JNK1 negatively controls anti-fungal innate immunity by suppressing CD23 expression

JNK1 negatively regulates the host anti-fungal innate immune responses in vivo

To investigate the role of JNK in fungal infection response, bone marrow-derived macrophages (BMDM) were stimulated with yeast or hyphae form of C. albicans, and the fungal cell wall component Zymosan or α-mannan. All these stimuli effectively induced JNK1/2 phosphorylation (Fig. 1a). To further characterize JNK function in the host immune response against fungal infection, we intravenously infected JNK1 knockout (KO), JNK2 KO, and wild type (WT) control mice with a sub-lethal dose of C. albicans. We found that JNK1 KO mice were more resistant to fungal infection compared with JNK2 KO or WT mice (Fig. 1b). Consistently, the fungal load in the kidneys was significantly lower (Fig. 1c and Supplementary Fig. 1a). JNK1 KO mice also manifested reduced renal inflammation and reduced numbers of C. albicans colony forming units (CFU) in the kidney (Fig. 1d, e and Supplementary Fig. 1b, c). The above findings were confirmed using two different doses of fungal infections in JNK1 KO and littermate heterozygous mice (Supplementary Fig. 1d, e). These data suggest that deficiency of JNK1 but not JNK2 in the host leads to a boost in antifungal immunity.

Myeloid lineage cells, including macrophage and DC, are key effector cells against fungal during the first few days after initial infection^3,26. JNK1 has been reported to be expressed on all of different tissue compartments²². To investigate the cellular basis of the JNK1-related antifungal effect, we generated bone marrow (BM)-chimeric mice by reconstituting lethally irradiated WT mice with syngeneic JNK1 KO BM, or JNK1 KO mice with WT BM. Loss of JNK1 in hematopoietic cells showed the similar phenotype with total JNK1 deficiency in response to C. albicans infection (Fig. 1f and Supplementary Fig. 1f, KO-WT vs. KO-KO). Hematopoietic cells contain both innate and adaptive immune cells. To determine the contribution of JNK1 in the adaptive immune system to the observed phenotype, we generated JNK1/Rag1-double knockout (KO) mice by crossing JNK1 KO mice with Rag1 KO mice. Notably, JNK1 deficiency in the absence of adaptive immune cells closely resembled the phenotype of whole-body JNK1 KO mice in response to C. albicans infection (Fig. 1g). Together, these data indicate that JNK1 negatively regulates the anti-fungal innate immune response in vivo.

JNK1 deficiency induce higher CD23 expression and nitric oxide production in vitro

Pro-inflammatory cytokines, such as IL-6 and TNFα, are reported to be key factors for the host innate immune system to fight against fungi^5,26. However, we did not detect significant changes in the levels of these pro-inflammatory cytokines post-infection in JNK1 KO mice compared with WT mice using Multiplex cytokine array analysis (Supplementary Fig. 2a), which we confirmed in vitro using BMDM stimulated with C. albicans (Supplementary Fig. 2b). Activation of p38 and ERK was also comparable in WT and JNK1 KO cells (Supplementary Fig. 2c). To identify gene(s) that may be responsible for resistance to fungal infection in JNK1 KO mice, we performed RNA-Seq analysis with WT and JNK1 KO BMDM after yeast form C. albicans stimulation (Supplementary Fig. 3a). Notably, we found a novel C-type lectin gene, Fcer2a, was significantly upregulated in stimulated JNK1 KO cells compared with WT control cells (Fig. 2a).

