replication, as indicated by reduced fungal loads (Figure 3E), restricted Candida dissemination and lower inflammation in kidneys. To verify that serum IL-1 Ra was indeed secreted by splenic CD169+ macrophages, we adopted a clodronate depletion protocol that allows selective targeting of marginal zone-resident macrophages by exploiting their distinct repopulation kinetics36 (Figure 1 1 H). Depletion of splenic marginal zone macrophages and marginal metallophilic macrophages reduced serum IL-1 Ra levels to that of uninfected mice (Figure 3H), and was associated with an increased recruitment of CD101 + neutrophils and of IL-1 p+neutrophils to kidney (Figures 3I, and 1 1 I-1 1 L). Collectively, these findings establish a direct correlation between IL-1 Ra expression in CD169+ macrophages of the splenic marginal zone and the production of serum IL-1 Ra following hematogenic dissemination of C. albicans.

[0089] Serum IL-1 Ra represents an innate immune checkpoint mediating impaired pathogen control and dysfunctional hyper-inflammation during invasive fungal infection. Our findings so far revealed that macrophage-produced IL-1 Ra impedes the efficient early containment of disseminated Candidiasis by restricting the tissue recruitment and antifungal capacity of neutrophils. They also indicated a substantial contribution of serum IL-1 Ra released from splenic CD169+ macrophages. To examine the impact of circulating IL-1 Ra on the immune response to Candida, we next reconstituted the pool of serum IL-1 Ra in IL-1 RaMafbmice with intravenous infusions of recombinant IL-1 Ra protein. We reasoned that since IL-1 RaMafbmice were deficient in both, circulating serum IL-1 Ra and IL- 1 Ra produced by macrophages within infected tissues, this approach would allow to selectively investigate the contribution of serum IL-1 Ra. Bolus injection of recombinant IL-1 Ra raised serum IL-1 Ra levels in Cand / da-infected IL-1 RaMafbmice initially above that of IL-1 Ra competent mice, but maintained wild type levels for up to 18 hours (Figures 4A, and 12A). We therefore administered two daily injections throughout the experiment to fully replenish serum IL-1 Ra in IL-1 RaMafbmice. Absence of IL-1 Ra in the splenic marginal zone following Candida infection or in peritoneal exudates during zymosan peritonitis (Figure 4B) confirmed that this regimen restored exclusively serum IL-1 Ra, but not the locally secreted cytokine. Reconstitution of serum IL-1 Ra almost completely reverted the protected phenotype of IL-1 RaMafbmice to that of IL-1 Rafl / flcontrols. For example, the enhanced neutrophil recruitment to zymosan priming observed in IL-1 RaMafbmice was abrogated upon replenishing their serum IL-1 Ra (Figure 4C). Likewise, reconstitution of serum IL-1 Ra restricted the fungicidal and phagocytic activities of neutrophils in IL-1 RaMafbmice to the level of IL-1 Ra-competent wild type mice. Moreover, the ex vivo effector functions of neutrophils from IL-1 RaMafbmice but not from IL-1 Ra-reconstituted IL-1 RaMafbmice were amplified by in vitro IL-1 £ stimulation, suggesting that absence of serum IL-1 Ra in vivo increased the sensitivity of neutrophils to local IL-1 £ stimulation (Figures 4D, and 4E). Indeed, when primary neutrophils were pre-pulsed with IL-1 Ra in vitro, this abolished their IL-1 R signaling upon subsequent exposure to IL-1 £ even after removal of unbound extracellular IL-1 Ra (Figure 4F). As could be expected from these results, IL-1 Ra-reconstituted IL-1 RaMafbmice no longer controlled Candida replication in their kidneys as efficiently as IL-1 RaMafbmice. Instead, they presented with higher fungal loads, increased Candida dissemination and stronger renal inflammation (Figures 4G-4I), and thus displayed a phenotype similar to that of IL-1 Rafl / flcontrols. This showed that the pronounced immediate resistance of IL-1 RaMafbmice to Candida was mainly attributable to the lack of serum IL-1 Ra in these mice, and emphasized the impact of serum IL-1 Ra on the early immune control of C. albicans. In addition, it suggested that targeted removal of IL-1 Ra might provide a valid strategy to harness the endogenous antifungal response during disseminated Candidiasis.

[0090] We therefore tested whether in vivo neutralization of IL-1 Ra prior to infection increased the ability of wild type mice to contain fungal infection (Figure 12B). Daily injections of a neutralizing antibody against murine IL-1 Ra efficiently depleted serum IL-1 Ra, yet did not affect IL-1 Ra protein expression in the splenic marginal zone (Figure 4J). Furthermore, preventive IL-1 Ra neutralization greatly improved the immune control of Candida infection, as illustrated by reduced fungal titers, limited Candida dissemination and lower tissue inflammation in infected kidneys (Figures 4K and 4L). We observed that serum concentrations of IL-1 Ra peaked on day 2 p.i. and returned to naive levels by day 7 p.i. (Figures 1 C and 4M). We hence scrutinized whether therapeutic IL-1 Ra neutralization protected against lethal C. albicans infection (Figure 12C). Indeed, IL-1 Ra neutralization with onset at day 2 p.i. reduced fungal titers and markedly enhanced the survival of wild type mice upon high dose C. albicans challenge (Figures 4M, 4N, and 40). The phenotypes of IL-1 RaLysM, IL-1 RaMafband IL-1 RaCD11cmice already revealed that an early suppression of fungal replication prevented the dysfunctional inflammatory response observed in wild type IL-1 Rafl / flcontrols (Figure 2K). These observations were confirmed following the depletion and reconstitution of serum IL-1 Ra. Whereas IL-1 RaMafbmice displayed very low pro-inflammatory cytokine levels in kidney, reconstitution of serum IL-1 Ra in these mice not only hampered their ability to restrict Candida proliferation (Figure 4G), but also caused renal hyper-inflammation similar to that seen in IL-1 Rafl / flmice (Figure 4L). Conversely, neutralization of IL-1 Ra promoted the rapid immune control of Candida in wild type mice, and was associated with greatly reduced inflammatory profiles in their kidneys (Figures 4K and 4M). These data directly link macrophage-produced IL-1 Ra to the ineffective immune control of invasive fungal infection and the resulting dysfunctional inflammatory response; and thereby suggest serum IL-1 Ra as a biomarker and potential therapeutic target for disseminated Candidiasis.

[0091] IL-1 Ra secreted by infiltrating macrophages limits pathogen elimination in infected tissue. Our findings demonstrate the significant impact of serum IL-1 Ra on the immune defense against Candida. Nevertheless, the transient protection observed in IL-1 RaCD11cmice indicated that later stages of the response were influenced by IL-1 Ra secreted from a second macrophage population sensitive to gene deletion mediated by Mafb-Cre but not CD11 c-Cre. We therefore interrogated the relevance of IL-1 Ra produced locally in infected tissues and characterized the IL-1 Ra-responses of kidney-infiltrating leukocytes. Neutrophils, inflammatory monocytes and kidney-resident macrophages comprised the main leukocyte populations present in kidneys on day 3 p.i. (Figure 2J). Analysis of FACS-purified cells from infected mice revealed highest mRNA expression of IL- 1 Ra in neutrophils, whereas all three populations expressed IL-1 p and IL-1 R1 at comparable levels (Figure 5A). Still, exposure of naive primary cells to Candida in vitro provoked much stronger IL- 1 Ra protein secretion from monocytes than from neutrophils. Simultaneous IL-i p cytokine stimulation affected IL-1 Ra secretion of neither monocytes nor neutrophils (Figure 5B). However, we found that type I IFN markedly augmented the Cand / da-elicited IL-1 Ra secretion in monocytes, yet did not affect IL-1 Ra release from neutrophils (Figure 5B). Likewise, neutrophils isolated from infected kidneys secreted low levels of IL-1 Ra irrespective of ex vivo exposure to Candida or type I IFN (Figure 5C). In contrast, inflammatory monocytes isolated from the same animals released substantial amounts of IL-1 Ra in response to Candida, which were further augmented by additional IFNp stimulation (Figure 5C). Western blot analysis revealed that neutrophils predominantly expressed the 16 kDa intracellular isoform of IL-1 Ra, which may explain the discrepant mRNA expression and cytokine secretion detected for these cells in response to Candida (Figure 5D). Taken together, these data identified inflammatory monocytes as main early IL-1 Ra producers in infected tissue and suggested the positive regulation of their response by type I IFN.

