key: cord-0884378-ep6rw9y9
authors: Dai, Chenyang; Me, Rao; Gao, Nan; Su, Guanyu; Wu, Xinyi; Yu, Fu-Shin X.
title: Role of IL-36γ/IL-36R Signaling in Corneal Innate Defense Against Candida albicans Keratitis
date: 2021-05-10
journal: Invest Ophthalmol Vis Sci
DOI: 10.1167/iovs.62.6.10
sha: 73cd67fdc21132351364bfc07f33da6f7e573f18
doc_id: 884378
cord_uid: ep6rw9y9

PURPOSE: Interleukin (IL)-36 cytokines have been shown to play either beneficial or detrimental roles in the infection of mucosal tissues in a pathogen-dependent manner, but their involvement in fungal keratitis remains elusive. We herein investigated their expression and function in mediating corneal innate immunity against Candida albicans infection. METHODS: Gene expression in mouse corneas with or without C. albicans infection was determined by regular RT- and real-time (q)-PCR, Western blot analysis, ELISA or proteome profile assay. The severity of C. albicans keratitis was assessed using clinical scoring, bacterial counting, and myeloperoxidase (MPO) activity as an indicator of neutrophil infiltration. IL36R knockout mice and IL-33-specific siRNA were used to assess the involvement IL-33 signaling in C. albicans–infected corneas. B6 CD11c–DTR mice and clodronate liposomes were used to define the involvement of dendritic cells (DCs) and macrophages in IL-36R signaling and C. albicans keratitis, respectively. RESULTS: IL-36γ were up-regulated in C57BL6 mouse corneas in response to C. albicans infection. IL-36 receptor-deficient mice display increased severity of keratitis, with a higher fungal load, MPO, and IL-1β levels, and lower soluble sIL-1Ra and calprotectin levels. Exogenous IL-36γ prevented fungal keratitis pathogenesis with lower fungal load and MPO activity, higher expression of sIL-1Ra and calprotectin, and lower expression of IL-1β, at mRNA or protein levels. Protein array analysis revealed that the expression of IL-33 and REG3G were related to IL-36/IL36R signaling, and siRNA downregulation of IL-33 increased the severity of C. albicans keratitis. Depletion of dendritic cells or macrophages resulted in severe C. albicans keratitis and yet exhibited minimal effects on exogenous IL-36γ-induced protection against C. albicans infection in B6 mouse corneas. CONCLUSIONS: IL-36/IL36R signaling plays a protective role in fungal keratitis by promoting AMP expression and by suppressing fungal infection–induced expression of proinflammatory cytokines in a dendritic cell- and macrophage-independent manner.

F ungal keratitis is an infectious keratitis that has replaced bacterial keratitis in some regions as the leading cause of corneal blindness. 1, 2 In developed countries, the main cause of fungal keratitis is the widespread use of contact lenses, with Candida albicans (CA) as a major pathogen. In recent years, with the abuse of antibiotics and glucocorticoids, the incidence of fungal keratitis has been increasing. [3] [4] [5] [6] Hence, studies aimed at understanding the pathogenesis of fungal keratitis are of great significance for the development of mechanism-based therapies to treat fungal keratitis.

The IL-36 family of cytokines are new members of the IL-1 superfamily and consists of three biologically similar agonists, IL-36α, IL-36β, and IL-36γ , and their antagonist IL-36Ra, which is similar to IL-1Ra. 7-10 IL-36 cytokines are expressed in epithelial barriers throughout the body, including skin, lung, gastrointestinal tract, and cornea. 11 These cytokines regulate the balance between proinflammatory and anti-inflammatory cytokine production at these tissue sites. 9, 11 IL-36 agonists exert their effects by binding to the specific receptor IL-36R (IL-1 Receptor-Like 2, encoded by IL1RL2 gene) dimerizing with IL-1RAcP (IL-1 receptor accessory protein, encoded by IL1RAP gene), with IL-36Ra or IL-38 as antagonists. 12, 13 In addition to mucosal epithelia cells, IL-36R is constitutively expressed in dendritic cells and macrophages, two cell populations that reside in naïve corneas, and this expression increases in response to injury or infection. 9, 14 Mutations in IL-36Ra (encoded by IL36RN) are associated with the severe episodic inflammatory skin disease known as generalized pustular psoriasis. 15, 16 IL-38 is another antagonist of both IL-36R and IL-1R. Dysfunction of IL-38 expression is implicated in the pathogenesis of various autoimmune diseases such as rheumatoid arthritis, psoriasis, and systemic lupus erythematosus. 17 Moreover, a significant increase in circulating and tissue levels of IL-36α was found to be associated with primary Sjogren's syndrome. 18 Collectively, these studies suggest an overall role of the IL-36 gene as-GGGAAAGCCACTGATTCAAA S100A8: s-TGCGATGGTGATAAAAGTGG as-GGCCAGAAGCTCTGCTACTC S100A9 s-GTGCGAAAAGATCTGCAAAA as-TCAGCTGCTTGTCTGCATTT β-actin: s-GACGGCCAGGTCATCACTATTG as-TCAGCTGCTTGTCTGCATTT Results are normalized with β-actin and presented as fold increase (mean ± SD) relative to the control, naïve corneas, set as a value of 1. Stars on top of columns are P value results: *P < 0.05, ***P < 0.001 determined by Student s t-test, N = 3. Western blotting assessing the expression of IL-36γ (C) and IL-36R (D) at indicated times with naïve corneas as the control. The band intensity of IL-36γ (C') and IL-36R (D') were quantitated by densitometric analysis and the results were presented as the ratio of IL-36γ or IL-36R versus β-actin, which served as protein loading controls. P values were generated using unpaired Student's t-test o of naïve versus 6, 9, 18, or 24 hpi, **P < 0.01, **P < 0.01, n = 3.