Fcer2a encodes the protein CD23 that was identified as the low affinity receptor for IgE ²⁷, and is also a C-type lectin receptor. It is located on chromosome 19p131 and forms a gene cluster with Cd209a, which encodes DC-SIGN²⁸. To validate the RNA-Seq data, we analyzed expression of a series of C-type lectin receptor genes by quantitative real time PCR, and found only Fcer2a was significantly induced in JNK1 KO BMDM cells (Supplementary Fig. 3b). We confirmed CD23 protein upregulation on the cell surface upon C. albicans stimulation by FACS (Fig. 2b). Since CD23 is also a C-type lectin receptor, we examined whether it can recognize surface components of fungi directly by performing a cellular binding assay. Indeed, we found that CD23 can bind both yeast and hyphae forms of C. albicans (Supplementary Fig. 4a). Since the C. albicans cell wall is mainly composed of α-mannan and β-glucan, we perform Atomic force microscopy (AFM) and ligand-binding assay to detect whether CD23-expressing cells or CD23 protein can bind with α-mannan and β-glucan directly. Indeed, we found that both cell surface expressed CD23 and purified soluble CD23 protein could effectively bind to α-mannan and β-glucan/Curdlan similarly to the known CLR receptors Dectin-1 and Dectin-3 (Fig. 2c, d and Supplementary Fig. 4b).

It has been reported that infection of human monocyte-derived macrophages with Mycobacterium avium induced membrane expression of CD23 ²⁹, and CD23 induces Nitric Oxide-Synthase (NOS) activity in monocytes ^30,31. Furthermore, CD23 and Nitric Oxide (NO) is involved in the process killing Leishmania and Mycobacterium avium by human macrophages^29,32,33. Notably, Nos2, which encodes the inducible NOS (iNOS), is highly differentially expressed between WT and JNK1 KO cells following C. albicans stimulation (Fig. 2a). Consistently, we found both α-mannan and β-glucan could stimulate CD23-overexpressing monocyte cell line, Raw264.7 cells, to produce more NO (Supplementary Fig. 4c). Importantly, JNK1 KO BMDM cells produced about three fold increased soluble NO in the supernatants compared with WT cells following C. albicans infection (Fig. 2e), which is consistent with the significant induction of Nos2 mRNA in JNK1 KO cells (Supplementary Fig. 5a). Furthermore, this elevated NO production in JNK1 KO cells is CD23 dependent, since JNK1 KO BMDM secreted much less NO upon treatment with p30A (Fig. 2f), a CD23-blocking peptide that effectively induce CD23 endocytosis from the cytoplasmic membrane³⁴. p30A treatment decreased both the infected WT and JNK1 KO BMDM cell surface CD23 expression efficiently (Supplementary Fig. 5b), and treated JNK1 KO BMDMs could not kill C. albicans as efficiently as those treated with a control peptide (Supplementary Fig. 5c). Using of a NO synthesis inhibitor, L-NAME, to block NO production, also abolished the in vitro killing by JNK1 KO BMDM cells (Supplementary Fig. 5d, e). Together, these results show that JNK1 deficiency leads to the up-regulation of CD23 and higher NO production in fungal infected cells, which contribute to the enhanced fungal killing effect in the absence of JNK1.

Elevated CD23 and Nitric Oxide is essential for JNK1-related antifungal response in vivo

To confirm the function of CD23 and NO in vivo, we collected kidney tissues from C. albicans-infected mice, and found that Nos2 and Fcer2a mRNA expression was significantly higher in JNK1 KO mice (Fig. 3a and Supplementary Fig. 6a). CD23 protein level in the infected kidney was also higher in JNK1 KO mice (Fig. 3b), and p30A treatment could efficiently inhibit CD23 expression in mice (Supplementary Fig. 6b). To evaluate whether CD23 is responsible for the fungal protective effect in JNK1 KO mice, both blocking peptide and CD23 KO mice were used. Fungal burden of p30A-treated JNK1 KO mice was much higher than control peptide treated mice (Fig. 3c and Supplementary Fig. 6c), which is consistent with our in vitro data. JNK1/CD23-double knockout mice were generated by crossing CD23 KO mice with JNK1 KO mice, and they were infected with C. albicans. In accordance with the p30A blocking data, the higher survival rate in JNK1-single KO mice was impaired in JNK1/CD23-double KO mice (Fig. 3d and Supplementary Fig. 6d). Interestingly, the JNK1/CD23-double KO mice died even faster than WT and CD23-single KO mice for unknown reasons.

