IL-1 is crucial for the immune defense against C. albicans

Once systemic infection is established, C. albicans penetrates and grows in various organs of the host. In murine systemic candidiasis, the primary targets of fungal growth are the kidneys and brain. C. albicans is also found in the liver and spleen, though to a lesser extent. Immediately after systemic infection with C. albicans, the expression of IL-1α, IL-1β, and IL-1R is rapidly upregulated in the kidneys (Fig. 1A), possibly suggesting a role in the host defense against fungal infection. Indeed, IL-1R-deficient mice show high susceptibility to C. albicans, indicated by rapid weight loss and approximately 1000-fold elevated fungal titers in the kidneys and brain, but not in the spleen or liver, three days post-infection with 10⁵ CFU C. albicans (Fig. 1B, C), when mice had to be euthanized to prevent undue suffering. Il1r1^-/- mice exhibit significantly increased fungal titers in the kidneys and brain starting at 48 hours post-infection (supplementary Fig. 1D); however, no differences compared to controls were observed at 24 h p.i. (Fig. 1D).

To determine which IL-1 cytokine, IL-1α or IL-1β, is more critical in the anti-fungal immune response, we infected Il1α ^-/- and Il1β ^-/- mice. Interestingly, the absence of IL-1α only slightly increased susceptibility to the fungus, and this was observed only in the brain. However, the absence of IL-1β significantly impacted immune defense and led to elevated fungal growth in both the kidney and brain (Fig. 1E), which is consistent with a 10⁷-fold higher induction of Il1b transcripts compared to Il1a transcripts during the first two days post-infection. This indicates a crucial role for IL-1β, but not for IL-1α, in the immune defense against C. albicans.

To visualize the infection in-vivo, we performed histological staining for fungi, using Modified Grocott Methanamine Silver (GMS) staining, in kidney and brain sections from day 2 infected Il1r1^-/- mice and Il1r1^+/- controls. Histopathological analysis of the kidney revealed dense clusters of C. albicans hyphae located in inflammatory foci in the Il1r1^-/- mice, while the control kidneys had only inflammatory foci, with almost no fungi (Fig. 1F). Interestingly, the analysis of the brain tissue of Il1r1^-/- mice showed that C. albicans not only creates dense clusters of hyphae but is also disseminated throughout the tissue (Fig. 1G, panel A). In contrast, barely any C. albicans were detectable in control brains at day 2 (Fig. 1G).

Our results suggest that Il1r1^-/- mice are highly susceptible to systemic candidiasis, especially in the kidney and brain.

Il1r1-deficient mice exhibit a hyper-inflammatory state upon C. albicans infection

The immune response to systemic candidiasis consists of the fungi being recognized by tissue-resident cells, who subsequently respond by immediately releasing chemokines. This is followed by the infiltration of leukocytes belonging to the innate immune system, such as neutrophils and monocytes²¹.

To obtain information about the inflammatory state of the Il1r1^-/- mice, we analyzed the cytokine and chemokine protein levels in lysates of the kidney and brain of WT and Il1r1^-/- mice 2.5 days post-infection (p.i.). The cytokine analysis revealed higher levels of IL-6, TNFα, and IL-23 in the infected kidneys of Il1r1^-/- mice compared to the kidneys of WT mice, and an even more striking difference was seen in the brain tissue of the Il1r1^-/- mice. The brain of infected Il1r1^-/- mice contained dramatically higher levels of IL-6, TNFα, and MCP-1, compared to the brains of WT mice (Fig. 2A). The chemokine analysis revealed elevated levels of CCL3, CCL4, CCL5, CXCL1 and CXCL13 in the kidney and brain tissues of Il1r1^-/- mice compared to controls. Additionally, levels of CXCL10 were up in the brain of Il1r1^-/- mice (Fig. 2A). These data demonstrate that Il1r1^-/- mice experience a hyperinflammatory state at this stage of the infection, indicating that IL-1R signaling is not necessary for the activation of a pro-inflammatory immune response during systemic C. albicans infection.

We next examined whether Il1r1^-/- mice display differences in infiltration of specific cell types into the infected tissues, which might explain the elevated susceptibility. Therefore, we characterized CD45⁺ cells in the kidney and in the brain of Il1r1^-/- mice and WT controls by high-dimensional flow cytometry at day 3 p.i.. As the phenotype is seen in the first days of infection, we focused on myeloid cells, which are known to infiltrate organs relatively early after initiating infection. To analyze the acquired data, we used an unbiased approach, including the dimensionality reduction algorithm “UMAP” and the clustering algorithm “Phenograph”²².