family as proinflammatory cytokines in the pathogenesis of autoimmune diseases. Because the deficiency of IL-36R antagonist appears to be associated with various autoimmune diseases, 15, 17, 19, 20 the underlying mechanism might be a dysfunction in controlling the basal activity of IL-36R signaling. This was supported by the finding that, unlike IL-1, IL-36 cytokines have basal expression in unchallenged tissues and cells. 11, 21 Infection has been shown to induce rapid upregulation of IL-36 cytokines in a Toll-like receptor (TLR)dependent manner. 22, 23 IL-36/IL-36R signaling may play a protective or detrimental role in a tissue and pathogendependent manner. 10, [24] [25] [26] [27] [28] [29] We recently reported that soluble IL-1Ra and IL-36Ra have opposing effects on Pseudomonas aeruginosa (PA) keratitis, with IL-1Ra improving and IL-36Ra worsening the outcome of PA keratitis. 11 Our data showed that the IL-1 and IL-36 families have opposing effects in mediating innate immunity FIGURE 2. IL-36R deficiency increases the severity of CA keratitis. The centers of WT and IL-36R −/− mouse corneas were gently scratched and inoculated with 1.0 × 10 5 cfu of CA. (A) Eyes were photographed and clinically scored at 1 and 3 dpi. The clinical scores were analyzed with nonparametric Mann-Whitney U test (N = 5) and presented as inserts (low right corner). ***P < 0.001, n = 6. (B, C) Six WT and IL-36 −/− were euthanized at 1 dpi and the corneas were excised and subjected to fungal plate counting, the results were presented as cfu CA per cornea (B) or to MPO determination, units/cornea (C). The data in B and C were presented as dot plots with the average of CFU or MPO units per cornea and error bars (mean ± SD). P values were generated using unpaired Student's t test. (***P < 0.001), N = 6. and inflammation. Moreover, IL-36/IL-36R signaling antagonizes IL-1/IL-1R signaling, resulting in a reduced severity of PA keratitis. 11 Others have reported that CA infection of the oral mucosa induces the production of IL-36 and that oral candidiasis in IL-36R −/− mice shows an increased fungal burden, indicating a role of IL-36 in mediating the innate protective response to CA. 10 IL-36R signaling has been shown to induce the expression of known IL-36-responsive genes encoding Il17c, and the antimicrobial peptides S100a8 and S100a9, 30 which form calprotectin that acts early in infection to restrict metal nutrients from CA. 31 Calprotectin was also reported in neutrophil extracellular traps and involved in host defense against CA in vitro and in vivo. 32 To date, the involvement and role of IL36 signaling in fungal keratitis remains to be determined.

In this study, we explored the function of IL-36/IL-36R signaling in mediating innate immune defense against CA infection in the corneas. We demonstrate that IL-36R activation improves the outcome of CA keratitis by inducing the expression of antimicrobial peptides and anti-inflammatory cytokines in a dendritic cell (DC)-and macrophage-independent manner. Our results suggest that IL-36 cytokines might be used as an adjunctive therapy in treating CA keratitis.

All animal studies were approved by Wayne State University (WSU) Institutional Animal Care and Use Committee (IACUC), and all investigations conformed to the regulations of the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and of the NIH. Wild-type and IL-36R −/− on C57BL6 (B6) background mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Knockout mouse genotyping was performed by TransnetYX, Inc., Cordova, TN, USA.

Mice were anesthetized by intraperitoneal injection of Ketamine (90 mg/kg) and Xylazine (10 mg/kg). 33 Corneas were scratched with a sterile 26-gauge needle to create three 1-mm incisions to break the epithelial barrier and inoculated with 1.0 × 10 5 CFU CA in 5 μL PBS.

Recombinant IL36γ 100 ng (R&D systems, Minneapolis, MN, USA) in 5 μL PBS was subconjunctivally injected in each cornea at one site four hours before CA inoculation. Dharmacon SMARTpool (a mixture of 4 siRNAs) ON-TARGETplus siRNAs were used to knockdown IL-33. Mice were subconjunctivally injected twice with 5 μL RNase-free water containing 50 pM siRNA at −24 and −6 hours before CA inoculated at 0 hpi.

Eyes were examined daily to monitor the disease progression with a dissection microscope and photographed. For clinical scores assessment, mice were examined at the indicated time to visually grade the disease severity. Ocular disease was graded in clinical scores ranging from 0 to 12. A total score of ≤5 indicated mild eye disease, 6 to 9 signaled moderate disease, and ≥9 severe disease.