Since iNOS expression is significantly elevated in response to fungal infection in JNK1 KO mice, we treated C. albicans-infected JNK1 KO mice with NOS1 specific inhibitor Spermidine and iNOS (NOS2) specific inhibitor SMT (Supplementary Fig. 6e). We found that SMT, but not Spermidine, significantly reduced survival rate of these mice (Fig. 3e). This iNOS specific effect was confirmed by using another inhibitor, L-NAME, which also reduced the survival rate of the infected JNK1 KO mice (Fig. 3f). These data indicate that CD23 induction and CD23-dependent elevation of nitric oxide are responsible for the enhanced anti-fungal immune response in JNK1 KO mice.

Monocytes/macrophages, dendritic cells, and neutrophils are all reported to be important for the anti-fungal immune response^18,19,35. We examined which type of cells show increased expression of CD23 and iNOS in C. albicans-infected mice, and found that Fcer2a and Nos2 expression were significantly induced in bone marrow-derived macrophage and DC, but not in neutrophils in JNK1 KO mice after fungal infection (Supplementary Figure 7a). The neutrophil number decreased in JNK1 KO mice after fungal infection (Supplementary Figure 7b, c), which may be due to the lower fungal burden and reduced inflammatory response seven days after initial infection. Notably, peritoneal macrophages of CD23 KO mice express significantly less iNOS mRNA then control littermates (Supplementary Figure 7d). The fungal load was much higher in the kidneys of CD23 KO mice compared with WT mice after C. albicans infection (Supplementary Figure 7e). ROS production and phagocytosis are also important processes in anti-fungal responses^19,36,37, and NO can interact with ROS and form peroxynitrite to efficiently kill fungi^26,38. We found that JNK1-deficient cells produced higher amounts of ROS upon C. albicans infection (Supplementary Figure 8a), but the phagocytosis ability between wildtype and JNK1-deficient cells were comparable (Supplementary Figure 8b, c).

JNK1 negatively regulates CD23 expression through Dectin-1-induced NFAT activation

We next investigated how JNK1 deficiency results in upregulated CD23 expression following fungal infection. It has been shown that JNK1-deficient T cells exhibit elevated NFAT activation upon stimulation³⁹, and NFAT bound to the CD23 promoter regulates its expression^40,41. Interestingly, C. albicans stimulation can also trigger NFAT activation in macrophages and dendritic cells^42,43. To determine the molecular mechanism by which CD23 expression is up-regulated following fungal infection, BMDM cells were stimulated with C. albicans and NFAT activation was examined. More NFATc1 translocated to the nucleus in JNK1 KO cells compared with WT cells upon C. albicans stimulation, although p65 nuclear translocation was comparable (Fig. 4a, b, c). Notably, JNK phosphorylation and NFAT activation was dependent on Dectin-1, since Dectin-1 KO BMDMs showed significantly reduced NFAT activation and JNK phosphorylation upon C. albicans yeast or Zymosan stimulation, whereas NF-κB subunit p65 translocation was not affected upon either α-mannan or Zymosan stimulation (Fig. 4d, e, and Supplementary Figure 9a, b, c, d). This result is consistent with the previous finding that Dectin-1 stimulation by C. albicans yeast or its ligand, Zymosan, triggers NFAT activation in macrophages⁴².

To further determine whether CD23 induction is through a NFAT-dependent mechanism, we used 11R-VIVIT to inhibit NFAT activation, and then examined the expression level of CD23 in BMDM cells following fungal infection. We found that CD23 expression in JNK1 KO cells was significantly reduced upon 11R-VIVIT and C. albicans treatment (Fig. 4f). However, TPCA-1, which inhibits the NF-?B activation through IKK, hardly influences the CD23 induction. Since two putative NFAT-binding sites (-1368, and -714) were found in the Fcer2a promoter, we performed a ChIP assay and found a high level of binding of NFATc1 to the Fcer2a promoter in JNK1 KO cells following C. albicans stimulation (Fig. 4g). To determine whether the elevated nuclear translocation of NFATc1 dictates transcription of Fcer2a genes, we cloned the genomic promoter (about 1500bp) of Fcer2a and performed a luciferase reporter assay. We examined luciferase reporter activity, and found that the CD23 activation could be substantially induced when both potential NFAT binding sites were present, while adding the NFAT inhibitor, 11R-VIVIT, completely diminished this activation (Fig. 4h, -1500). Deletion of the -1368 NFAT site severely impaired CD23 promoter activation, but additional deletion of the -714 NFAT site did not further reduce the promoter activity (Fig. 4h, -750 and -480). These data indicate that NFATc1 regulates CD23 expression through directly binding to the -1368 binding site in CD23 promoter.