The Phenograph algorithm identified 25 cell clusters in the kidney and 23 cell clusters in the brain (Fig. 2B, C). To annotate the identified cell types, we grouped the clusters identified in the kidney and in the brain into 8 cell types including neutrophils, eosinophils, cDC1, cDC2, Ly6C⁺ monocytes, MHCII⁺Ly6C^–CD11b⁺ monocyte-derived inflammatory macrophages (Mo-Mac), CD11b^low kidney macrophages and brain resident CD45^lowCD64⁺ microglia (Fig. 2B, C).

Consistent with the overshooting inflammatory response, we found that Il1r1^-/- mice exhibited significantly elevated numbers of neutrophils, Ly6C⁺ monocytes, and Ly6C^– monocytes, both in the kidney and in the brain, (Fig. 2D, E). In contrast, Mo-Mac numbers were down in both organs. CD45^lowCD64⁺ microglia were reduced in the brain, whereas CD11b^low macrophages in the kidney were unaffected. Analysis using manual gating (supplementary Fig. 1A) and definition of the cell types using the same marker panel reproduced the results reliably (quantification of multiple experiments in supplementary Fig. 1B). Notably, our panel at this point included only the markers CD45 and CD64 to define microglia, and it might not reflect all microglia and resident immune populations of the brain. Later analysis, using a more detailed antibody panel, allowed better characterization of the brain immune populations and astrocytes (supplementary Fig. 2A–D, discussed below).

No differences in the number of tissue myeloid cell types in the kidney and brain were observed prior to infection in naïve mice, excluding the role of IL-1R signaling in the steady state (supplementary Fig. 1C).

Thus, consistent with elevated levels of several chemokines that are known to act on migration and/or polarization of myeloid cells, neutrophils, Ly6C⁺ and Ly6C^– monocytes were massively increased in brain and kidney. Considering that neutrophils and inflammatory monocytes are fungicidal, increased Candida growth in the presence of elevated numbers of neutrophils may indicate that they are dysfunctional.

Neutrophils and inflammatory monocytes are functionality intact in Il1r1-deficient mice

To explore whether IL-1R signaling is vital for the ability to kill Candida, we assessed the functionality of neutrophils and monocytes from Il1r1^-/- and WT mice. Phagocytes eliminate engulfed fungi, using both oxidative and non-oxidative killing mechanisms. The inducible nitric oxide synthase (iNOS) is an enzyme involved in producing reactive nitrogen intermediates (RNI), which have direct candidacidal activity²³. Interestingly, the frequency of iNOS⁺Ly6C⁺ monocytes was dramatically increased in the kidney and brain of Il1r1^-/- compared to WT mice (Fig. 3A), suggesting that the nitrosative killing capacity in the organ is increased rather than impaired by the lack of IL-1R, again consistent with elevated inflammatory signals and fungal burden in the KO mice.

To assess whether Il1r1^-/- neutrophils have impaired candidacidal activity, we isolated neutrophils from blood and infected kidneys at 3 days p.i. and performed an ex-vivo killing assay with C. albicans yeast and hyphae. Figure 3B shows neutrophils from infected WT and Il1r1^-/- mice displayed comparably potent Candida killing. However, to control Candida hyphae in-vivo, neutrophils mainly use NETs^24,25,26. Thus, we assessed the capability of neutrophils from Il1r1^-/- mice to produce NETs in response to a stimulus. Neutrophils isolated from bone marrow of WT and Il1r1^-/- mice had comparable capacity to produce NETs in response to PMA, as well as in response to C. albicans (Fig. 3C). Notably, IL-1b stimulation did not affect the NET formation in WT or in Il1r1^-/- mice. Next, we sought to determine whether neutrophils in the Il1r1^-/- mice can produce NETs in response to Candida hyphae in vivo. For this, we used immunohistochemistry and stained consecutive slides of brain of infected Il1r1^+/- and Il1r1^-/- mice, at day 2 p.i., using anti-Ly-6G antibody and anti-Citrullinated Histone H3 antibody (specific for NETs) (Fig. 3D). This analysis confirmed the elevated neutrophil numbers in the Il1r1^-/- mice compared to WT mice and showed citrullinated Histone H3 (Cit H3) staining surrounding the neutrophil clusters in WT and in Il1r1^-/- mice, indicating that NET mediated Candida killing is not affected in Il1r1^-/- mice.