The corneas were excised, minced, and homogenized in 100 μL PBS with a Qiagen Micro Tissue Grinder (Hilden, Germany). The homogenates were divided into two parts. The first part was subjected to plate counting. Aliquots (50 μL) of serial dilutions were plated onto Salouraud agar medium plates in triplicates, and colonies were counted Figure 2 . (A) Corneal epithelial cells were collected at 6 hpi and subjected to qPCR analysis for the expression of S100A8, A9, IL-1β, and soluble IL-1Ra. The results of qPCR are presented as fold increase compared to the naïve, WT corneas, set as value 1 after normalization with the level of β-actin as the internal control. NS, no statistical significance. P values were generated using unpaired Student's t-test, **P < 0.01, ***P < 0.001, n = 3. (B) Whole corneas were dissected and processed for ELISA analysis of calprotectin and IL-1β at 1 dpi. The results of ELISA are presented as pg/μg total protein. P values were generated using unpaired Student's t-test, **P < 0.01, ***P < 0.001, N = 3. UD, undetectable. Data are presented as mean ± SD and representative of two independent experiments. on the third day. The results were expressed as the mean number of CFU/cornea ± standard error. Our previous study showed that by 3 dpi there was no recoverable live CA while inflammation was still lingering up to seven days 34 ; hence, we only performed fungal load determination at 1, but not 3 dpi. The second part of the homogenates was mixed with 5 μL of 1% SDS and 10% Triton-X 100. For MPO assay, 30 μL homogenates was mixed with 270 μL of hexadecyltrimethylammonium bromide (HTAB) buffer (0.5% HTAB in 50 mM phosphate buffer, pH 6.0). The samples were then subjected to three freeze-thaw cycles, followed by centrifugation at 16,000g for 20 minutes. The supernatant 20 μL was mixed with 180 μL of 50 mM phosphate buffer (pH 6.0) containing 16.7 mg/mL O,O-dianisidine hydrochloride and 0.0005% hydrogen peroxide at a 1:30 ratio in a well of 96-well plate. The change in absorbance at 460 nm was monitored continuously for five minutes in a Synergy2 Microplate reader (BioTek, Winooski, VT, USA). The results were expressed in units of MPO activity/cornea. One unit of MPO activity corresponded to approximately 2.0 × 10 5 PMN. For ELISA, protein concentration was first determined and using Micro BCA protein assay kit (Thermo Fischer Scientific, Waltham, MA, USA) and 1 μg of total protein was used for ELISA assay for IL1β and S100A8/9 (R&D Systems).

For RT-PCR, mouse corneas or epithelia were collected for RNA preparation using RNeasy Mini Kit (Qiagen) followed by cDNA was generated with oligo (dT) primer and realtime PCR or regular PCR. For quantitative PCR (qPCR), cDNA was amplified using StepOnePlus Real-Time PCR system (Applied Biosystems, University Park, IL, USA) with the SYBR Green PCR Master Mix (Alkali Scientific, Fort Lauderdale, FL, USA). Data were analyzed using CT method with β-actin as the internal control. RT-PCR products were subjected to electrophoresis on 2% agarose gels containing ethidium bromide. Stained gels were captured by using a digital camera. The primer pairs used for PCR were listed in the Table. ) and IL-36γ pretreated corneas at 1 dpi. The data are presented as total number of CA or MPO units per cornea and are representative of two independent experiments. The indicated P values were generated using unpaired Student's t-test, **P < 0.01, ***P < 0.001, N = 5.

Mouse corneal samples were lysed with RIPA buffer and protein concentration was determined by BCA assay. For proteome profiling, 200ug total protein from 4 corneas in each group was detected by mouse protein array kit (R&D Systems). Another portion of the protein samples were separated by SDS-PAGE and electrically transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% BSA and subsequently incubated with primary and secondary antibodies. Antibodies used: anti-mouse IL36γ (ab233420), 36R (ab180894) from Abcam, IL-33 (R&D); anti-mouse Reg3g (Thermo Fisher Scientific); anti-β-actin (A1978; Sigma-Aldrich Corp., St. Louis, MO, USA) was used as the loading control.

CD11c-diphtheria toxin (DT) receptor mice express a transgene encoding a DTR-EGFP fusion protein under the control of the murine CD11c promoter, which makes them sensitive to DT. 35 B6-DTR mice were depleted of their DCs using 50 ng of DT in 5 μL of PBS, administered subconjunctivally 24 hours before CA inoculation. WT B6 mice injected with 50 ng DT were used as controls. To deplete macrophages, 1 μg of Clophosome, neutral clodronate liposomes (F70101C-N; FormuMax Scientific, Inc., Sunnyvale, CA, USA), or control liposomes (F70101-N), were subconjunctivally injected 24 hours before CA inoculation. Complete depletion of residential macrophages was observed one and two days later using immunohistochemistry with F4/80 antibody (data not shown).

Data were presented as means ± SD. Statistical differences among three or more groups were identified using oneway ANOVA, followed by a Bonferroni's post test to determine statistically significant differences. Analysis of clinical scores was performed by a nonparametric Mann-Whitney U test. Differences were considered statistically significant at P < 0.05.

As a first step to understanding the role of IL-36 cytokines in CA (strain SC5314) keratitis, 36 we assessed the expression pattern of IL-36 cytokines and their receptor. Our previous study showed that, unlike IL-1β and soluble IL-1Ra, IL-36R ligands have basal expression in naïve corneas. Hence, we first investigated IL-36α and -36γ expression using regular PCR that allows detection of the basal expression. Figure 1A shows the mRNA expression of IL-36α and -γ in B6 mouse corneas using RT-PCR at 0, 6, 9, 18, and 24 hpi. CA infection augmented IL-36α expression 9 hpi and 18hpi and receded at 24 hpi. IL-36γ expression was markedly increased at 9 and 18 hpi and remained elevated at 24 hpi (Fig. 1A) . In contrast, epithelial scratch wound, a sham control, did not change the DNA band intensity of IL-36γ (Fig. 1A ) . The band intensities of IL-36α and 36γ in Figure 1A were the result of more cycles of PCR amplification, compared to that in Figure 1A , at corresponding times. The mRNA expression was also quantitated by real-time PCR (Fig. 1B ) and the results confirmed the infection-induced expression of IL-36α and IL-36γ in CECs collected at 6 hpi. IL-36α expression was elevated approximately threefold (3.14 ± 0.94) and IL-36γ by fourfold (3.95 ± 0.48), whereas no significant increase was detected for IL-36β. The expressions of IL-36γ and IL-36R at the protein levels were assessed by Western blot (Figs. 1C and 1D), followed by Image J determination of pixel values normalized to that of actin (Figs. 1C and 1D ) . CA infection induced significant upregulation of IL-36γ and IL-36R started at 9 hpi.