JNK inhibitors promote the anti-fungal immune response in vivo and in vitro

SP600125 is one of the most extensively used ATP-competitive JNK inhibitors and several studies using SP600125 have demonstrated its potential for therapeutic intervention by directly inhibiting JNK ^24,25,44. To investigate whether JNK inhibitors can efficiently boost the anti-fungal innate immune response, wild-type mice were infected with C. albicans and then treated with two different doses of SP600125. Administration of both doses of SP600125 increased the survival rate compared with the control mice (Fig. 5a). Consistently, JNK inhibitor treatment also significantly reduced the fungal burden (Fig. 5b), and increased NOS2 and CD23 expression in the kidney of infected mice (Fig. 5c, d). Moreover, SP600125 specifically inhibited JNK activation and had no additional effect on phosphorylation of other MAPK kinases (p38 and ERK) (Supplementary Fig. 10a, b). To confirm the anti-fungal effect of the JNK inhibitor in vitro, BMDM cells were pretreated with SP600125 and then stimulated with C. albicans. Cell surface staining showed that significantly higher CD23 expression was induced upon JNK inhibition (Supplementary Fig. 10c). Consistently, SP600125-treated cells produced more nitric oxide (Supplementary Fig. 10d) and more efficiently killed fungi (Supplementary Fig. 10e).

To further confirm the anti-fungal effect of JNK inhibitor in vivo, we use another JNK inhibitor, JNK-IN-8 ⁴⁵, which specially targets JNK activation (Supplementary Fig. 10a, b), to treat the clinical C. albicans strain sc5314-infected mice. Strikingly, JNK-IN-8 shows even better anti-fungal protective effect than SP600125 (Fig. 5e). These results demonstrate that JNK inhibitors can promote anti-fungal immune responses both in vivo and in vitro.

JNK1 is a potential therapeutic target for antifungal infection

To investigate whether JNK inhibitor has a therapeutic benefit in fungal infection, we infected wildtype mice with C. albicans by intravenous injection, and 24 hours later, injected JNK-IN-8 via the tail vein. We found that a significantly higher fungal burden in kidneys of solvent-treated mice than those of mice treated with the JNK inhibitor (Fig. 5f). To further confirm the therapeutic effect of the JNK inhibitor, we first intravenously infected wildtype mice with C. albicans, and 24 hours later, treated the mice intraperitoneally with SP600125 or control solvent daily for four continuous days, and monitored the mortality of these mice. We found that most of the control mice died within ten days of infection, but mice treated with JNK inhibitor were significantly more resistant to infection (Fig. 5g). These results indicate that JNK inhibitors may serve as potent therapeutic drugs for enhancing host innate immunity against fungal infection.

To test the relevance of our results in mice to humans, we infected the human monocytic cell line THP-1 and human PBMC-derived monocytes with C. albicans. Both THP-1 and human primary cells treated with JNK inhibitor showed significant upregulation of CD23 and iNOS, and killed fungi more efficiently in vitro (Fig. 6a, b). Thus, we conclude that JNK1 deficiency leads to the up-regulation of C-type lectin receptor CD23 through Dectin-1 dependent NFAT hyper-activation, and the elevated CD23 expression induces the cells to produce more nitric oxide by directly recognizing the fungal cell wall components, which will efficiently eliminate the fungal infection (Fig. 6c). Therefore, the JNK inhibitor enhances antifungal immunity and may serve as a potential drug to protect from lethal C. albicans sepsis in clinic.