[0092] Intracellular mRNA staining by Prime Flow analysis confirmed that inflammatory monocytes represented more than 50% of total IL-1 Ra mRNA-positive leukocytes on day 2 p.i. (Figure 5E). However, a contribution of monocyte-secreted IL-1 Ra to the enhanced protection seen in IL- 1 RaMafbor IL-1 RaCD11cmice seemed unlikely, as both strains contained wild type proportions of IL- 1 Ra mRNA-positive monocytes (Figure 5F), and given that EYFPMafband EYFPCD11creporter mice displayed negligible Cre-activity in kidney-infiltrating monocytes (Figures 5G, and 5H,). Nevertheless, we observed the appearance of a second Ly-6Clo MHC II+ F4 / 80+ IL-1 Ra- producting population, presumably monocyte-derived macrophages, that comprised 40% of IL-1 Ra producers by day 5 p.i. (Figures 5I and 5J). This subset exhibited higher Cre-activity in EYFPMafbthan in EYFPCD11creporter mice, suggesting that IL-1 Ra would be more efficiently deleted in these cells in IL-1 RaMafbmice (Figure 5J). Provided that type I IFN augmented IL-1 Ra secretion from monocytes, we examined its impact on the macrophage-produced IL-1 Ra response. Type I IFN was not required to induce serum IL-1 Ra in response to Candida infection, as mice deficient in IFNAR signaling (IFNAR' / _) showed wild type IL-1 Ra expression in spleen and serum (Figure 5K), and controlled fungal replication in kidney equally well as controls (Figures 5L and 12E). Type I IFN preferentially boosted IL-1 Ra secretion in the subset of MafbCre-marked monocyte-derived macrophages, which we had identified as key IL-1 Ra producers. In particular, amongst all CD11 c+ macrophages isolated from infected kidneys of EYFPMafbmice on day 5 p.i., only the EYFP+ MafbCre-marked population showed substantial IL-1 Ra secretion in response to Candida, which was amplified by concomitant IFN-£ stimulation (Figure 5M). In summary, these results highlight the impact of IL-1 Ra secreted by monocyte-derived macrophages in infected tissue. Moreover, they suggest that the particularly efficient gene targeting of this type I IFN-sensitive population in IL-1 RaMafbmice contributes to their sustained long-term protection against Candida.

[0093] Type I IFN amplifies the macrophage IL-1 Ra response and exacerbates fungal sepsis. Prior viremia can predispose hospitalized patients to secondary invasive fungal infections, and our data demonstrated that type I IFN signaling potentiates the Candida-induced IL-1 Ra secretion from macrophages; We therefore evaluated whether IFN-I responses elicited in context of viral infection could induce IL-1 Ra as a permissive factor for subsequent invasive Candidiasis. Intravenous injection of IFN-£ confirmed its ability to directly elicit a serum IL-1 Ra response (Figure 6A). Moreover, i.v. administration of the synthetic TLR3 agonist polyinosinic-polycytidylic acid (PIC) - a well-established method to induce robust IFN-I production in mice - rapidly triggered expression of IL-1 Ra mRNA and protein in spleen, and induced substantial levels of circulating IL-1 Ra in serum (Figures 6B and 6C). However, PIC elicited no such IL-1 Ra production in IL-1 RaMafbmice, suggesting that fungal infection and IFN-I stimulated the identical macrophage population to release IL-1 Ra (Figures 6C). Moreover, PIC treatment considerably amplified the Candida-induced IL-1 Ra response in spleen and serum (Figure 6D), and as a result rendered mice highly susceptible to fungal infection. In particular, PIC-treated mice showed greatly increased morbidity upon infection with 2.5x105 CFU Candida and had to be prematurely removed from the experiment, while this fungal dose was tolerated in absence of PIC-induced IFN-I (Figure 6E). In contrast, PIC-elicited IFN-I failed to trigger detectable IL-1 Ra production and did not aggravate disease severity in Candida-infected IL-1 RaMafbmice (Figure 6E). This confirmed the IFN-driven amplification of macrophage-produced IL-1 Ra as underlying mechanism for the enhanced susceptibility to Candida infection following PIC-injection in wild type mice. Co-injection of PIC also exacerbated the disease severity in IL-1 Ra+ / +mice upon infection with a lower Candida inoculum (Figure 6F). Whereas we detected minimal levels of serum IL-1 Ra in mice receiving 105 CFU Candida alone, the simultaneous induction of IFN-I strongly augmented the serum IL-1 Ra response, interfered with the early immune control of C. albicans and increased fungal titers in kidney by two orders of magnitude (Figure 6F). Confirming these observations, infection with microbial pathogens known to stimulate IFN-I production in vivo, such as lymphocytic choriomeningitis virus (LCMV), vesicular stomatitis virus (VSV), vaccinia virus (W) or Listeria monocytogenes, triggered IL-1 Ra expression in spleen, albeit to different degrees (not shown). Particularly the potent IFN-I inducers LCMV and VSV elicited strong serum IL-1 Ra production from MafbCre-marked macrophages (Figure 6G). LCMV represents a well-characterized experimental model that recapitulates relevant aspects of systemic viral infection in human patients, and we therefore further interrogated the impact of IFN- augmented IL-1 Ra production on susceptibility to fungal bloodstream dissemination in the context of LCMV-WE infection. Similar to our findings with PIC-induced IFN-I, a high dose LCMV coinfection massively exacerbated the morbidity of Candida-infected mice; and fungal doses tolerated in control mice without LCMV co-infection were no longer contained and caused exacerbated morbidity (Figure 6H). Although low dose LCMV-infected mice were able to control a lower Candida inoculum (105 CFU) until day 3 p.i., they nevertheless exhibited very high levels of serum IL-1 Ra (Figure 6I) and showed uncontrolled fungal dissemination in their kidneys (Figure 6J). The aggravating effect of viral infection was strictly type-l IFN-dependent, as co-infected IFNAR- deficient mice completely lacked the increased IL-1 Ra responses in spleen and serum; and their fungal titers that were comparable to mice infected with C. albicans alone (Figures 7A and 7B). Accordingly, kidneys of co-infected IFNAR-deficient mice contained isolated infectious foci and thereby resembled those of singly C. albicans infected controls, whereas kidneys of co-infected wild type mice displayed unrestricted fungal dissemination. Transcripts of IFN-regulated genes Ifitl, Isg15, and Mx1 revealed that C. albicans infection per se triggered considerable IFNAR-signaling in spleen and kidney, which was further enhanced by virally induced IFN-I (Figures 7C and 7D). Still, the Il1rn expression in context of C. albicans infection appeared to be IFN-independent, whereas its viral amplification was partially IFNAR-dependent (Figures 7C and 7D). Co-infection of IL-1 RaMafbmice confirmed that LCMV-induced IFNAR-signaling elicited serum IL-1 Ra production from MafbCre-marked macrophages (Figure 7E). Furthermore, it revealed that virally induced IFN- I exacerbated Candida infection in part by amplifying the macrophage-produced IL-1 Ra response, but also through IFNAR-dependent mechanisms unrelated to macrophage-produced IL-1 Ra (Figure 7F). Altogether, these observations demonstrate that type I IFN elicited during viral infection strongly increases the susceptibility to Candida bloodstream infection with detrimental consequences for host survival, and implicate macrophage-produced IL-1 Ra in this process.

[0094] Discussion

[0095] Due to the high disease-related mortality and limited treatment options, invasive fungal infections remain an urgent and inadequately addressed medical problem. Our study uncovers a disease mechanism that contributes to the high disease susceptibility of disseminated candidiasis and its known amplification by viral co-infections. We identify macrophage-produced serum IL-1 Ra as a disease-promoting innate immune-checkpoint that can be inhibited to protect against fatal C. albicans sepsis in the mouse model. These findings are of immediate importance to our understanding of the pathogenesis of invasive fungal infections and may open new avenues for their treatment.