Having shown the infection-associated upregulation of IL-36 cytokines, we next investigated the effects of IL-36R signaling on the pathogenesis of CA-keratitis, using IL-36R −/− B6 mice. 14,37 At 1dpi, IL-36R deficiency increased the severity of CA keratitis, including elevated clinical scores (5.25 ± 0.96 vs. 9.0 ± 0.82, Fig. 2A) , fungal burden (2.05 × 10 3 vs. The P values were generated using unpaired Student's t test. BSA-and IL-36γ -treated corneas were compared to each other as uninfected or infected at 6 hpi, respectively. **P < 0.01, ***P < 0.001. Data are representative of three independent experiments with three corneas per group (mean ± SD).

3.46 × 10 3 , Fig. 2B ), and MPO levels (33 vs. 44.9 U/cornea, Fig. 2C ), compared to the wildtype (WT) control. At 3 dpi, IL-36R-deficient mice had significantly increased keratitis severity, with higher clinical scores, 11 ± 0.82, indicative of perforation. There was heavy infiltration into the aqueous humor and clear signs of corneal melting in IL-36R −/− corneas ( Fig. 2A, white arrows) .

To investigate the mechanism of IL36/IL-36R action in CA keratitis, we collected corneal epithelia at 6hpi and whole corneas at 18hpi and assessed the levels of mRNA and protein of IL-1β, IL-Ra, S100A8, and S100A9 of WT and IL-36 R −/− B6 mice by qPCR and ELISA, respectively. Figure  3A shows that in infected WT corneas, all four genes were increased to a different extent compared to the control, naive corneas (value as 1). IL-36R deficiency significantly suppressed infection-induced expression of sIL-1Ra (61.06 vs. 12.59-fold; PCR primers were designed to amplify only soluble isoform of IL-1Ra), S100A8 (25.65 vs. 14.75 fold), and S100A9 (8.0 vs. 4.4 fold) but further augmented the expression of IL-1β (32.53-vs. 118.6-fold). We selected IL-1β and calprotectin (S100A8/A9) for ELISA analyses of mouse corneas (Fig. 3B) . There was no detectable IL-1β and calprotectin in the naïve and IL-36R−/− mouse corneas; infection markedly upregulated expression of both IL-1β (46.17 pg/μg total proteins) and calprotectin (345.5 pg/μg). IL-36Rdeficiency greatly upregulated IL-1β (158.7 vs. 46.17 pg/μg) but markedly downregulated calprotectin (72.51 vs. 345.5 pg/μg) expression in B6 mouse corneas extracted at 18 hpi.

Having shown that proper IL-36R signaling is necessary for the corneal innate immune response to PA infection, we next investigated whether exogenous IL-36γ has positive effects on ameliorating the pathogenesis of CA keratitis. Murine recombinant IL-36γ (100 ng/eye) or BSA control was subconjunctivally injected four hours before CA inoculation. Although the infected WT corneas had typical pathological characteristics of CA keratitis, IL-36γ treatment resulted in decreased opacification with lower clinical scores (Fig. 4B) , lower fungal burden at 1 dpi (1.64 × 10 3 vs. 0.092 × 10 3 per cornea), and greatly reduced but still-measurable MPO activity (28.65U vs. 5.9U per cornea, Fig. 4D ).

To identify downstream effectors of IL-36R signaling, we next assessed the effects of exogenous IL-36γ on the expression IL-1β, IL-1Ra, of S100A8, and A9 (Fig. 5) . In uninfected B6 mouse corneas, IL-36γ stimulated the expression of IL-1Ra, S100A8, and S100A9 at the mRNA level (7.02-, 17.95-, and 6.06-fold, respectively). In CA-infected corneas, treatment with IL-36γ downregulated IL-1β expression (12.56vs. 35.61-fold) while elevating the expression of S100A8 (52.2-vs. 21.73-fold), S100A9 (10.34-vs. 4.52-fold), and sIL-1Ra (156.4-vs. 45.34-fold) at 1 dpi (Fig. 5A) . At the protein levels (Fig. 5B) , IL-36γ treatment resulted in a large amount of calprotectin (255.5 ± 13.7 pg/ug) whereas IL-1β was undetectable in uninfected corneas. In CA-infected corneas, IL-36γ markedly lowered the levels of IL-1β (11.3 ± 4.1 from 46.2 ± 3.9 pg/μg) and moderately but significantly upregulated the levels of calprotectin (335.5 ± 28.2 from 286.1 ± 7.3 pg/μg).