Together, these data demonstrate that the fungicidal activity of neutrophils and iNOS expression by monocytes are not affected in Il1r1^-/- mice, indicating that their severe susceptibility is less likely to be explained by defects in these well-established antifungal immune effector mechanisms.

To explain the high number of neutrophils seen in Il1r1^-/- mice, we infected WT mice with increasing infection doses of C. albicans (supplementary Fig. 3A). As expected, we observed a positive correlation between the dose of infection and neutrophil numbers in the kidney and the brain at 3 days p.i. This correlation suggests that the observed numbers of neutrophils in Il1r1^-/- mice at the height of infection is due to the severe fungal burden in these mice.

Delayed neutrophil recruitment to the brain but not the kidney upon C. albicans infection of Il1r1-deficient mice

Flow cytometry analysis of the brain at 48 h p.i., using a wider antibody panel, revealed significantly reduced numbers of both “activated” (Dectin⁺) and “homeostatic” microglia, as well as CD206⁺ border-associated macrophages and neutrophils in Il1r1^-/- mice, compared to Il1r^+/- littermates (supplementary Fig. 2A–C). At this time point (48 h post-infection), there was already a considerably higher fungal load in the brains of Il1r1^-/- mice (supplementary Fig. 1D), which could indicate that microglia and border-associated macrophages might be especially vulnerable and die due to infection. Interestingly, we observed a significantly increased frequency of ACSA-2^low astrocytes and decreased ACSA-2^high astrocytes at 48 h p.i. Decreased ACSA-2^high astrocytes were already observed in naïve Il1r1^-/- mice compared to controls (supplementary Fig. 2D), indicating that steady state IL-1 production promotes the development or maintenance of the ACSA-2^high population. The heightened inflammation at 48 h p.i. may explain the increase in astrocyte numbers.

Since fungal titer in Il1r1^-/- mice started to differ from WT mice already at 24 h p.i. (Fig. 1D), we suspected that a crucial role of IL-1R signaling may be instigated very early after infection. Indeed, Ly6C⁺ monocyte and neutrophil recruitment to the brain were strikingly reduced in Il1r1^-/- mice at 8 h, 16 h and 24 h p.i. (Fig. 3E–G). Notably, bulk RNA sequencing of the brain at 8 h p.i. showed significantly lower expression of molecules responsible for adhesion and extravasation into the tissue, such as Icam, Lcn2, Sele and Vcam1 (supplementary Fig. 2E–H). Moreover, expression of the chemokines Ccl1 and Csf1, which are known to promote monocyte recruitment and differentiation, was substantially reduced, as were the RNA and protein levels of CXCL1, a potent chemoattractant of neutrophils (Fig. 3H, supplementary Fig. 2 E–H).

These data suggest that the increased fungal titer in the brain of Il1r1^-/- mice results from impaired monocyte and neutrophil recruitment due to reduced production of these chemokines.

On the other hand, in the kidneys of Il1r1^-/- mice, compared to controls, we observed no differences in the number of monocytes and only a modest reduction of neutrophils at 8 h but not at 16 h and 24 h p.i. (Fig. 3F), which is unlikely to explain excessive Candida growth in this organ.

IL-1 prevents severe susceptibility to C. albicans by acting on non-hematopoietic cells

Depending on the context, Il1r1 is expressed by many immune and non-hematopoietic cell types^27,28,29. We wanted to examine which Il1r1-expressing cells are crucial for the defense against C. albicans. First, to assess the role of IL-1R in hematopoietic and non-hematopoietic cells, we used reciprocal bone marrow chimeras that were generated by reconstitution of sub-lethally irradiated WT mice with Il1r1^-/- bone marrow (Il1r1^-/- - > WT) and vice versa (WT -> Il1r1^-/-). WT mice reconstituted with WT bone marrow (WT - > WT) and Il1r1^-/- mice with Il1r1^-/- bone marrow (Il1r1^-/- -> Il1r1^-/-) were used as controls. Interestingly, weight loss and fungal burden in mice with complete IL-1R deficiency (Il1r1^-/- -> Il1r1^-/-) was phenocopied by animals lacking IL-1R exclusively on non-hematopoietic cells (WT -> Il1r1^-/-) (Fig. 4A). Surprisingly, mice lacking IL-1R exclusively in bone-marrow-derived leukocytes (Il1r1^-/- - > WT) had only a very slight susceptibility compared to their WT - > WT counterparts. This suggests that IL-1R-signaling in radioresistant non-hematopoietic cells is crucial for the early defense against C. albicans. However, as irradiation does not deplete brain resident microglia, we used the conditional KO mice vav^creIl1r1^fl/fl, which have a deletion in the Il1r1 gene locus in all hematopoietic cells, including brain microglia (supplementary Fig. 5A). A statistically significant elevated fungal load was only seen in the brain (Fig. 4B). However, the differences in fungal titer were much smaller compared to Il1r1^-/- mice (i.e., 10-fold vs > 10,000-fold) (Fig. 1C), and similar to the Il1r1^-/-→WT group from the bone marrow chimera experiment (Fig. 4A).