Antibodies and reagents

Antibodies against phospho-p38 (4631), phospho-ERK (9101), phospho-JNK (9251), and total p38 (9212), total JNK (9252) were purchased from Cell Signaling Technology; antibodies against p65 (sc-8008), PCNA (proliferating cell nuclear antigen) (sc-56), NFAT-c1 (sc-7294), NFAT-c2 (sc-7296), CD23 (sc-271900), ERK (sc-154), I?Bα (sc-371), and tubulin (sc-8035) were from Santa Cruz Biotechnology. Dectin-1 and Dectin-3 monoclonal antibody was generated by using the extracellular domain of Dectin-1or Dectin-3 as immunogens, which was described before¹¹. TPCA-1 (T1452) and α-mannan (M3640) were purchased from Sigma. Zymosan (#tlrl-zyn) was got from Invivogen. NFAT inhibitor 11R-VIVIT (#13855), JNK inhibitor SP600125 (#10010466), NO inhibitor L-NAME (#80210) were got from Cayman chemical. JNK inhibitor JNK-IN-8 (#s4901) was purchased from Selleck. NOS1 inhibitor Spermidine (#s0010), iNOS inhibitor S-Methylisothiourea hemisulfate salt (SMT, #s0008) were from Beyotime Biotechnology. PerCP-Cyanine5.5 anti-mouse CD11b antibody (#45-0112-82) was purchased from eBioscience, FITC anti-mouse F4/80 (#123108) and PE anti-mouse CD23 (#101608) antibodies were from Biolegend, APC-Cy7 anti-mouse CD11b was from BD Pharmingen. FITC-conjugated goat anti-mouse IgG (H+L) secondary antibody was purchased from Invitrogen (#62-6511). CD23 blocking peptide (p30A, FHENWPS) and control peptide (pCtr, SFNYNYA) were synthetized by Peptide 2.0 Inc. (Chantilly, VA, USA) and GL Biochem Ltd. (Shanghai, China).

Expression plasmids

Mouse CD23, Dectin-1 and Dectin-3 were amplified by PCR with cDNA of mouse BMDM as template. PCR amplifying fragment was inserted into a lentivirus vector, pRV3, between the ClaI and EcoRI sites. Extracellular domain of CD23, Dectin-1 and Dectin-3 were amplified by PCR with mouse BMDM as template. PCR amplifying fragments were inserted into an expression vector, pET28a, between the EcoRI and XhoI sites. The PCR primers used were listed in Supplementary Table 1.

Mice

JNK1 knockout mice, JNK2-knockout mice, and CD23-knockout mice were purchased from the Jackson Laboratory. To obtain JNK1^−/−/CD23^−/− or JNK1^−/−/Rag^−/− mice, JNK1^−/− mice were paired with CD23^−/− or Rag^−/− mice, and subsequent intercross of their offspring led to the generation of the double knock-out mice. Wildtype C57BL/6 mice were purchased from Jackson Laboratory and breed in the facility. All mice were housed in the specific pathogen-free animal facility at MD Anderson Cancer Center and Tsinghua University. In all experiments described here, sex- and age-matched mice were used. All animal experiments were performed in compliance with institutional guidelines and according to the protocol approved by the Institutional Animal Care and Use Committee of The University of Texas MD Anderson Cancer Center and Tsinghua University.

Bone marrow-chimeric mice

Six-week-old recipient mice were lethally irradiated by X-ray (550 rad × 2), and then intravenously transferred with 5×10⁶ bone-marrow leukocytes from indicated donors. Chimaeras were used for further experiments 7–8 weeks after the initial reconstitution.

Murine Systemic Candidiasis Model

For in vivo C. albicans infection, mice were injected via lateral tail veins with 200 ul of a suspension containing different doses of C. albicans (SC5314) in sterile PBS (Hyclone). Mouse survival rates were monitored following infection. Fungal loading was assessed by plating a series of diluted solutions of homogenized kidneys on the YPD plate.