[0096] Our results emphasize the critical role of serum IL-1 Ra during invasive Candidiasis. Hepatocyte- produced serum IL-1 Ra has been described in various inflammatory conditions. In contrast, we identify splenic CD169+ macrophages as main producers of serum IL-1 Ra during fungal infection. While serum IL-1 Ra may be beneficial by preventing excessive IL-1 signaling in bacterial sepsis, we find it is deleterious during invasive Candidiasis. Genetic ablation of macrophage-produced IL- 1 Ra and liposomal depletion of marginal macrophages blunted serum IL-1 Ra levels and enhanced protective neutrophil responses, thereby highlighting the impact of CD169+ macrophage-derived IL-1 Ra. Marginal zone macrophages sense blood-borne pathogens, including Candida, to instruct the induction of innate and adaptive immunity. G-CSF released by CD169+ macrophages upon splenic colonization promotes neutrophil dysfunction in fungal sepsis. We detected no effects on splenic neutrophils in IL-1 Ra-deficient mice (Figures 2F, 2G, 2H, 2I and 10A), but could modulate neutrophil functionality and renal hyperinflammation by reconstituting or neutralizing IL-1 Ra. This suggests CD169+ macrophage-derived IL-1 Ra inhibits antifungal immunity by restricting the IL-1 - driven maturation and rapid tissue recruitment of highly fungicidal neutrophils (Figures 2 and 10A). We detected a second wave of IL-1 Ra produced by macrophages in infected tissue; and efficient targeting of this population may contribute to the superior resistance of IL-1 RaMafbmice to Candida. Besides macrophages, kidney-infiltrating neutrophils and monocytes exhibited considerable IL-1 Ra expression, while that of dendritic cells or kidney-resident macrophages appeared negligible. Neutrophils contained mainly an intracellular IL-1 Ra isoform and secreted limited amounts of IL- 1 Ra in vitro. This suggests that neutrophil-expressed IL-1 Ra is predominantly biologically active following its release upon neutrophil death. Unlike neutrophils, monocytes secreted substantial quantities of IL-1 Ra, which were further augmented by IFN-I stimulation. The Cre-drivers used here did not delete IL-1 Ra in monocytes (Figure 5); and we cannot conclude to which extent monocyte- secreted IL-1 Ra impacts the immune defense against disseminated Candida. However, the fact that we observed the strong protective effect of removing macrophage-expressed IL-1 Ra despite the presence of monocyte-expressed IL-1 Ra argues for a predominant role of macrophage- secreted IL-1 Ra.

[0097] Our study identified neutrophils as key defense mechanism regulated by macrophage-secreted IL- 1 Ra, and demonstrated that the enhanced maturation, tissue recruitment and functionality of neutrophils mediate the protective effect of IL-1 Ra removal. Neutrophil effector pathways including phagocytosis, ROS production, and NET formation are indispensable for systemic antifungal immunity. Our data suggest the exposure of neutrophils to IL-1 Ra in blood determines their IL-1 £ responsiveness following recruitment to inflamed tissue (Figure 4). IL-1 programs neutrophil clustering behavior and their execution of defense mechanisms during antifungal responses. Hence, the higher sensitivity to IL-1 £ stimulation (Figure 4) and increased IL-1 £ production (Figure 2) observed for neutrophils in absence of IL-1 Ra may enhance such neutrophil-directed activities of IL-1 . Very likely also increased IL-1 signaling in other cell types, including endothelial cells, added to the superior neutrophil responses in IL-1 RaMafbmice. Enhanced IL-1 signaling may also protect against C. albicans by promoting granulopoiesis and via neutrophil-independent mechanisms.

[0098] We found that macrophage-secreted IL-1 Ra was positively regulated by IFN-I. Type I IFN-signaling has been linked to the host defense against Candida in human patients. However, its role in protective immunity remains controversial, as both positive and negative disease outcomes were reported in IFNAR-deficient mice. We observed no significant effects of IFNAR-deficiency on Candida monoinfection, which might signal differences in genetic backgrounds or microbiota composition between these studies. Candida stimulates IFN-I secretion from conventional dendritic cells, which increases their fungicidal capacity and licenses monocytes to promote protective NK cell and neutrophil responses. Conversely, type I IFN may aggravate the severity of Candida infection by inhibiting inflammasome activation and generation of bioactive IL-1 , and by promoting hyper-inflammatory renal injury. In addition, IFN-induced factor IFIT2 limits the ROS production and fungicidal activity of leukocytes. We demonstrate that IFN-I inhibits protective IL1 -signaling by augmenting IL-1 Ra secretion from macrophages (Figures 6 and 7). This implicates IL-1 Ra in the reciprocal crosstalk between type I IFN-driven and IL-1 -driven inflammation in invasive Candidiasis, as proposed for bacterial infections. Candida elicited IFNAR-independent IL-1 Ra production, yet additional IFN-l-signaling severely amplified the Candida-induced IL-1 Ra response. IFN-I is induced by numerous pathogens and prior viremia constitutes a risk factor for invasive Candidiasis. We show that viral co-infection drastically aggravated the disease mortality via the IFN- dependent augmentation of IL-1 Ra production in Mafb-Cre-marked macrophages, which provides a potential disease mechanism for secondary Candidiasis or polymicrobial sepsis. Targeting IL-1 Ra in such IFN-l / IL-1 crosstalk might prove advantageous, as it would enhance protective IL-1 R-signaling by increasing the potency of physiologically secreted, endogenous IL-1 without hindering beneficial defense mechanisms induced through type I IFN.

[0099] IL-1 -mediated inflammation is absolutely essential to control Candida bloodstream infection, as is unequivocally shown by the fact that defects throughout the IL-1 pathway drastically increase the disease mortality. Yet, lack of physiological balancing of IL-1 by the IL-1 Ra due to genetic deficiency or neutralizing anti-IL1 Ra autoantibodies results in uncontrolled IL-1 -signaling and a multi-organ inflammatory syndrome. An impaired IL-1 / IL-1 Ra balance has been linked to hyper- inflammatory states, and anakinra is considered for bacterial sepsis. In contrast, we did not observe exacerbated multi-organ inflammation in IL-1 Ra-deficient IL-1 RaMafbmice or following IL-1 Ra neutralization during Candida infection. Instead, removal of IL-1 Ra not only resulted in faster pathogen clearance, but, surprisingly, also in the rapid and paradoxical attenuation of the pathogenic hyper-inflammation observed in mice expressing functional IL-1 Ra. Accordingly, the dysfunctional inflammatory response during Candida sepsis is not caused by excessive IL-1 signaling, but by a failure to eliminate the pathogen. Therapeutic neutralization substantially improved the survival of wild type mice, which should encourage the development of more efficient IL-1 Ra-targeted approaches. We presume that enhancing IL-1-driven mechanisms by neutralizing IL-1 Ra could be beneficial in patients with active fungal replication, but might not be suitable for inflammatory conditions triggered by residual fungal antigen. For example, IL-1 oc elicited neutrophilic inflammation is required for clearance of pulmonary Aspergillus infection, but worsens disease outcomes in Aspergillus-induced asthma. Thus, increased neutrophil recruitment following IL-1 Ra neutralization could promote resistance to invasive fungal infection, but may also sustain pathogenic inflammation to non-replicating fungal components. Whether such mechanisms contribute to the immune reconstitution inflammatory syndrome (IRIS) in chronic disseminated candidiasis remains to be investigated.

[0100] In conclusion, by ablating the endogenous IL-1 inhibitor IL-1 Ra instead of the IL-1 cytokines or their receptor our study allowed both, to highlight the impact of physiological IL-1 responses by enhancing their potency, as well as to gain valuable insights into their regulation via the IL-1 Ra expressed by different subsets of immune cells. While this approach confirmed the beneficial role of IL-1 in antifungal defense, it also exposed the detrimental consequences of macrophage- secreted IL-1 Ra for the ability to contain bloodstream Candida infection. Moreover, it revealed that the increased inflammation observed during invasive candidiasis does not reflect excessive signaling via the IL-1 R, but the unsuccessful elimination of the fungal pathogen. Collectively, these observations suggest serum IL-1 Ra as a future biomarker and potential therapeutic target for invasive candidiasis.

[0101] Description of the figures

[0102] Figure 1. Ablation of macrophage-produced IL-1Ra protects against invasive fungal infection

[0103] (A) mRNA expression of IL-1 |3, IL-1 Ra and IL-1 R in kidney at indicated days p.i. (n = 4-8 mice / timepoint, two pooled experiments).

[0104] (B) Quantification of serum IL-1 Ra at indicated days p.i. (n = 12-20 mice / timepoint).

[0105] (C) IL-1 Ra production by indicated myeloid cell subsets upon stimulation with LPS, heat-killed C. albicans yeast or hyphae in vitro.

[0106] (D) Generation of conditional IL-1 Ra-deficient lines.

[0107] (E-G) Candida titers in kidneys of IL-1 RaLysM, IL-1 RaMafb, IL-1 RaCD11cand wild type IL-1 Rafl / flmice on days 3 and 7 p.i. (n > 7 mice / group, three pooled experiments).