To further understand the mechanism of action of IL-36R signaling, we used the proteome profile assay, consist-ing of 111 soluble mouse proteins including cytokines, chemokines, and growth factors, to identify downstream modulators in CA-infected corneas. Figure 6A shows the clinical phenotypes of CA-keratitis. As expected, exogenous IL-36 (100 ng/eye) and IL-36R deficiency decreased and increased the severity of CA-keratitis, respectively. Among 111 mouse proteins, staining intensities of IL-33 and Reg3g appeared to be associated with changes in IL-36 signaling: infection-increased dot intensities were suppressed by IL-36γ and augmented in IL-36R-deficient corneas of B6 mice (Fig. 6B ). Western blotting (Fig. 6C ) and image analysis (Fig. 6D ) confirmed the expression patterns on IL-33 and Reg3g determined by proteome profile assay. Although basal expression of IL-33 was observed in naïve corneas, Reg3g levels were undetectable. CA induced the upregulations of both IL-33 and Reg3g. In IL-36γ pretreated corneas the protein levels of both IL-33 and Reg3g were lower, whereas in IL-36R-deficient mice they were higher than that of the control, infected corneas.

Both IL-33 and human REG3A are known to play a role in tissue inflammation and repair. 38, 39 Human REG3A (human regenerating family member 3 alpha) is a potent ROS scavenger with antioxidant and anti-gram-positive bacterium activities. 40 Mice have two homologues of human REG3A, Reg3g (also anti-gram-positive bacterium activity due to peptidoglycan carbohydrate binding) and Reg3b (anti-gram-negative bacterium by binding to lipopolysaccharide). 38 Hence, we focused on the role of IL-33, a member of IL-1 superfamily of cytokines, in our fungal keratitis model. Figures 7A and 7B show pathology and clinical scores of CA keratitis in control and IL-33-specific siRNAtreated corneas, with two micrographs for each condition. At 1 dpi, opacification was observed along the scratch line, with heavier opacification in IL-33-specific versus control, nonspecific siRNA-treated corneas. At 3 dpi, IL-33 siRNAtreated corneas had larger and denser opacification, with FIGURE 7. Downregulation of IL-33 increases severity of CA keratitis. WT B6 mice were subconjunctivally injected with IL-33 specific or the control, nonspecific siRNA (50 pmol in 5 μL RNase free water) at −1 dpi and at −6 hpi. Corneas were scratched and inoculated with 1.0 × 10 5 cfu of CA at 0 hpi. (A) Micrographs of nonspecific siRNA (the control) and IL-33 siRNA-treated corneas photographed at indicated times, two corneas each presented. (B) Clinical scores of for the severity of keratitis were analyzed using nonparametric Mann-Whitney U test (**P < 0.01) at 1, 3, and 5 dpi. At 5 dpi, corneas were excised and subjected to MPO determination. This was analyzed using unpaired Student's t-test and presented as MPO units per cornea (C). The corneas were also subjected to qPCR analysis for the expression of IL-36α and IFN-γ and analyzed with one-way ANOVA (D), NL, naïve cornea, N = 5 each, *P < 0.05, **P < 0.01. several regions of corneal melting, compared to the control. At 5 dpi, the control siRNA-treated corneas mostly recovered with minimal signs of inflammation, whereas IL-33 siRNAtreated corneas remained opaque with large melting areas. At 5dpi, MPO activity was significantly higher in IL-33 siRNAtreated corneas compared to control siRNA-treated corneas (Fig. 7C) . Downregulation of IL-33 significantly suppressed IL-36α while increasing IFN-γ expression in CA-infected corneas (Fig. 7D ).

A previous study showed that IL-36R ligands are more potent than IL-1β in inducing DC activation, cytokine expression, and induction of the differentiation of T-lymphocyte into TH1 cytokine-producing cells in vitro. 14 To determine whether the protective effects of IL-36R signaling on CA keratitis is through DCs, we depleted DCs locally through subconjunctival injection of DT into CD11c-DTR mice, with WT B6 mice receiving the same amount of DT as the control. Local depletion of DCs markedly increased the severity of CA keratitis, including increased corneal opacification, clinical scores (Fig. 8A) , fungal load (5062 ± 303 vs. 1696 ± 287 cfu/cornea) (Fig. 8B) , and MPO activity (46 ± 5 vs. 18.7 ± 3.14 units/corneas) (Fig. 8C ) at 1 dpi, compared to DT-injected WT control mice. Prophylactic application of 100 ng IL-36γ greatly suppressed the progression of CA keratitis with reduced clinical scores, decreased size of opacity (Fig. 8A) , barely detectable levels of CA (16.7 ± 3.79 cfu/cornea) (Fig. 8B) , and diminished MPO activity (3.81 ± 1.05 units/cornea, 12.1-fold decrease compared to DCdepleted, untreated corneas; Fig. 8C ). Having shown that IL-36γ -induced protection is DCindependent, we next investigated the role of macrophages in CA keratitis and IL-36γ -induced protection. Mice were treated subconjunctivally with liposomes containing clodronate to deplete corneal macrophages. Control mice were treated with liposomes containing PBS. Mice treated with PBS-liposomes exhibited similar pathology to nonpretreated corneas at 1 dpi (1695 ± 278 cfu/cornea and 17.8 ± 2.86 MPO units/cornea) and presence of IL-36γ protected these corneas from CA infection (Fig. 9A) with no detectable cfu and 0.85 ± 0.05 units/cornea MPO (Figs. 9B and 9C). Mice treated with clodronate-containing liposomes had more severe keratitis than the control (Fig. 9A) , with 3461 ± 575 cfu and 31.89 ± 3.51 MPO units per corneas (Figs. 9B and 9C ). Prophylactic application of 100 ng IL-36γ significantly reduced the severity of CA keratitis in macrophage-depleted corneas (Fig. 9A ) with 308 ± 140 cfu and 8.99 ± 3.09 MPO unit per cornea, a 3.55-fold decrease compared to macrophage-depleted, untreated corneas (Figs. 9B and 9C ).