From these data, we conclude that the defense against systemic candidiasis strongly depends on Il1r1 expression in non-hematopoietic cells, while IL-1R signaling in hematopoietic cells plays a less critical role.

IL-1R-signaling in kidney endothelial cells contributes to the control of C. albicans independent of neutrophils

IL-1R triggering induces a signaling cascade resulting in NF-κB activation³⁰. To identify the non-hematopoietic cell types in the kidney and brain that contribute to IL-1R-mediated defense against C. albicans, we measured NF-κB activation in response to IL-1β stimulation as a proxy for IL-1R expression. To this end, we used recently generated NF-κB reporter (KappaBle) mice, allowing single-cell detection of NF-κB transcriptional activity monitored by expression of a destabilized EGFP³¹. Intravenous injection of IL-1β into naïve KappaBle mice resulted in a significant elevation of GFP expression after 4–5 h in kidney and brain cells compared to PBS-treated KappaBle controls. The GFP-positive cells in the kidney could be characterized as CD45^–CD31⁺ endothelial cells and CD45^–EpCam⁺ epithelial cells, as well as other non-hematopoietic CD45^– cells that are CD31⁺EpCam⁺ double-positive and CD31^–EpCam^– double negative (Fig. 4C and supplementary Fig. 4A). In the brain, we found IL-1R-signaling mediated NF-κB activation in border-associated macrophages (BAM) (GFP⁺CD45⁺CD206⁺), astrocytes (GFP⁺CD45^–ACSA-2⁺) and in undefined CD45^–CD31^–EpCam^– cells, but not in endothelial and epithelial cells (Fig. 4D and supplementary Fig. 4B, C).

To assess whether IL-1R-signaling in endothelial cells is critical for protection from systemic candidiasis, we generated mice allowing inducible deletion of Il1r1, specifically in endothelial cells. To this end, we crossed Il1r1^flox mice³² to Pdgfb^icreERT2 mice³³. We treated Pdgfb^icreERT2Il1r1^fl/fl mice and Il1r1^fl/fl littermates with Tamoxifen on five consecutive days and infected the mice with C. albicans four days after the last treatment. We confirmed efficient deletion of the Il1r1 gene in endothelial cells from the kidney and the brain by qPCR (supplementary Fig. 5B). On day 3 p.i., the fungal burden was up to 10-fold higher in the kidneys of Pdgfb^creIl1r1^fl/fl mice compared to their Il1r1^fl/fl littermates. Intriguingly, a similar effect was not seen in the brain (Fig. 4E). Like global Il1r1^-/- mice, at 3 days p.i., Pdgfb^icreERT2Il1r1^fl/fl mice showed elevated number of neutrophils and inflammatory monocytes in the kidney, relatively to their Il1r1^fl/fl littermates (Supplementary Fig. 5C). These results demonstrate that lack of IL-1R on endothelial cells contributes to dramatically elevated (up to 1000-fold) fungal load observed in the kidney of global Il1r1^-/- mice at day 3 p.i.