[0108] (H-K) Quantification of histopathological analysis (H-J) and IL-1 Ra-positive areas (K).

[0109] (L) Plasma creatinine and BUN concentrations of indicated strains at day 2 p.i. Dashed lines indicate naive baselines, (n = 7-13 mice / group, two pooled experiments).

[0110] (M) Quantification of renal Kim-1 and Lcn2 mRNA expression, (n = 4-9 mice / group, two pooled experiments).

[0111] Error bars represent mean ±SEM.

[0112] Figure 2. Macrophage-produced IL-1 Ra prevents rapid neutrophil recruitment and inflammation resolution (A-B) Analysis of blood leukocytes in indicated strains on day 2 p.i. for Ly-6G fluorescence intensity

[0113] (A) of neutrophils, and frequencies of mature neutrophils, immature neutrophils, and monocytes

[0114] (B). (n = 6-8 mice / group, two pooled experiments).

[0115] (C) Absolute neutrophil and monocyte counts in peritoneal lavages of indicated mice at 18 h post zymosan injection.

[0116] (D,E) Fungicidal activity (D) and IL-1 £ secretion (E) of kidney neutrophils purified from indicated mice on day 2 p.i. (n = 6 mice / group).

[0117] (F-l) Frequencies of CD101+ROShi(F), pro-IL-1 p+(G), CD63+(H), and MPO+(I) neutrophils in blood, spleen and kidney of indicated strains on day 2 p.i. (n = 4 mice / group, representative experiment of two).

[0118] (J) Characterization of renal inflammation in indicated mice on day 2 p.i. by absolute counts of leukocyte subsets (J) and cytokine concentrations (K). (n = 6 mice / group).

[0119] Error bars represent mean ±SEM.

[0120] Figure 3. Serum IL-1Ra is produced by CD169+marginal zone macrophages

[0121] (A-B) IL-1 Ra expression in serum (A,B) of indicated mice on day 3 p.i. (A) or 5 h post LPS injection

[0122] (B). Scale bars, 200pm. (n = 6-15 mice / group, three experiments pooled).

[0123] (C) Cartoon depicting EYFP-reporter used in (F).

[0124] (D,E) Analysis of splenectomised (SE) and control (ctrl) mice on day 3 p.i. for serum IL-1 Ra (D), and Candida titers (E).

[0125] (F,G) Histopathological evaluation of fungal dissemination (F) and inflammation (G) (n= 4-6 mice / group, representative experiment).

[0126] (H , I ) Analysis of clodronate liposome-treated (CL) and control (ctrl) mice on day 3 p.i. for serum IL- 1 Ra (H), and CD101+and pro-IL-1 £+in kidney (I).

[0127] Error bars represent mean ±SEM.

[0128] Figure 4. Serum IL-1Ra mediates the impaired pathogen control and dysfunctional hyperinflammation during disseminated Candida infection

[0129] (A,B) Analysis of IL-1 Ra reconstitution in IL-1 RaMafbmice in serum (A) during Candida infection, and in peritoneal lavage (B) during zymosan-induced peritonitis. (A, n = 7 mice / group; C, n = 9 mice / group).

[0130] (C-E) Characterization of peritoneal infiltrates of IL-1 Ra-reconstituted IL-1 RaMafbmice at 18 h post i.p. zymosan by absolute counts (C), fungicidal neutrophil activity (D) and phagocytosis (E). (n = 6 mice / group, two pooled experiments).

[0131] (F) Western blot analysis of IL-1 R-signaling in neutrophils with or without prior IL-1 Ra pulse treatment. (G,H) Characterization of IL-1 Ra-reconstituted mice at day 3 p.i. by fungal titers (G), cellular infiltrates (H) (n = 7 mice / group, two pooled experiments).

[0132] (I) Histopathological quantification of fungal replication and inflammation.

[0133] (J-L) Preventive IL-1 Ra neutralization (NT) at day 3 post Candida infection examined by IL-1 Ra expression in serum (J) and fungal kidney titers (K). Scale bar, 1000pm. (L) Histopathological quantification of Candida replication and inflammation (n = 6-10 mice / group, three pooled experiments).

[0134] (M-O) Therapeutic IL-1 Ra neutralization in Candida-infected wild type mice assessed by serum IL- 1 Ra (M), renal Candida titers (N), and survival probability (O). (n = 6-7 mice / group, two pooled experiments).

[0135] (P,Q) Inflammatory profiles in kidneys of indicated mice following reconstitution (P) or neutralization (Q) of IL-1 Ra as determined by cytokine array, (n = 5-7 mice / group).

[0136] Error bars represent mean ±SEM.

[0137] Figure 5. IL-1Ra from infiltrating macrophages limits pathogen elimination in infected tissue

[0138] (A) mRNA expression of IL-1 family genes in indicated cell subsets on day 2 p.i.

[0139] (B,C) IL-1 Ra production by neutrophils and monocytes stimulated with Candida in presence or absence of IL-10 or IFN-0 in vitro. Cells were purified from naive mice (B) or on day 2 p.i. (C). (B, duplicate cultures, n = 4 mice; C, n = 4-7 mice).

[0140] (D) Western blot analysis of IL-1 Ra isoforms in Cand / da-stimulated neutrophils and monocytes.

[0141] (E) Frequencies of neutrophils and monocytes amongst IL-1 Ra mRNA-expressing kidney leukocytes at day 2 p.i. (n = 7 mice / group, two pooled experiments).

[0142] (F) Frequencies of IL-1 Ra mRNA-expressing cells within indicated immune cell subsets of IL- 1 Rafl / fl, IL-1 RaMafband IL-1 RaCD11cmice at day 2 p.i. (n = 7 mice / group, two experiments).

[0143] (G-J) Analysis of Cre-expression in IL-1 Ra mRNA-expressing leukocytes of EYFPMafband EYFPCD11cmice on days 2 (G-l) and 5 (J) p.i. Expression of EYFP and IL-1 Ra mRNA in neutrophils or monocytes from EYFPMafb(G) and EYFPCD11c(H) mice assessed by flow cytometry and quantification, (n = 4-8 mice / group).

[0144] (I, J) Frequency of EYFP-expression amongst IL-1 Ra mRNA-expressing Ly6G' CD11 b+cells of EYFPMafband EYFPCD11cmice at days 2 (I) and 5 (J) p.i. (n = 4-8 mice / group).

[0145] (K,L) Characterization of IFNAR' / _mice by IL-1 Ra expression in serum (K) and fungal burden in kidney (L) on day 3 p.i. (n= 4-8 mice / group).

[0146] (M) IL-1 Ra production by EYFPMafbmacrophages FACS-purified ex vivo on day 5 p.i. and stimulated in vitro as indicated, (n = 6 mice / group, two pooled experiments).

[0147] Error bars represent mean ±SEM. Figure 6. Type I IFN amplifies the macrophage IL-1 Ra response and exacerbates fungal sepsis

[0148] (A,B) In vivo induction of IL-1 Ra by type I IFN as evaluated by serum IL-1 Ra (A) and kidney mRNA expression (B) following injection of IFN-p or PIC, respectively, (n = 7 mice / group, two pooled experiments).

[0149] (C,D) PIC-induced IL-1 Ra expression in serum (C,D) and of IL-1 Rafl / fland IL-1 RaMafbmice determined 5 h post PIC injection (C) or 48 h after additional Candida infection (D). (C, n = 4-7; B, n = 3; E, n = 3-6 mice / group).

[0150] (E) Survival probability of IL-1 Rafl / fland IL-1 RaMafbmice injected with Candida and PIC as indicated, (n = 6 mice / group, two pooled experiments).

[0151] (F) Fungal burden (F) and in kidneys of mice treated as in (E) evaluated on day 2 p.i. (n = 6 mice / group).

[0152] (G) Serum IL-1 Ra levels of IL-1 Rafl / fland IL-1 RaMafbmice infected. Dashed line indicates naive baseline, (n = 3-7 mice / group, single experiment).

[0153] (H) Survival probability of indicated groups of infected mice, (n = 4 mice / group).

[0154] (I, J) Characterization of co-infected mice at day 3 p.i. by IL-1 Ra expression in serum (I) and Candida titers in kidney (J) (n = 4 mice / group, representative experiment).

[0155] Error bars represent mean ±SEM.