We previously demonstrated that IL-1Ra and IL-36Ra have opposing effects on the outcome of P. aeruginosa keratitis. 11 In this study, we assessed the expression and functions of the IL-36γ /IL-36R axis and observed CA infection-induced upregulations in expression of IL-36α, IL-36γ , as well as their receptor IL-36R in B6 mouse corneas. IL-36R knockout greatly exacerbated CA keratitis, resulting in corneal perforation at 3 dpi. Worsened outcome of CA keratitis in IL-36 −/− mice may be related to a significant upregulation of proinflammatory IL-1β and to the suppressed expressions of antiinflammatory sIL-1RA and antimicrobial peptides, S100A8, A9 (calprotectin). Moreover, recombinant IL-36γ exhibited opposing effects compared to IL-36R deficiency: improved outcome of keratitis, reduced IL-1β, and increased sIL-1RA, S100A8 and A9 expression in infected corneas. Interestingly, IL-36γ also stimulated upregulation of calprotectin in noninfected corneas, suggesting a potential role of IL-36/IL-36R in corneal homeostasis and resistance to environmental opportunistic pathogens such as CA. We also showed CA infection induced IL-36/IL36R-related upregulation of IL-33 and Reg3g at both mRNA and protein levels. Surprisingly, downregulation of IL-33 increased the severity of CA keratitis, FIGURE 9 . Macrophage depletion increases the severity of but exhibits minimal effects on IL-36γ -induced protection against CA keratitis. B6 mice were subcutaneously injected with 1 μg of neutral clodronate liposomes or control liposomes at −1 dpi, followed by injection of IL-36γ at −4 hpi. Corneas were scratched and inoculated with 1.0 × 10 5 cfu of CA at 0 hpi. (A) Micrographs of liposome treated B6 corneas at 1 dpi with or without exogenous IL-36γ ; clinical scores were presented in the representative corneas. The diseased corneas were excised and subjected to fungal plate counting (B) and MPO determination (C) and analyzed using unpaired Student's t-test, n = 5 each, ***P < 0.001. suggesting a positive role of this unique member of the IL-1 family. Finally, DC and macrophage depletion greatly increased the severity but failed to overcome IL-36γ -induced protection against CA infection of B6 mouse corneas. Taken together, our results indicate that the IL-36γ /IL-36R axis plays a protective role in CA keratitis through upregulating AMPs and downregulating the inflammatory response, and consequently improves the outcome of CA keratitis.

IL-36 cytokines, unlike members of the IL-1 subfamily, are constitutively expressed at basal levels in mucosal epithelia, including CECs, as well as in monocytes/macrophages and T-lymphocytes. 13, [41] [42] [43] In keratinocytes, IL-36Ra was found to be constitutively expressed, whereas IL-36α, IL-36β and IL-36γ were rapidly induced after stimulation with TNFα. 44, 45 Our data showed that 36α and 36γ were detectable by RT-PCR in naïve B6 mouse corneas and infection induced their expression. Moreover, we detected elevated expressions of IL-36γ and IL-36R at the protein levels in a time-dependent manner in CA-infected corneas. Increased expression of IL-36R during CA infection may be due to increased infiltration of immune cells, such as dendritic cells and macrophages, but not neutrophils. 46 These results show that B6 mouse corneas increase IL-36/IL-36R signaling in response to CA infection, suggesting that IL-36 might be involved in corneal innate immunity against CA infection.