Bone marrow-derived macrophage (BMDM) preparation

Primary cultures of BMDMs from indicated mice were prepared as previously described¹¹. Briefly, bone marrow cells were harvested from the femurs and tibias of mice. Erythrocytes were removed from cells by using a hypotonic solution. Cells were cultured for 7 days in Dulbecco's modified Eagle medium containing 20% fetal bovine serum, 55 µM β-mercaptoethanol, streptomycin (100 µg/ml), penicillin (100 U/ml), and 30% conditioned medium from L929 cells expressing macrophage colony stimulating factor. After 6–7 days of culturing, flow cytometry analysis indicated that the harvested cell population contained above 97% CD11b⁺ F4/80⁺cells.

Isolation of neutrophils, monocytes, macrophages, and dendritic cells

Bone Marrow and splenic neutrophils, monocytes, macrophages, and dendritic cells were purified by CD45 MACS beads (Miltenyi, #130-052-301) first, and then FACS-sorting labeling with antibodies specific for CD11b (BD, #557396), CD11c (BD, #550261), Ly-6G (eBioscience, #48-5931-82), and Ly-6C (BD, #561237). Purified neutrophils, monocytes, macrophages, and dendritic cells were cultured in RPMI medium.

Immunoblotting assay

BMDMs were stimulated and lysed in lysis buffer (150 mM NaCl, 50 mM HEPES (pH 7.4), 1 mM EDTA, 1% Nonidet P-40, protease inhibitors). Nuclear extracts or total cell lysates were subjected to SDS-PAGE and then blotted using indicated antibodies.

Quantitative PCR

Total RNA was isolated using TRIZOL (Invitrogen) and reverse transcribed using SuperScriptIII (Invitrogen). Quantitative PCR was performed in triplicates using Power SYBR Green PCR Master Mix (Applied Biosystems). The amounts of transcript were normalized to GAPDH. Melting curves were run to ensure amplification of a single product. Primers used were listed in Supplementary Table 1.

RNA-seq

The RNA-sequencing was performed by Novogene (Beijing, China). Sequencing libraries were generated using NEBNext Ultra™ Directional RNA Library Prep Kit for Illumina; clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina); sequencing was done on an Illumina Hiseq platform and paired-end reads were generated. The filtered reads were aligned to the mouse most recent genome reference mm10 by using TopHat (version 2.1.0). After the alignment, the BAM files of each individual alignment were used to analyze genes differential expression by using Cufflinks (version 2.2.1). The heatmaps were generated by using gplots package in R (version 3.2.3). The RNA-Seq data is being deposited in NCBI Gene Expression Omnibus and the accession GEO number is GSE83265.

Chromatin immunoprecipitation assay

The Chromatin immunoprecipitation (ChIP) assay was performed with a ChIP kit (Active Motif, Cat#53009) as previously described before⁴⁹. The antibodies used for ChIP are as follows: NF?B-p65 (sc-109), NFATc1 (sc-7294). The resulting DNA was analyzed by real-time PCR. The PCR primers used were listed in Supplementary Table 1.

Cytokine Measurement

Cytokine panel in the mouse serum were detected with Milliplex Map kit (Millipore, #MCYTOMAG-70K). All assays were performed according to the manufacturer’s protocols, and the MFIs were detected by the Luminex 200 system and were analyzed with Bio-Plex software (Bio-Rad). TNF-α, IL-6, IL-1β levels in the supernatant of the cells were measured with Ready-SET-GO enzyme-linked immunosorbent assay (ELISA) kits (eBioscience). All samples were measured in triplicate according to the manufacturer’s protocol.