[0156] Figure 7. Virus-induced exacerbation of fungal dissemination critically depends on type I IFN and macrophage-produced IL-1 Ra

[0157] (A-D) IFNAR_ / _and wild type mice infected with Candida (Ca) or co-infected with LCMV and Candida (LCMV+Ca) were analysed by PAS and IL-1 Ra staining of serum IL-1 Ra (A), fungal kidney titers (B), and mRNA expression of IFN-stimulated genes in spleen (C) and kidney (D) on day 3 p.i. (n = 5-7 mice / group, two pooled experiments). (E,F) Serum IL-1 Ra levels (E) and kidney Candida titers (F) of IL-1 Rafl / fland IL-1 RaMafbmice infected as in (A-E). (n = 4-5 mice / group, single experiment). Error bars represent mean ±SEM.

[0158] Figure 8. Efficiency and specificity of C re-drivers used in this study. Related to Figure 1.

[0159] (A) Efficiency of Cre-mediated IL-1 Ra deletion in indicated cell subsets of IL-1 RaLysM, IL- 1 RaMafb, IL-1 RaCD11cmice and their respective IL-1 Rafl / fllittermate controls was assessed in vitro. BM- derived macrophages, BM neutrophils, and splenic DCs were stimulated with 10 ng / ml LPS for 24 h. Production of IL-1 Ra was then assessed in culture supernatants by ELISA or in whole cell lysates by western blot analysis. Representative western blot are shown.

[0160] (B) Flow cytometry analysis of EYFP Cre-reporter expression in the indicated immune cell subsets isolated from BM, spleen and kidney from naive EYFPLysM, EYFPMafb, and EYFPCD11cmice and their respective Cre-negative EYFPLSL littermate controls. (C) Body weight of IL-1 RaLysM, IL- 1 RaMafb, IL-1 RaCD11cand their respective IL-1 Rafl / fllittermate controls infected with C. albicans. Body weight at day 3 and 6 p.i. is expressed as percentage of the initial weight before infection (day 0). Dots represent single mice. Panel A shows pooled data from three experiments with at least six mice per group. Panel B show pooled data from one experiment with two mice per group. Bars represent mean ± SEM.

[0161] Figure 9. Inflammatory responses of conditional IL-1 Ra-deficicent mice infected with C. albicans. Related to Figure 2.

[0162] (A-B) Absolute counts of neutrophils and monocytes were determined by flow cytometry in lavage (A) and blood (B) from IL-1Rafl / fl, IL-1 RaLysM, IL-1 RaMafband IL-1 RaCD11cmice at 6 and 18 h after i.p. instillation of zymosan A. Dots represent individual mice. Data representative for two experiments is shown. Bars represent mean ±SEM.

[0163] Figure 10. Early neutrophil recruitment and functionality in IL-1 RaMafb mice infected with C. albicans. Related to Figure 2.

[0164] (A) Kinetics of neutrophil recruitment in spleens and kidneys of IL-1 Rafl / fland IL-1 RaMafbmice at day 1 , 2 and 3 post C. albicans infection. Representative data from two experiments with four mice per group.

[0165] (B-C) Reactive oxygen species (ROS) production by neutrophils measured by luminol-enhanced chemiluminescence assay. (B) Neutrophils isolated from kidneys of IL-1 Rafl / fl and IL-1 RaMafbmice at day 2 post C. albicans infection. (C) Neutrophils isolated from lavages of IL-1 Rafl / fland IL-1 RaMafbmice at 18 h post i.p. zymosan A injection.

[0166] (D) Fungal titers in spleens of IL-1 RaLysM, IL-1 RaMafb, IL-1 RaCD11cand their respective IL-1 Rafl / fllittermate controls at day 2 p.i. with C. albicans. Data are pooled from two experiments with eight mice per group. Bars represent mean ±SEM.

[0167] Figure 11. The inhibitory effect of IL-1 Ra is cell-extrinsic and mediated by IL-1 Ra secreted from splenic marginal zone macrophages. Related to Figure 3.

[0168] (A) Frequencies of EYFP+ cells in indicated cell subsets in blood and peritoneal lavages of EYFPLSL, EYFPLysM, EYFPMafb, and EYFPCD11cmice at 18 h post i.p. instillation of zymosan A.

[0169] (B) Scheme for the generation of mixed BM chimeras (BMCs). Congenic CD45.1 + IL-1 Ra+ / +recipients were reconstituted with equal ratios of CD45.1 + IL-1 RA+ / +wild type BM (as internal reference present in all chimeras) and the respective CD45.2+ experimental BM from IL-1 RaMafb, IL-1 RaLysM, or control IL-1 Rafl / flmice.

[0170] (C-E) Analysis of immune cell subsets in mixed BMCs on day 3 post Candida infection. (C) Absolute counts of total CD45+ neutrophils, inflammatory monocytes and kidney-resident macrophages in the kidneys of indicated mixed BMC as determined by flow cytometry. (D) Frequencies of CD45.2 / 2+ cells (closed bars) and CD45.1 / 2+ cells (open bars) amongst neutrophils and monocytes in infected kidneys of indicated mixed BMC mice on day 3 pi. Frequencies of CD45.2 / 2+ cells were normalized to the mean frequency of CD45.1 / 2+ B220+ cells in BM to normalize for potential differences in BM reconstitution efficiency in the different recipient mice. (E) Phagocytosis activity of neutrophils isolated from kidneys of infected IL-1 Rafl / flBMC, IL-1 RaLysMBMC and IL- 1 RaMafbBMC was measured in vitro as the uptake of pHrodo green zymosan particles following staining for CD11 b, Ly6G, CD45.1 and CD45.2 and flow cytometry analysis.

[0171] (F) Generation of chimeric mice: BM cells isolated from one donor strain (either IL-1 Rafl / flor IL- 1 RaMafb) were transplanted to irradiated recipient wild type mice (C57BL / 6), rested for at least 8 weeks, and then infected with C. albicans.

[0172] (G) Kidney fungal burden IL-1 Rafl / fl>B6 and IL-1 RaMafb>B6 BMC mice at day 3 and day 7 post Candida infection.

[0173] (H) Scheme of selective depletion of marginal zone-resident macrophages in C57BL / 6 mice using clodronate liposomes (CL), followed by C. albicans infection. Mice were sacrificed 3 days after infection.

[0174] (I-L) Absolute counts of neutrophils and monocytes in infected kidneys (I) and spleens (J) of control and CL-treated mice on day 3 post C. albicans infection. (K, L) Absolute numbers of CD101 -positive mature neutrophils (K) and activated pro-IL-1 p-positive neutrophils (L) in spleens were assessed by flow cytometry. Panel A shows pooled data from at least three experiments with at least eight mice per group. Panels C-E show pooled data from two experiments with five mice per group. Panel G shows pooled data from two experiments with four mice per group. Data in figures l-L were pooled from two experiments with twelve mice per group. Bars represent mean ± SEM.

[0175] Figure 12. Neutralization of macrophage-produced serum IL-1 Ra improves early pathogen control and prevents renal hyperinflammation. Related to Figure 4 and Figure 5.

[0176] (A) Experimental scheme for rlL-1 Ra reconstitution.

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

[0178] (C) Experimental design for therapeutic IL-1 Ra neutralization. Monoclonal IL-1 Ra antibody was given intravenously from day 2 to day 6 p.i. Mice were injected with 3.5x105 CFU for assessing survival and with 2x105 for examination of fungal titers on day 5 and day 7 experiments.

[0179] (D) Kaplan-Meier survival plots of C. a / b / cans-challenged IL-1 RaMafbmice and IL-1 Rafl / flcontrols. Data were pooled from two experiments with at least seven mice per group.