How might IL-36R mediate the innate immune response to CA infection? Various studies have shown that IL-36R activation induces the release of antimicrobial proteins such as human beta-defensins 2 and 3, LL37, S100A7, and more importantly S100A8/A9. 30, [47] [48] [49] In an oral candidiasis model, IL-36R −/− mice show an increased fungal burden and reduced IL-23 gene expression. 9,10,26,50 Previously, we found that IL-36R antagonist (IL-36Ra) downregulation and exogenous IL-36γ prevented corneal infection while exogenous IL-36Ra augmented the pathogenesis of P. aeruginosakeratitis. 11 Our current study provides evidence that IL-36R signaling influences the outcome of CA keratitis by suppressing the expression of proinflammatory cytokines IL-1β and promoting the expression of IL-Ra, S100A8, and S100A9. Furthermore, our data show that IL-36R deficiency enhanced innate immune defense and reduced severity of CA keratitis. IL-36γ -deficient mice infected with Streptococcus pneumoniae exhibited diminished lung bacterial clearance, increased bacterial dissemination and a higher mortality rate. 26, 51 Our results are consistent with the protective role of IL-36γ in corneal innate defense against fungal infection. IL-36γ significantly stimulates the expression of S100A8, S100A9, and sIL-1RA, but not IL-1β, in uninfected corneas in vivo. To our knowledge, this is the first report of IL-36-stimulated upregulation of S100A8/A9 or calprotectin in a mucosal surface with or without infection. Because calprotectin possesses potent anti-CA activity, 30-32 its upregulation may represent an underlying mechanism for IL-36/IL-36R to play a protective role in CA keratitis. Although the activation of IL-1β, a master pro-inflammatory cytokine, is most stringently regulated, its final activities are likely determined by the ratio of IL-1β/sIL-1Ra. [52] [53] [54] Hence, the differential effects of IL-36γ on the expression of IL-1β and sIL-1Ra in naïve and infected corneas is consistent with our recently-suggested notion that the IL-36 family of cytokines, as a whole, antagonize IL-1R in the corneas in response to microbial infection. Because blocking IL-1R activation has been used to treat a broad spectrum of diseases, such as gout, rheumatoid arthritis, type 2 diabetes, atherosclerosis, and acute myocardial infarction, 55 activation of IL-36R signaling may have syngeneic effects with Anakinra, a human recombinant IL-1Ra clinically used to treat aforementioned inflammatory diseases, 56 as well as COVID-19. 57, 58 Using a mouse proteomic array, we discovered that among others, IL-36 had a significant impact on the expression of IL-33 and Reg3g in CA-infected corneas. Reg3γ is a secreted, C-type lectin and has been shown to be expressed in the cornea exposed to pathogens or after injury. [59] [60] [61] Reg3γ -deficient mice have increased mucosal inflammatory responses to microbiota. 62 Compared to IL-33, a member of the IL-1 superfamily, expression of REG3γ , an AMP, is more dramatically altered in IL-36-modulated CA-infected corneas at 1 dpi, suggesting an important role of IL-36R signaling in corneal innate immunity, as demonstrated for oral candidiasis. 10 However, because murine REG3γ is one of two homologs of human REG3A, 63 we focused on IL-33. The siRNA-mediated downregulation of IL-33 greatly worsened the severity of CA keratitis for up to 5 dpi, whereas the control corneas with nonspecific siRNA almost completely recovered. This result contradicts the expectation that IL-33, as a downstream effector of IL-36R signaling, would play a detrimental role in CA keratitis. Combined with the fact that the effects of IL-36R signaling on IL-33 expression were moderate, we suggest that the expression or the levels of IL-33 at 1 dpi may be related to the severity of CA keratitis rather than depending on IL-36 signaling in CA-infected corneas. Hence, IL-36 and IL-33 may function in parallel in the cornea to mediate different cellular responses to fungal infection. One potential mechanism for the observed effects of IL-33 in CA keratitis is that IL-33 acts as an alarmin cytokine to mediate innate immunity in the corneas. 64 The role and the mechanism of action of IL-33 as an alarmin or as a cytokine in innate immunity of the corneas warrant further investigation.

DCs are known to express IL-36R highly and, compared to epithelial cells, are more sensitive to IL-36 than IL-1β. 14 IL-36 was shown to stimulate DC activation and the expression of inflammatory cytokines such as IL-1β, IL-12, IL-23, IL-6 and chemokines, including CCL1, CXCL1 and GM-CSF in an IL-36R-dependent manner. 46, 65 Surprisingly, although local depletion of DC exacerbated CA keratitis, it exhibited few, if any, effects on IL-36γ -induced innate protection with total eradication of inoculated CA at 1 dpi. Similarly, local depletion of macrophages also resulted in severe CA keratitis and the presence of IL-36γ protected macrophage-depleted corneas from CA infection with no detectable cfu. Comparing MPO (Fig. 8C and Fig. 9C ) activities suggests that there are more neutrophils in macrophagedepleted corneas than in DT-depleted corneas. This may be due to the higher phagocytic capacity of macrophages than that of DCs in removing damaged, dead, and dying neutrophils and/or apoptotic bodies (efferocytosis). 66 The fact that depletion of both DCs and macrophages in IL-36γ pretreated corneas results in the eradication of invading CA suggests that autocrine activation of IL-36γ /IL-36R in epithelial cells may be the underlying cause for IL-36γinduced protection against CA keratitis. In IL-36γ pretreated corneas, presence of calprotectin and other AMPs such as Reg3γ and/or CXCL10 33 may eliminate invading CA within the epithelial layer before they penetrate the basement membrane, which functions as a barrier for pathogens to reach the stroma, as we have shown for P. aeruginosa infection. 67 In summary, the IL-36 family plays an important role in CA corneal infection by contributing to innate immunity in the corneas. IL-36/IL-36R signaling alters the balance of IL-1β and sIL-1Ra and increases the secretion of the antimicrobial peptide calprotectin and Reg3γ . Activation of IL-36/IL-36R may be used as a means to control fungal and other causes of microbial keratitis.

Corneal blindness: a global perspective

Fungal keratitis

Fungal keratitis in Melbourne

Microbial keratitis in South India: influence of risk factors, climate, and geographical variation

Genetically regulated filamentation contributes to Candida albicans virulence during corneal infection

Fungal keratitis in London: microbiological and clinical evaluation

IL1HY1: A novel interleukin-1 receptor antagonist gene

IL-36 cytokines: regulators of inflammatory responses and their emerging role in immunology of reproduction

Overlapping roles for interleukin-36 cytokines in protective host defense against murine Legionella pneumophila pneumonia

IL-36 and IL-1/IL-17 drive immunity to oral candidiasis via parallel mechanisms

Opposing effects of IL-1Ra and IL-36Ra on innate immune response to Pseudomonas aeruginosa infection in C57BL/6 mouse corneas

Two novel IL-1 family members, IL-1 delta and IL-1 epsilon, function as an antagonist and agonist of NF-kappa B activation through the orphan IL-1 receptor-related protein 2