Since we have established that IL-1R is needed for the very early recruitment of neutrophils to the site of infection (Fig. 3E), we were interested in investigating whether this process depends on endothelial IL-1R. Therefore, we quantified neutrophil infiltration at 8 h p.i. in Pdgfb^icreERT2Il1r1^fl/fl mice, compared to their cre negative littermates and to Il1r1^-/- mice. Surprisingly, neutrophil infiltration in Pdgfb^icreERT2Il1r1^fl/fl mice was comparable to the Il1r1^fl/fl mice (Fig. 4F), suggesting that neutrophil recruitment upon Candida infection is independent of endothelial IL-1R. We next treated naive Il1r1^fl/fl and Pdgfb^icreERT2Il1r1^fl/fl mice with IL-1β intravenously and quantified the myeloid cells in the kidney and the brain after 4 h. Interestingly, IL-1β treatment was sufficient to induce neutrophil infiltration to the kidney and brain of wild-type (Il1r1^fl/fl) mice, and a comparable recruitment was observed in Pdgfb^icreERT2Il1r1^fl/fl mice (Fig. 4G), suggesting that the recruitment of myeloid cells is independent of endothelial expression of IL-1R.

These data suggest that IL-1R signaling in kidney endothelial cells contributes to fungal defense independent of neutrophil recruitment. However, IL-1R signaling in other non-hematopoietic cells in the kidney and brain is involved in protecting against Candida overgrowth.

C. albicans infection induces massive transcriptional upregulation of uniform pathways across all kidney cell types in the absence of IL-1R

To uncover the transcriptional response to C. albicans infection in WT mice and differential gene expression in the absence of IL-1R at the single cell level, we performed single-nucleus RNA sequencing (snRNA-seq) on the kidneys of Il1r1^+/- and Il1r1^-/- mice at 8 h p.i. and naïve controls. We opted for snRNA-seq as this method has been shown to supply comparable gene detection to single-cell RNAseq³⁴, while reducing transcriptional biases that originate in the tissue dissociation process and allowing better annotation of rare kidney cell types³⁵. We sequenced a total of 69,820 kidney cells. Following filtering steps, 47,243 cells passed QC with ~2270 genes and ~4070 reads on average per cell (Supplementary Fig. 6A, B, Methods QC). Using previously published kidney datasets^35,36, we annotated our dataset to the 20 unique kidney cell types recapitulating most of the nephron-forming cells (Fig. 5A; Supplementary Fig. 6C, D). We did not observe differences in the abundance of a particular cell type, except for an increased proportion of ascending-loop of Henle/proximal tubule cells (CTAL-PT cells). This cell cluster showed similarity both to the proximal tubules and thick ascending loop of Henle in the cortex (Fig. 5A; Supplementary Fig. 6C). Using data imputation, we were able to detect expression of the Il1r1 gene in at least 5 cell types, with the highest levels in mesangial cells (MC), endothelial cells (EC), connective tubule (CNT) cells, principal cells (PC), and intercalated cells (Supplementary Fig. 6E). However, due to limited sensitivity of snRNA-seq and low abundance detection of IL-1R in the dataset, IL-1R is probably expressed at a low level in other kidney cell types.

Notably, Candida infection in WT mice resulted in relatively minor transcriptional changes (~50 genes up or down), with endothelial cells and cell types localized in the loop of Henle, showing more inhibition of gene expression (Fig. 5B). In contrast, infection of Il1r1^-/- mice induced a massive upregulation of genes in every kidney cell type compared to infected WT and naïve Il1r1^-/- mice, in particular the cells in the proximal tubule (i.e., PT-S1 to S3) and in the loop of Henle (i.e., CTAL1/2), which showed upregulation of 400–500 genes (Figs. 5B, 6A). Genes upregulated in the infected Il1r1^-/- mice were highly overlapping between different kidney cell types, suggesting that in these mice, infection causes an upregulation of non-cell-specific gene programs. In contrast, downregulated genes upon infection were rather cell-specific (Fig. 5C).

To determine whether our observations of the transcriptional response could be secondary to kidney damage, we analyzed the kidney function over the course of infection. We collected blood and urine of infected Il1r1^-/- and control mice at 10 h, 24 h and 48 h p.i. (Fig. 5D, E). The analysis revealed high blood urea and macroalbuminuria (ACR > 300 mg/g) at 48 h p.i. in the Il1r1^-/- mice, indicating that they suffered from severe kidney damage at that time point. However, no signs of kidney damage were observed at 10 h p.i., and only slight microalbuminuria (ACR > 30 mg/g) was seen at 24 h p.i. in the Il1r1^-/- mice. Additionally, the fungal burden in the kidneys of Il1r1^-/- and control mice at 8 h p.i. is not different (supplementary Fig. 7A). These results confirm that the transcriptional phenotype we observe at 8 h p.i. is likely a direct consequence of the absence of IL-1R and the presence of C. albicans, and it is not secondary to kidney damage, or differences in fungal burden.