[0180] (E) WT and IFNAR-deficient mice were infected with 1x105 CFU C. albicans for 7 days. Fungal burden in kidneys was assessed by CFU. Data depicts result of one experiment with four mice per group. EXPERIMENTAL MODELS

[0181] Animals

[0182] Mice were bred and maintained in specific-pathogen-free (SPF) facilities at the Institute of Pathology of the University of Bern. All procedures were performed in accordance with ethical guidelines and approved animal license protocols of the Canton of Bern (BE3 / 18 and BE31 / 2021 ). Mice were housed with a 12 h light / dark cycle, regulated temperature and humidity, and provided unlimited 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-1 RaLysMmice had been generated by crossing IL-1 Rafl / fl(111 rntml ,1Cga) mice to LysM-Cre (Lyz2tm1 (cre)lfo / J) mice and were kindly provided by Marc Donath (Department of Biomedicine, University Hospital Basel). To obtain IL-1 RaMafband IL-1 RaCD11cmice, we crossed IL-1 Rafl / flmice with Mafb-Cre or CD11 c-Cre (B6.Cg-Tg(ltgax-cre)1-1 Reiz) mice, respectively. CD11 c-Cre mice were a gift from Manfred Kopf (Institute of Molecular Health Sciences, ETH Zurich). In general, IL-1 Rafl / flCre-negative littermates served as controls; for some experiments IL-1 Rawt / wtCre-positive mice were used as controls and yielded wild type results. R26R-EYFP mice (termed EYFPLSLhere) were crossed with LysM-Cre, Mafb-Cre, or CD11 c-Cre mice to create the respective EYFP-Cre-reporter strains, which we referred to as EYFPLysM, EYFPMafb, or EYFPCD11chere. All mouse strains were backcrossed onto C57BL / 6 for more than 10 generations or had been generated on a C57BL / 6 background. Age- and sex-matched animals were randomly assigned to experimental groups. To generate bone marrow (BM) chimeras (BMC), six-week-old recipient mice were lethally y-irradiated using a GammaCell X40 irradiator 24 hours prior to intravenous injection of 5x106donor BM cells. Mice received sulfamethoxazole and trimethoprim via the drinking water for two weeks, and were rested for at least eight weeks to allow BM reconstitution before performing the experiments.

[0183] Candida albicans and inflammation models

[0184] C. albicans (SC5413) was grown in YPD medium (BD Difco™ YPD Broth, BD Sciences) at 30°C for 18 hours. C. albicans yeast cells were washed twice in sterile PBS and counted using a hemocytometer. To obtain C. albicans hyphae, washed C. albicans yeast were cultured in RPMI 1640 (10% FBS) at 37°C for 4 h. Heat-killed yeast and hyphae were prepared by incubation at 72°C for 1 h, and complete inactivation was confirmed by plating on YPD agar plates at 30°C for 48 h. For in vivo experiments, eight- to ten-week-old mice were infected intravenously with 0.5 - 2.5x105colony forming units (CFU) C. albicans via the lateral tail vein. To determine fungal organ titers, mice were euthanized, and organs were weighed and homogenized in 0.5% NP- 40 water with a TissueLyser II (Qiagen) at 25 Hz for 2x 3 min. Serial dilutions of tissue homogenates in PBS were then plated onto YPD agar, incubated for 24 - 48 h, and fungal burden was calculated as CFU C. albicans / g tissue. To assess the effect of type I IFN on IL- 1 Ra production and C. albicans infection in vivo, mice were injected with 2 pg of mouse recombinant IFN-£ (Biolegend) alone, and with the synthetic TLR3 ligand polyinosinic- polycytidylic acid (PIC, InvivoGen) either alone or 5 hours prior to C. albicans infection. Similarly, mice were infected with 1x104or 2x106plaque forming units (pfu) lymphocytic choriomeningitis virus (LCMV) strain WE, either alone or one day before infection with C. albicans. Production of IL-1 Ra in spleen and serum was also assessed at day 1 p.i. with 106pfu vesicular stomatitis virus (VSV Indiana), 2x106pfu vaccinia virus (W) or 3000 CFU Listeria monocytogenes (strain 10403S). Pathogens were diluted from frozen viral stocks grown in MDCK cells (W), BHK21 cells (LCMV WE), Vero cells (VSV), or prepared freshly as overnight cultures in brain heart infusion broth (Listeria). To evaluate early cell recruitment induced by fungal components, mice were intraperitoneally injected with 1 mg of Zymosan A from Saccharomyces cerevisiae (Sigma) freshly dissolved in sterile PBS. At 6 h and 18 h post injection, blood and peritoneal exudates were collected. Serum and peritoneal lavage fluid were stored at -80°C prior to IL-1 Ra measurement by ELISA. Leukocytes in blood and peritoneal lavage were characterized by flow cytometry. Peritoneal neutrophils were purified from lavage using magnetic bead-based isolation before assessing C. albicans killing and phagocytosis in vitro. To assess LPS-induced IL-1 Ra expression, mice were intraperitoneally injected with ultra-pure LPS 0111 :B4 (150 pg / kg, Sigma) and D-galalactosamine (800 mg / kg, Carbosynth Ltd.), and serum and organs were collected 5 h later.

[0185] METHODS DETAILS

[0186] In vivo manipulation of splenic macrophages

[0187] Splenectomy was performed under isoflurane anesthesia in a laminar flow bench. The abdominal skin and peritoneal membrane were opened by two small incisions to expose the spleen. The spleen was removed by cauterizing the splenic arteries and veins at the splenic hilum. The peritoneal membrane was closed with absorbable suture and skin incision was closed with wound clips. After the surgery, mice were allowed to recover under a heating lamp and rested for eight weeks before C. albicans infection. To selectively deplete macrophages of the splenic marginal zone, mice were intravenously injected with commercial clodronate liposomes (Liposoma) at 1 mg per animal, while control mice received an equal volume of sterile PBS. Ten days later, all mice were infected with 2.5 x105CFU C. albicans. Serum and organs were harvested at day 3 p.i. for analysis.

[0188] In vivo reconstitution and neutralization of IL-1 Ra

[0189] Recombinant human IL-1 Ra (anakinra) was a gift from Marianne Boni-Schnetzler (Department of Biomedicine, University Hospital Basel). IL-1 RaMafbmice received recombinant IL-1 Ra (500 pg per mouse) intraperitoneally twice daily on days 0, 1 , and 2 post C. albicans infection. Nonreconstituted IL-1 Rafl / fland IL-1 RaMafbmice in these experiments received injections of an equal volume of sterile PBS. The hybridoma producing a neutralizing anti-mouse IL-1 Ra antibody was generously provided by Naofumi Mukaida (Kanazawa University, Japan) and Russell Vance (University of California, Berkeley, USA). Monoclonal antibody was produced and purified in-house using protein G resin (GenScript). For preventive IL-1 Ra neutralization in vivo, mice received intraperitoneal injections of either neutralizing anti-IL-1 Ra antibody or control Ultra-LEAF purified armenian hamster IgG isotype antibody (BioLegend) on days -1 , 1 and 2 of infection. For therapeutic IL-1 Ra neutralization, mice were administered intravenously anti-IL-1 Ra or control antibody on days 2 to 6 post C. albicans infection.

[0190] Isolation of leukocyte populations

[0191] Leukocytes were isolated from kidneys by a modification of the protocol of Swamydas et al. Kidneys were dissected into 1 mm3pieces using a scalpel, digested in 6 ml of serum-free RPMI 1640 medium with 0.2 mg / ml of Liberase™ (Roche) and 0.2 mg / ml DNase I (Sigma) for 45 min at 37°C. At the end of the incubation, an equal volume of complete RPMI 1640 medium was added; the cell suspension was filtered through a 40 pm cell strainer, and washed twice in PBS. The pellet was then suspended in 40% Percoll (Sigma), gently overlaid onto 70% Percoll and centrifuged at 880 g at room temperature for 30 min. Splenic single cell suspensions were obtained by enzymatic digestion with 2 mg / ml of type IV collagenase (Worthington) and 0.2 mg / ml DNAse I (Sigma) for 45 min at 37°C. Neutrophils were purified using the EasySep™ Mouse Neutrophil Enrichment Kit (StemCell Technologies) supplemented with biotinylated antibodies (all BioLegend) as following: anti-CD3e (3.45 pg / ml), anti-B220 (2.5 pg / ml), anti-TER119 (0.25 pg / ml), anti-F4 / 80 (3.45 pg / ml), anti-CD11 c (3.45 pg / ml), anti-CD19 (3.45 pg / ml), anti-NK1.1 (3.45 pg / ml) and anti-CD317 (2.5 pg / ml). Monocytes were isolated using the EasySep™ Mouse Monocyte Isolation Kit (StemCell Technologies) using biotinylated antibodies (all BioLegend) at the following final concentrations: anti-CD3e (3.45 pg / ml), anti-B220 (2.5 pg / ml), anti-TER119 (0.25 pg / ml), anti-F4 / 80 (3.45 pg / ml), anti-CD19 (3.45 pg / ml), anti-NK1.1 (3.45pg / ml) and anti- CD317 (2.5 pg / ml). For the analysis of the gene expression or in vitro IL-1 Ra production of individual leukocyte subsets, single cell suspensions were first prepared from infected organs as described above. Following cell surface staining to identify leukocyte subsets, cells of individual mice were FACS-purified on a MoFlo Astrios EQ sorter (Beckman Coulter) by the flow cytometry core facility at the Department of Biomedical Research, University of Bern. Cells were sorted into pre-warmed complete medium. BM cells were collected by flushing femur and tibia from both hind legs with sterile PBS. Erythrocytes were lysed using ACK lysis buffer, before primary BM neutrophils and BM monocytes were isolated using the EasySep™ Mouse Neutrophil Enrichment Kit and EasySep™ Mouse Monocyte Isolation Kit as stated above. For in vitro analysis of LPS- induced IL-1 Ra production, splenic macrophages and splenic dendritic cells were purified from single cell suspensions using magnetic anti-CD11 b MicroBeads and CD11 c MicroBeads (Miltenyi Biotec) according to the manufacturer’s instructions. BM-derived macrophages we generated by culturing BM cells in complete RPMI 1640 medium supplemented with 10% L929 cell supernatant as a source of M-CSF. Medium was exchanged every 3 days, and BM-derived macrophages were harvested for experiments on day 7. Peritoneal macrophages were harvested 4 days after intraperitoneal instillation of 1 ml 3.8% thioglycollate (Becton Dickinson AG). Lavage cells were washed with PBS and cultured overnight in the presence or absence of 10 ng / ml LPS (InvivoGen). IL-1 Ra production was then assessed in culture supernatants by ELISA or in cell lysates by western blot.