Luciferase reporter assay

Luciferase assay were performed as described previously⁴⁹. Briefly, 293T cells were transfected with 200ng of luciferase (firefly) reporter plasmid pGL3 containing the different length of CD23 promoter together with 4ng of EF1α promoter-dependent Renilla luciferase reporter. The transfected cells were cultured in DMEM containing 10% FBS for about 24 hours. These cells were treated with PMA (200ng/ml)/ Inomycine (500ng/ml) for another 6 hours. NFAT inhibitor 11R-VIVIT (10uM) was added 20 minutes prior to PMA/Inomycine stimulation. Resulted cells were harvested and lysed. Firefly and renilla luciferase activity was measured with a Dual-Luciferase Reporter system (Promega) and renilla luciferase was used to normalize transfection efficiency and luciferase activity.

Immunofluorescence staining

Indicated cells were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.25% Triton X-100 and blocked with 10% goat serum. NFAT-c1 (Sant Cruz, sc-109) and p65 (Sant Cruz, sc-7294) were used as the first antibodies, Cy3-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, 715-166-150) and Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, 711-546-152) were used as the secondary antibodies. DAPI (Beyotime Biotech) was counterstained to label the cell nuclear. Fluorescence was detected using a Zeiss LSM780 confocal laser scanning microscope.

Histopathology

For histopathology analyses, kidneys were fixed in 4% Paraformaldehyde solution, processed according to standard procedures, embedded in paraffin, and sectioned. 5um thick sections were stained with haematoxylin and eosin (H&E), periodic-acid-Schiff (PAS), Gomori Methenamine Silver (GMS), or anti-Ly-6G (MDL, MD6477-020). Stained slides were scanned using a Microscope (OLYMPUS, IX73) and evaluated for severity of inflammation and intra-lesional fungal burden as described previously¹⁹. Renal inflammation was scored based upon H&E and PAS stainings (proportion of renal parenchyma and/or pelvis involved by tubulointerstitial nephritis and/or pyelonephritis) as not significant (score 0), less than 10% (score 1), 10–25% (score 2), 25–50% (score 3), or greater than 50% (score 4). The intra-lesional fungal burden was based upon PAS and GMS stainings as not significant (score 0), scant presence in less than 10% of inflammatory foci (score 1), mild-to-moderate presence in 10–25% of inflammatory foci (score 2), moderate-to-significant presence in 25–50% of inflammatory foci (score 3), or significant presence in more than 50% of inflammatory foci (score 4). ImageJ software was applied to evaluate the extent of infiltration by Ly-6G+ cells in affected kidneys.

Detection of reactive oxygen (ROS) production

The production of ROS was assayed as described with minor modification³⁶. Briefly, 2 × 10⁵ BMDM cells were washed with PBS twice and re-suspended in DMEM containing 10uM DCFH-DA. Cells were incubated at 37 °C for 30 min, after incubation cells were washed with DMEM five times to remove the nonspecific binding. Then cells infected with heated killed C. albicans at MOI = 10 for different time points, respectively. The relative amount of ROS generated was detected every 10 min by flow cytometry measuring the mean fluorescence intensity (MFI) in FL1 channel.

Phagocytosis of C. albicans and in vitro fungal killing assay

The phagocytosis assay was performed as described³⁷ with minor modification. Briefly, 5 × 10⁵ FACS-sorted cells were dispensed into 24-well plate and incubated at 37 °C for 2 hours to create a monolayer of phagocytes, followed by gently washing with culture medium to remove non-adherent cells. Resulting cells were incubated with 5×10⁵ CFU C. albicans (MOI=1) at 37 °C for 15 minutes, supernatants were collected and monolayers were washed gently with DMEM to remove uningested microorganisms. The supernatant and well washings, containing the non-phagocytized C. albicans were combined and plated in serial dilutions on YPD agar plates incubated overnight at 30 °C. The percentage of phagocytized microorganisms was defined as (1- (number of uningested CFU / CFU at the start of incubation)) × 100. For phagocytosis of C. albicans by BMDM cells, GFP-labelled C. albicans yeast was used⁵⁰. BMDM cells were stained with APC-labelled CD11b antibody (Biolegend, #101211), and then co-cultured with UV crosslinked GFP-labelled C. albicans (MOI=5) for 1 hour at 37 °C. Adherent fungal cells were quenched with trypan blue, and gated CD11b⁺ cells phagocytosis rate was determined by flow cytometry with GFP positive. In vitro fungal killing assay was performed as described before with minor modification¹⁴. Briefly, cells were allowed to interact for 30 min at 30 °C with live C. albicans (Ratio: 0.4 yeast per macrophage). Unbound particles were removed and cells were returned to the incubator for 4 hour to allow fungal killing. Control plates were kept at 4 °C to provide a measure of live fungi in the wells. After incubation, medium was removed and cells were lysed by incubation for 5 min at 25 °C with water at pH 11. Lysis buffer was neutralized with excess PBS and CFU was determined by plating on YPD agar incubated overnight at 30 °C.