Dysregulated metabolism in kidney cells from C. albicans infected Il1r1-deficient mice

Analysis of the differentially expressed genes and pathways upon infection in Il1r1^-/- and in control mice using Metascape³⁷ uncovered an interesting transcriptional response in mice lacking IL-1R (Fig. 6A). While Candida infection of Il1r1-sufficient mice mainly results in downregulation of pathways such as respiratory electron transport and sodium transport in most cell types, infection of Il1r1-deficient mice results in a strong upregulation of aerobic respiration in all cell types, except for macrophages (Fig. 6A). Additionally, there is what seems to be a metabolic crisis, as indicated by the upregulation of ribosomal genes, aerobic respiration, carbon and amino-acid metabolism, glycolysis and gluconeogenesis, oxidative stress and redox pathway, fatty acid degradation and other (Fig. 6A, C supplementary Fig. 7B, C). These observations were consistent with the analysis of signature scores of KEGG metabolic pathways (Fig. 6C, supplementary Fig. 7C). Oxidative phosphorylation is one of the highest upregulated pathways. It is markedly up in all the kidney cell types except macrophages in infected Il1r1^-/- mice (Fig. 6C). A multitude of genes encoding for subunits of complexes I, III, IV, and V of the electron transport chain (ETC) were up in all kidney cells within 8 h after infection of Il1r1^-/- mice compared to controls (supplementary Fig. 7E). In addition, we see elevation in genes involved in glycolysis (Fig. 6D) as well as genes involved in targeting and import of proteins into the mitochondria, such as Timm50³⁸ and Tomm20³⁹; Prohibitin (Phb), which forms a complex on the mitochondrial membrane and functions as a protein stabilizing chaperon^40,41; and the mitochondrial citrate carrier Slc25a1 (Fig. 6E). These observations indicate an activation of mitochondrial activity and respiration.

Pathway activity analysis with PROGENy database (Fig. 6B) showed reduced activity of a few main pathways upon infection of Il1r1^-/- mice, such as NF-κB, Wnt, and PI3K. A reduction in NF-κB pathway activity is consistent with the absence of IL-1R and is observed to a significant extent in 11 cell types out of 18, suggesting the importance of IL-1R in most kidney cell types.

Interestingly, this analysis highlighted an increase in the hypoxia pathway for the majority of the cell types in the infected Il1r1^-/- mice (Fig. 6B). Transcription factor specific analysis using DoRothEA spotlighted increased HIF-1α, PPARα, Myc and Sp1 regulon activity in Il1r1^-/- kidney cells (Supplementary Fig. 7D), suggesting elevated transcriptional regulation of hypoxia-related target genes.

Under conditions of very low oxygen content in a tissue (hypoxia), cells increase glycolysis to promote survival. This metabolic switch is initiated by stabilization and nuclear translocation of hypoxia inducible factor 1 (HIF-1), which induces upregulation of several enzymes of glycolysis⁴². Indeed, all kidney cell types from Candida-infected Il1r1-deficient mice showed strikingly increased transcription of almost every enzyme involved in the breakdown of glucose to pyruvate including 6-phophofructokinase l (Pfkl), aldolase A, B and C (Aldoa, Aldob, Aldoc), triosephoshphate isomerase (Tpi1), glyceraldehyde 3-phosphate dehydrogenase (Gapdh), phosphoglycerate kinase (Pgk1), phosphoglycerate mutase (Pgam1), enolase (Eno1), and pyruvate kinase M (Pkm) (Fig. 6D). Moreover, there was a transcriptional increase of lactate dehydrogenase (Ldha) mediating the conversion of pyruvate to lactate and back. Notably, promotor and enhancer regions of the genes Pfkl, Aldoa, Pgk1, Eno1, and Ldha contain HIF-1 binding sites (HRE), suggesting that HIF directly upregulates them in response to hypoxia^43,44.

Interestingly, in parallel to the ramp-up of glycolysis genes, there was a significant increase in the transcription of pyruvate dehydrogenases a and b (Pdha1 and Pdhb) and dihydrolipoamide dehydrogenase (Dld), which together catalyze the conversion of pyruvate to acetyl-CoA and CO₂ in the mitochondria, thereby providing a substrate for the TCA cycle. The purpose of hypoxia-driven HIF-1α guided transcriptional activity is to reduce oxygen consumption by the cell via inhibition of oxidative phosphorylation and boosting of glycolysis for energy needs. In the absence of Il1r1^-/-, however, HIF-1α is activated but seems insufficient for inhibiting oxidative phosphorylation.