[0192] Flow Cytometry analysis

[0193] Single cell suspensions were prepared as described above and stained with live / dead dye (Invitrogen) and anti-mouse CD16 / 32 antibody (BioLegend) for 15 min at 4°C. Immune cell subsets were characterized using antibodies against CD90, CD19, CD49b, CD45, CD11 b, CD11 c, F4 / 80, Ly-6C, Ly-6G, l-A / l-E, and CD115 (all BioLegend), as well as against CD101 (eBioscience™) and Siglec F (Miltenyi Biotec). Neutrophils, resident macrophages, Ly-6hlmonocytes, Ly-6Cl0monocytes and DCs were defined as CD45+CD11 b+Ly-6Ghl, CD45+CD11 b+F4 / 80+, CD45+CD11 b+F4 / 80’ MHCIL Ly6Chi, CD45+CD11 b+F4 / 80’ MHCIL Ly6C CD11 c+, and CD45+CD11 b+F4 / 80' MHC+CD11 c+, respectively. Blood mature neutrophils, immature neutrophils and monocytes were identified as CD11 b+CD115- Ly6G+CD101+, CD11 b+CD115’ Ly6G+CD10T and CD11 b+CD115+Ly6C+, respectively. For phenotypic characterization of neutrophils, cells were stained with antibodies against CD45.2, CD11 b, Ly-6G, CD63 (all BioLegend), CD101 and pro-IL-1 beta (both from eBioscience™) and MPO (Hycult Biotech). Cell populations were positive for CD90, CD19, CD49b, CD11 c, F4 / 80, l-A / l-E and Siglec-F, were excluded from the flow cytometry analysis. Cells were fixed in 4% paraformaldehyde (PFA) for 5 min before acquisition. Intracellular IL-1 Ra transcripts in kidney-infiltrating leukocytes were revealed using the PrimeFlow™ RNA assay (Thermo Fisher) according to the manufacturer’s instructions. After cell surface staining for CD45.2, CD11 b, CD11 c, MHC II, F4 / 80, Ly-6C and Ly- 6G, cells were fixed and permeabilized, followed by the hybridization with the IHrn probe, signal amplification and fluorescence labeling. All samples were acquired on a LSRII cytometer (BD Biosciences) and analyzed using FlowJo (BD Biosciences).

[0194] Measurement of reactive oxygen species

[0195] Freshly purified neutrophils (106per well) were equilibrated for 60 min at 37°C before staining with 5 pM 2’,7’-dichlorofluorescein (DCF) in serum-free RPMI for 30 min, followed by exposure to heat-killed C. albicans (MOI 3) in the presence or absence of IL-10 (20 ng / ml) for additional 30 min. Cells were then stained with live / dead fluorescent dye and antibodies against CD45.2, CD11 b, Ly-6G, and Ly6C. ROS production was quantified in leukocytes isolated from the blood, spleen and kidneys of C. albicans infected mice. Cells were incubated for 30 min at 37°C with Dihydrorhodamine 123 (Sigma Aldrich) before staining with live / dead fluorescent dye and antibodies against CD45.2, CD11 b, Ly-6G, Ly-6C, and CD101 . Frequencies of ROS producing neutrophils were analyzed using FlowJo (BD Biosciences).

[0196] C. albicans killing assay

[0197] To test fungicidal capacity, 5 x104neutrophils were incubated with 2 x 104C. albicans for 3 h. Cells were then lysed with 1 % NP-40 water and serial dilutions of the lysates were plated on YPD agar. Colonies were counted after 24 h at 30°C. Fungicidal capacity was expressed as the percentage of C. albicans inoculum detected in control wells without leukocytes, that was killed in presence of neutrophils.

[0198] Phagocytosis assay

[0199] Phagocytosis activity was measured using pHrodo™ Green Zymosan Bioparticles (Thermo Fisher) according to the manufacturer’s instructions. Neutrophils were purified from peritoneal cavities of Zymosan A-primed mice, plated in black clear bottom 96-well plates (Corning) at 105cells / well in 100 pl Opti-MEM® (Gibco), and equilibrated at 37°C for 1 h. Culture medium was then replaced by 100 pl of 0.5 mg / ml pHrodo™ Green Zymosan Bioparticles in uptake buffer and incubation continued for 2 h at 37°C in the presence or absence of IL-10 (20 ng / ml). Bioparticles in pH 5.0 buffer served as positive control. Bioparticle-free wells and wells containing Bioparticles in uptake buffer served as background and negative control. Fluorescence intensity was assessed with excitation at 490 nm and emission at 535 nm using an Infinite M200 pro Tecan microplate reader. Net phagocytosis was calculated by correcting for fluorescence intensity of background and negative controls. Phagocytosis is expressed as percentage of net fluorescence intensity in experimental wells as compared to the net positive control.

[0200] Blood sampling

[0201] Blood samples were collected terminally from the vena cava into syringes containing heparin (for creatinine and blood urea nitrogen) or EDTA (for cytokine analyses) as anticoagulant. All samples were kept on ice while handling. Blood was transferred to blood collection tubes (BD microtainer) for serum isolation or 1 .5 ml reaction tubes for plasma collection and centrifuged for 10 min at 2000 g at 4°C. 100 pl of resulting supernatants were transferred into clean polypropylene tubes and processed directly or stored at -80°C until analysis. Measurement of plasma creatinine and blood urea nitrogen concentrations was performed by the Center for Laboratory Medicine at the University Hospital Bern.

[0202] Analysis of cytokine responses

[0203] Cytokine concentrations in serum, peritoneal lavages or cell culture supernatants were determined by ELISA. Production of IL-1 Ra and G-CSF in serum and culture supernatant were measured using the mouse IL-1 ra / IL-1 F3 DuoSet kit and the mouse G-CSF DuoSet kit (RnD Systems), according to the manufacturer’s guidelines. Mouse IL-10 and IL-6 were quantified using sandwich ELISA with an anti-IL-101 biotinylated anti-IL-10 or anti-IL-6 I biotinylated anti-IL- 6 antibody pairs (all from eBioscience), respectively, followed by detection with streptavidinalkaline phosphatase (Southern Biotech) and para-nitrophenylphosphate. Optical densities were read on an Infinite M200 pro Tecan microplate reader and concentrations were calculated using recombinant standards for mouse IL-10 (RnD Systems) and IL-6 (eBioscience), respectively. Cytokine profiles in infected kidneys were characterized with a Mouse Cytokine / Chemokine 31- Plex Discovery Assay® Array (Eve Technologies). In brief, infected kidneys were snap-frozen, homogenized in modified RIPA buffer, and tissue homogenates were stored at -80°C until analysis. Western blot analysis

[0204] Purified leukocyte subsets were lysed in modified RIPA buffer (50 mM Tris HCI pH 7.4, 150 mM NaCI, 1 % NP-40, 0.5% Triton X100, 1 mM EDTA, 0.5% sodium deoxycholate, 0.1 % SDS, and 10 mM NaF), supplemented with protease inhibitors (Roche), phenylmethylsulfonylfluoride, and phosphatase inhibitor cocktail 2 and 3 (Sigma Aldrich). Protein extracts were normalized using the Pierce® BCA Protein Assay Kit (Thermo Fisher); and 25 pg of protein were assessed by reducing SDS-PAGE (10-12%). After transfer onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories), immunoblotting was performed with anti-IL-1 Ra (Thermo Fisher), anti- p38, anti-phospho-p38 (both Cell Signaling), and anti-p actin (Santa Cruz Biotechnology) primary antibodies and corresponding HRP-conjugated secondary antibodies (Santa Cruz), followed by development in SuperSignal™ West Pico chemiluminescent substrate. Staining was visualized using a ChemiDoc™ MP Imaging System (Bio-Rad Laboratories).