Nitric Oxide Concentration Assay

Indicated cell culture supernatants were collected and Nitrite concentration was measured by a Nitric Oxide Assay Kit (Cat#S0021) from Beyotime Biotechnology (Shanghai, China) based on the Griess reaction. All samples were measured in triplicate according to the manufacturer’s protocol.

Ligand binding assay

Ligand binding assay was performed as previously described¹¹. In brief, ELISA plates were coated with α-mannan or β-glucan (40 mg/ml) overnight and then added with 100 ul/well renatured protein of recombinant protein at indicated concentrations. Bound proteins were detected by their respective mouse monoclonal antibodies following by HRP-conjugated goat anti-mouse IgG secondary antibody (ESAYBIO, Beijing. #BE0102). Tetramethylbenzidine (TMB) substrate solution from eBioscience ELISA kit was used and the reaction was stopped by 2N H₂SO₄. OD450 was read on a SpectraMax Plus 384 Microplate Reader.

Cellular binding assay

The cellular binding assay was performed as described with minor modification^14,51. Briefly, RAW264.7 cells stably expressing Flag-tagged CD23, Dectin-1 or Dectin-3 were co-cultured with GFP labelled yeast (MOI=5) or hyphae (MOI=0.1) form of C. albicans for 10 minutes, and uncombined yeast (for yeast form binding) or Raw cells (for hyphae form binding) were removed by four times washing with medium. Then cells were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.25% Triton X-100 and blocked with 10% goat serum. Flag (Abmart, #M20008) were used as the first antibodies, Cy3-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, 715-166-150) were used as the secondary antibodies. DAPI (Beyotime Biotech) was counterstained to label the cell nuclear. Fluorescence was detected using a Zeiss LSM780 confocal laser scanning microscope.

Atomic force microscopy (AFM)

The experiments were performed as described before⁵² using JPK Cellhesion unit (JPK, Berlin, Germany) with minor modification. (Set point: 0.5 nN, pulling length: 50um, delay mode: constant force, contact time: 5s). Probes (Nanoworld, #ARROW-TL-50) were pre-coated with α-mannan (20 mg/ml) or Curdlan (10 mg/ml) for overnight. NIH-3T3 cells stably expressing CD23, Dectin-1, Dectin-3, or control vector were assayed by the pre-treated probe. The max force of each touch was normalized and analyzed using JPK image processing software.

Isolation of human monocyte-derived monocytes (MDM)

Human MDMs were generated as previously described with minor modification¹⁸. In brief, peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated from heparinized blood on Lymphoprep™ (Stemcell, #07851), a Ficoll-sodium based Density Gradient Medium. Monocytes were then separated by the Monocyte Isolation Kit II (Miltenyi Biotec, #130-091-153) and cultured in RPMI 1640 medium at 37 °C. The protocol was approved by the Ethics Committee of Tsinghua University.

Statistical analysis

All values in the paper are given as mean ± SEM, unless stated otherwise. All in vitro experiments were reproduced at least 3 independent times and all in vivo experiments were reproduced more than twice unless stated otherwise. Statistical significance was calculated by Two-tailed unpaired t test, Multiple t test, or Log-rank (Mantel-Cox) test using GraphPad Prism software. Statistical significance was set based on the P value. n.s. P > 0.05, *P < 0.05, ** P < 0.01, *** P < 0.001.