IL-1Rrp2 expression and IL-1F9 (IL-1H1) actions in brain cells

IL-36R ligands are potent regulators of dendritic and T cells

Mutations in IL36RN/IL1F5 are associated with the severe episodic inflammatory skin disease known as generalized pustular psoriasis

A tale of two sisters: identical IL36RN mutations and discordant phenotypes

IL-38: A New Player in Inflammatory Autoimmune Disorders

Interleukin-36alpha axis is modulated in patients with primary Sjogren's syndrome

Mutation analysis of IL36RN gene in Japanese patients with palmoplantar pustulosis

New aspect of allergic contact dermatitis, an inflammatory skin disorder mediated by mast cells: can IL-38 help? Med Hypotheses

Function and regulation of IL-36 signaling in inflammatory diseases and cancer development

The IL-36 receptor pathway regulates Aspergillus fumigatus-induced Th1 and Th17 responses

Role of interleukin 36γ in host defense against tuberculosis

Commensal Bacteroidetes protect against Klebsiella pneumoniae colonization and transmission through IL-36 signalling

IL-36 promotes antiviral immunity by boosting sensitivity to IFN-alpha/beta in IRF1 dependent and independent manners

IL-36γ is a crucial proximal component of protective type-1-mediated lung mucosal immunity in Gram-positive and -negative bacterial pneumonia

Interleukin-36γ and IL-36 receptor signaling mediate impaired host immunity and lung injury in cytotoxic Pseudomonas aeruginosa pulmonary infection: role of prostaglandin E2

IL-36 receptor deletion attenuates lung injury and decreases mortality in murine influenza pneumonia

Limited contribution of IL-36 versus IL-1 and TNF pathways in host response to mycobacterial infection

Keratinocyte interleukin-36 receptor expression orchestrates psoriasiform inflammation in mice

Role of calprotectin in withholding zinc and copper from Candida albicans

Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans

Flagellin-induced expression of CXCL10 mediates direct fungal killing and recruitment of NK cells to the cornea in response to Candida albicans infection

CXCL10 suppression of hem-and lymph-angiogenesis in inflamed corneas through MMP13

Dendritic cellepithelium interplay is a determinant factor for corneal epithelial wound repair

Morphogenic and genetic differences between Candida albicans strains are associated with keratomycosis virulence

IL-36R signalling activates intestinal epithelial cells and fibroblasts and promotes mucosal healing in vivo

Enteric delivery of regenerating family member 3 alpha alters the intestinal microbiota and controls inflammation in mice with colitis

Interleukin-33 attenuates sepsis by enhancing neutrophil influx to the site of infection

Hyperglycaemia inhibits REG3A expression to exacerbate TLR3-mediated skin inflammation in diabetes

Regulation and function of the IL-1 family cytokine IL-1F9 in human bronchial epithelial cells

Four new members expand the interleukin-1 superfamily

Regulation and function of interleukin-36 cytokines

Identification and gene organization of three novel members of the IL-1 family on human chromosome 2

Regulation and function of interleukin-36 cytokines in homeostasis and pathological conditions

The significance of IL-36 hyperactivation and IL-36R targeting in psoriasis

IL-1F5, -F6, -F8, and -F9: a novel IL-1 family signaling system that is active in psoriasis and promotes keratinocyte antimicrobial peptide expression

Phagocytosis of Aspergillus fumigatus conidia by primary nasal epithelial cells in vitro

Inter-regulation of Th17 cytokines and the IL-36 cytokines in vitro and in vivo: implications in psoriasis pathogenesis

IL-36alpha expression is elevated in ulcerative colitis and promotes colonic inflammation

IL-36γ is secreted in microparticles and exosomes by lung macrophages in response to bacteria and bacterial components

Targeting imbalance between IL-1β and IL-1 receptor antagonist ameliorates delayed epithelium wound healing in diabetic mouse corneas

The interleukin-1 family: back to the future

A clinical perspective of IL-1beta as the gatekeeper of inflammation

Anakinra Therapy for Non-cancer Inflammatory Diseases

Benefits of anakinra versus TNF inhibitors in rheumatoid arthritis and type 2 diabetes: long-term findings from participants furtherly followed-up in the TRACK study, a multicentre, open-label, randomised, controlled trial

An open label trial of anakinra to prevent respiratory failure in COVID-19

Efficacy of early antiinflammatory treatment with high doses of intravenous anakinra with or without glucocorticoids in patients with severe COVID-19 pneumonia

Toll-like receptor signalling in the intestinal epithelium: how bacterial recognition shapes intestinal function

Acceleration of pancreatic tumorigenesis under immunosuppressive microenvironment induced by Reg3g overexpression

Genome-wide expression differences in anti-Vegf and dexamethasone treatment of inflammatory angiogenesis in the rat cornea

REG3γ -deficient mice have altered mucus distribution and increased mucosal inflammatory responses to the microbiota and enteric pathogens in the ileum

Innate Stat3-mediated induction of the antimicrobial protein Reg3γ is required for host defense against MRSA pneumonia

IL-33: an alarmin cytokine with crucial roles in innate immunity, inflammation and allergy

IL-36 promotes myeloid cell infiltration, activation, and inflammatory activity in skin

The clearance of dead cells by efferocytosis

Matrix metalloproteinase-13 as a target for suppressing corneal ulceration caused by Pseudomonas aeruginosa infection

The authors thank Patrick Lee for proofreading the manuscript.