[0205] Immunohistochemistry and immunofluorescence

[0206] Organs were dissected and fixed in 4% paraformaldehyde for 6 to 8 h, followed by standard paraffin embedding. Tissue sections (2.5 pm) were stained on an Immunostainer Leica Bond RX (Leica Biosystems) with periodic-acid Schiff (PAS), with methenamine silver (Grocott), or with anti-IL-1 Ra, anti-IL-1 a, anti-IL-1 , anti-IL-1 R1 (all R&D Systems), anti-F4 / 80 (Bio-Rad Laboratories), anti-CD68, anti-Ly-6G, anti-CCR2, anti-lba1 , anti-Histone H3 (citrulline R2+R8+R17) (all from Abeam), anti-MPO (Agilent) or anti-GFP (Novus) antibodies. Embedding, sectioning and staining were performed by the Translational Research Unit of the Institute of Pathology at the University of Bern. Images were obtained with a Pannoramic 250 scanner (3DHistech) before analysis. Histological grading was performed on Periodic acid-Schiff (PAS) stained sections by a trained pathologist blinded to the identity of the specimens. Inflammation and C. albicans dissemination were evaluated separately for the tubulointerstitial compartment and glomeruli. Scores ranging from 0 to 3 were defined for each category and criterion, as described below. The final score of each section represents the sum of the scores for the tubulointerstitial compartment and the glomeruli.

[0207] Spleen tissue was frozen in OCT medium and cut (5 pm) on a Leica CM1950 cryostat. Acetone- fixed cryosections were incubated in PBS containing 10% goat serum and 0.1 % Triton X-100 (Sigma-Aldrich) to block nonspecific binding for 1 h, before staining with anti-mouse MARCO (BioRad Laboratories) and anti-mouse CD169 (BioLegend) antibodies overnight. Rinsed sections were stained with DAPI (Sigma Aldrich) for 20 minutes, and then mounted with Dako Fluorescence Mounting Medium (Dako). Fluorescent images were acquired using a Pannoramic 250 flash II slide scanner (3DHistech). IL-1 Ra-positive areas in stained kidney sections were quantified using ImageScope (v12.4.0.5043). To visualize the colocalization of marginal zone macrophages and IL-1 Ra, spleen tissue was frozen in OCT medium and cut (10 pm) on a Leica CM1950 cryostat. PFA-fixed (4%) cryosections treated with multistaining buffer (Lunaphore) were stained using the LabSat technology (Lunaphore). In the first cycle anti-mouse CD169 (Biolegend), anti-mouse IL1 RA (R & D Systems) with secondary rabbit anti-goat Alexa Fluor 546 antibody (Invitrogen) and DAPI were stained and imaged. After applying the quenching buffer (Lunaphore), in the second cycle, MARCO (Bio-Rad Laboratories) with corresponding secondary antibody (PE goat anti-rat Ig, SouthernBiotech) and DAPI was stained and imaged.

[0208] Quantitative reverse transcription PCR

[0209] Kidneys were disrupted in 0.5 to 1 ml of TRIzol Reagent (Ambion Life Technologies) with stainless steel beads (5 mm; Qiagen) in a TissueLyser II (Qiagen) for 2 cycles of 2 min at 25Hz. Total mRNA was isolated according to manufacturer’s instructions. Contaminating DNA was digested by RNase-free DNase (Life Technologies), mRNA concentrations were measured with a NanoDrop™ One spectrophotometer (Thermo Fisher) and 1 pg mRNA / reaction was reverse- transcribed using GoScript™ Reverse Transcriptase (Promega) in presence of RNase inhibitor (BioLabs). Quantitative PCR was performed using KAPA SYBR® FAST qPCR Master Mix (2X) Kit (Sigma) on a StepOnePlus™ Real-Time PCR System (Thermo Fisher), and expression was normalized to G6pdx or Actb expression. For FACS-sorted cells, RNA was purified using the ReliaPrep™ RNA Miniprep Systems (Promega) following the manufacturers’ instructions.

[0210] RNA-sequencing and bioinformatics analysis

[0211] Kidneys of infected mice were harvested at day 2 p.i., snap-frozen in liquid nitrogen before homogenization in TRI Reagent® (Zymo Research) in BashingBeat™ lysis tubes (Zymo Research) with a TissueLyser II (Qiagen) for 3 cycles of 1 min at 30Hz. Total RNA was extracted using the Direct-zol™ RNA miniprep Plus kit (Zymo Research) and stored at -80°C until use. The quantity and quality of the purified total RNA was assessed using a Qubit 4.0 fluorometer with the Qubit RNA BR Assay Kit (Thermo Fisher) and an Advanced Analytical Fragment Analyzer System using a Fragment Analyzer RNA Kit (Agilent), respectively. Two hundred ng of input RNA was first depleted of ribosomal RNA and globin mRNA using RiboCop for HMR+ Globin Depletion Kit (Lexogen) following the manufacturer’s instructions. Thereafter, cDNA libraries were generated using a CORALL Total RNA-Seq V2 library Prep. kit with UDIs 12nt set A1 (Lexogen) according to the protocol for long insert sizes. The quantity and length of the cDNA libraries were investigated using the Qubit 4.0 fluorometer and Advanced Analytical Fragment Analyzer System as above. Library quantification was also determined using a JetSeq library Quantification Lo- ROX kit (Bioline) following the manufacturer’s instructions. Equimolar-pooled cDNA libraries were sequenced paired-end using an illumina NovaSeq 6000 S1 Reagent Kit v1 .5 (300 cycles) on an illumina NovaSeq 6000 instrument (illumina). The run produced between 50-60 million reads / sample. The quality of the sequencing run was assessed using illumina Sequencing Analysis Viewer (illumina, version 2.4.7) and all base call files were demultiplexed and converted into FASTQ files using illumina bcl2fastq conversion software v2.20. The quality control assessments, generation of libraries and sequencing was carried out at the Next Generation Sequencing Platform, University of Bern. From the raw sequencing reads, adapters and poly(A) sequences were removed with cutadapt (v3.4). After that, 12 bp UMI sequences were extracted and high error rate sequences were removed with UMI-tools (v1 .2.2) according to the suggestions of the manufacturer of the library preparation kit. Reads were aligned with hisat2 (v2.2.1 ) to the ensemble mouse genome (GRCm38.p6). Read alignments were deduplicated with UMI-tools (v1 .2.2). The counting of fragments aligning to genic features was performed with featureCounts (v2.0.1 ) based on the ensembl mouse gene annotation v102. DESeq2 was used to calculate differential gene expression out of the raw count matrices. Gene set enrichment analysis (GSEA) was performed with the R package clusterProfiler based on gene ordering according to the test statistic acquired from the differential gene expression results of DESeq. Gene Ontology overrepresentation analysis was performed with the R package topGO on genes which were differentially expressed with an adjusted p-value smaller than 0.05. Heatmap visualizations were generated with the R package ComplexHeatmap. All downstream analyses in R were performed with R version 4.1 .0.

[0212] QUANTIFICATION AND STATISTICAL ANALYSIS

[0213] Unless data of individual mice are shown, data represent mean ± SEM. Statistical analyses were performed using the GraphPad Prism software. Two-tailed Student’s t-test, one-way AN OVA with a Bartlett’s multiple comparisons test or two-way ANOVA with a Tukey’s multiple comparisons test were applied to compare two or several groups as specified in figure legends. Statistical significance was considered as p <0.05. Asterisks denote statistical significance (*, p <0.05; **, p <0.01 ; ***, p <0.001 ; ****, p <0.0001 ).

Claims

Claims1 . A pharmaceutical composition comprising an agent capable of inhibiting IL-1 Ra activity, for use in 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 infection by Candida albicans.

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

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

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

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

7. The pharmaceutical composition for use according to claim 5 or 6, wherein the oligonucleotide agent is selected from an antisense oligonucleotide, a gapmer, an siRNA and a shRNA.

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