To assess the possibility that IL-1R intrinsically influences kidney cell metabolism, we used a human kidney podocyte cell line (differentiated AB8/13 cells^45,46). We performed an ATP assay on the Agilent Seahorse XF96 analyzer, which allows the precise quantification of the rate of adenosine triphosphate (ATP) production from glycolysis and mitochondria in live cells⁴⁷. Before analysis, we treated the cells overnight with either IL-1β or the IL-1R antagonist Anakinra (Fig. 7A, B). The assay revealed that podocytes increase the glycolytic source of ATP upon IL-1β treatment, while Anakinra treatment caused a significant increase of the mitochondrial (oxidative) source of ATP in the cells (Fig. 7C). Quantification of the mitochondrial ATP fraction over glycolytic ATP fraction demonstrated a significant effect of IL-1R signaling on the cell ATP production. In other words, while activation of IL-1R causes increased glycolytic ATP, blocking of IL-1R (as is happening in Il1r1^-/- mice) causes increased respiratory ATP.

These results demonstrate that IL-1R intrinsically influences kidney cell metabolism, specifically shifting away from respiratory ATP production. Respectively, in Il1r1^-/- mice the kidney cells seem to respond to C. albicans infection by increased oxidative respiration.

IL-1R1 protects from rapid kidney hypoxia upon C. albicans infection

The kidney is one of the best-perfused organs. However, it is not very efficient in extracting oxygen from the blood (~10–20% only from delivered oxygen)⁴⁸. This makes the kidney highly vulnerable to hypoxia. To confirm hypoxia in the kidney, we used Pimonidazole, which creates bonds with cellular macromolecules when the oxygen levels are under 1.3%. We injected Pimonidazole into Il1r1^-/- mice and Il1r1^+/- controls 24 h p.i. and visualized hypoxia in the kidneys. We used anti-pimonidazole and fluorescent secondary antibodies to visualize the Pimonidazole-positive areas. We then used a C. albicans-specific antibody to visualize the fungus. Since both antibodies have been generated in the same species, we could not co-stain the same slides. Thus, we stained subsequent slides (10μm apart) and approximated the co-localization of hypoxia and C. albicans (Fig. 7D). Consistent with the transcriptional analysis, the kidneys of Il1r1^-/- mice had significantly more hypoxic areas than the Il1r1^+/- controls (Fig. 7E).

Interestingly, we found co-localization of hypoxic regions and infection foci (Fig. 7D) more in the Il1r1^-/- mice than in the Il1r1^+/- controls. This observation suggests that C. albicans itself could contribute to the creation of hypoxia by overconsumption of oxygen. However, since IL-1R seems to prevent most of the hypoxia, as seen in Il1r1^+/- control mice, we believe that the kidney cells have a significant role in its exacerbation. We suggest that the metabolic response of the kidney cells to the infection in the absence of IL-1R exacerbates the depletion of oxygen in the infection foci and contributes to the creation of local hypoxia, which in turn activates HIF-1α. The HIF-1α activation, however, is not enough to combat the hypoxia.

Hypoxia promotes fungal invasiveness

C. albicans rapidly changes its morphology in response to environmental cues such as nutrient availability, temperature, CO₂ and O₂ levels. At higher temperatures (37 °C, compared to room temperature) C. albicans readily creates hyphae and upregulates virulence genes⁴⁹. This possibly happens when the fungi enter the host environment. CO₂ and oxygen availability have also been shown to affect C. albicans virulence and hyphae formation^50,51.

We asked whether the hypoxic environment in the kidneys of Il1r1^-/- mice during candidiasis can promote fungal hyphae formation. Since Pimonidazole staining is positive at oxygen levels below 1.3%, we decided to grow C. albicans in YPD agar in similar levels of hypoxia (1% oxygen) or normoxia in 37 °C, 5% CO2, and visualized the colonies. As predicted, while in normoxic conditions C. albicans did not create any hyphae, hypoxia promoted significant hyphae formation (Fig. 7F).

Since hyphae formation is crucial for C. albicans’ pathogenicity⁵², we suggest that the hypoxia that is created in the infection foci in the absence of IL-1R results in higher invasiveness of C. albicans, exacerbating the infection.