Opposing Effects of IL-1Ra and IL-36Ra on Innate Immune

Opposing Effects of IL-1Ra and IL-36Ra on Innate Immune Response to Pseudomonas aeruginosa Infection in C57BL/6 Mouse Corneas This information is current as of September 29, 2021. Nan Gao, Rao Me, Chenyang Dai, Berhane Seyoum and Fu-shin X. Yu J Immunol 2018; 201:688-699; Prepublished online 11 June 2018; doi: 10.4049/jimmunol.1800046 Downloaded from http://www.jimmunol.org/content/201/2/688

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Opposing Effects of IL-1Ra and IL-36Ra on Innate Immune Response to Pseudomonas aeruginosa Infection in C57BL/6 Mouse Corneas

Nan Gao,*,† Rao Me,*,† Chenyang Dai,*,†,‡ Berhane Seyoum,x and Fu-shin X. Yu*,†

Pseudomonas aeruginosa keratitis is characterized by severe corneal ulceration and may lead to blindness if not treated properly in a timely manner. Although the roles of the IL-1 subfamily of cytokines are well established, as a newly discovered subfamily, IL- 36 cytokine regulation, immunological relevance, and relation with IL-1 cytokines in host defense remain largely unknown. In this study, we showed that P. aeruginosa infection induces the expression of IL-36a and IL-36g, as well as IL-1b and secreted IL-1Ra (sIL-1Ra), but not IL-36Ra. Downregulation of IL-1Ra increases, whereas downregulation of IL-36Ra decreases the severity of P. aeruginosa keratitis. IL-1R and IL-36Ra downregulation have opposing effects on the expression of IL-1b, sIL-1Ra, IL-36g, Downloaded from S100A8, and CXCL10 and on the inﬁltration of innate immune cells. Administration of recombinant IL-1Ra improved, whereas IL-36Ra worsened the outcome of P. aeruginosa keratitis. Local application of IL-36g stimulated the expression of innate defense molecules S100A9, mouse b-defensin 3, but suppressed IL-1b expression in B6 mouse corneas. IL-36g diminished the severity of P. aeruginosa keratitis, and its protective effects were abolished in the presence of S100A9 neutralizing Ab and partially affected by CXCL10 and CXCR3 neutralizations. Thus, our data reveal that IL-1Ra and IL-36Ra have opposing effects on the outcome of P. aeruginosa keratitis and suggest that IL-36 agonists may be used as an alternative therapeutic to IL-1b–neutralizing reagents in http://www.jimmunol.org/ controlling microbial keratitis and other mucosal infections. The Journal of Immunology, 2018, 201: 688–699.

he IL-1 family of cytokines are fundamental regulators of the ratio of active IL-1b and its natural inhibitor, soluble IL-1Ra the innate immune response and are known to be involved (3, 6, 7). Disturbance in the IL-1b/sIL-1Ra balance usually re- T in multiple inflammatory diseases (1, 2). The family sults in inflammation diseases (8–11). More importantly, human contains seven ligands with agonistic (IL-1a, IL-1b, IL-18, IL-33, recombinant IL-1Ra (anakinra), a biologic designed to treat IL-36a, IL-36b, and IL-36g), three ligands with antagonistic rheumatoid arthritis, has been found to be effective in treating a (IL-1Ra, IL-36Ra, and IL-38), and one ligand with anti- broadening list of inflammatory diseases, cardiovascular diseases inflammatory activity (IL-37) (3, 4). The two large subfamilies, (12, 13), and type 1 and 2 diabetes (14, 15). by guest on September 29, 2021 IL-1 and IL-36, are located within a gene cluster: IL1A–IL1B– IL-36 cytokines have attracted much attention ever since IL137–IL36G–IL36A–IL36B–IL36RA–IL38–IL1RN, suggesting a IL-36RN mutations were found to cause generalized pustular close evolutionary relationship. IL-1b is considered to be a key psoriasis, the most severe form involving not only skin inflammation orchestrator of innate immune responses (3). IL-1b activation is but also systemic symptoms such as fever and malaise (16–19). regulated at the transcriptional, posttranslational processing, and Binding of IL-36 agonists to IL-36R recruits IL-1 receptor acces- secretion levels (5). However, regardless of how it is released or how sory protein (IL-1RAcP), a shared accessory protein with IL-1R the precursor is cleaved, the final activity of IL-1b is determined by and IL-33R. This ultimately results in the activation of NF-kB and MAPKs through MyD88 pathway (3, 20). The fact that IL-36R has *Department of Ophthalmology, Wayne State University School of Medicine, three agonists, IL-36a, IL-36b, and IL-36g, and two antagonists, Detroit, MI 48201; †Department of Anatomy and Cell Biology, Wayne State Univer- IL-36Ra and IL-38 (21–24), underpins the importance of fine- sity School of Medicine, Detroit, MI 48201; ‡Department of Ophthalmology, Qilu Hospital of Shandong University, Jinan, Shandong Province 250014, China; and tuning the IL-36R signaling pathway. In host defense, although xDivision of Endocrinology, Department of Internal Medicine, Wayne State Univer- the roles of the IL-1b and IL-1R pathways are well established, the sity School of Medicine, Detroit, MI 48201 regulation of IL-36 cytokines and their immunological relevance ORCIDs: 0000-0003-0087-5096 (B.S.); 0000-0002-8517-2738 (F.-s.X.Y.). remain largely unknown. Recently, IL-36 expression and signaling Received for publication January 10, 2018. Accepted for publication May 16, 2018. have been linked to viral (25), mycobacterial (26, 27), Gram- This work was supported by the National Eye Institute at the National Institutes of negative, and Gram-positive (28) bacterial infections of the lung. Health (Grants R01 EY017960 and R01 EY010869 to F.-s.X.Y. and Grant p30 The IL-36/IL-36R axis has been shown to play contradicting roles EY04078 to Wayne State University) and Research to Prevent Blindness (to Mark Juzych, Chair, Kresge Eye Institute). in host response to mycobacterial infection (26, 27). It is also Portions of this work were presented at the Annual Meeting of the Association for shown to play a protective role in Gram-positive Streptococcus Research in Vision and Ophthalmology, May 5–9, 2013, Seattle, WA. pneumoniae or Gram-negative Klebsiella pneumoniae pneumonia Address correspondence and reprint requests to Dr. Fu-Shin X. Yu, Kresge Eye by driving protective type-1 responses and classical macrophage Institute, Wayne State University School of Medicine, 4717 Street Antoine Boule- activation in the lung (28). Interestingly, the same group, using vard, Room K417, Detroit, MI 48201. E-mail address: [email protected] IL-36g and IL-36R knockout mice, showed that the IL-36 signal- Abbreviations used in this article: BM, basement membrane; CEC, corneal epithelial cell; CS, clinical score; CT, control; dpi, d postinfection; HCEC, human corneal ing pathway impaired host immunity and exuberated lung injury in epithelial cell; H-K, heat-killed; hpi, h postinfection; MPO, myeloperoxidase; NL, cytotoxic Pseudomonas aeruginosa pulmonary infection (29). The naive cornea; PMN, polymorphonuclear leukocyte; qPCR, quantitative PCR; involvement of IL-36/IL-36R in mediating tissue/cell responses to sIL-1Ra, secreted IL-1Ra; siRNA, small interfering RNA. infection in other mucosal tissues, including the cornea, have not Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 been reported to date. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800046 The Journal of Immunology 689

Bacterial keratitis is a sight-threatening disease, commonly incisions to break the epithelial barrier and were inoculated with 1.0 3 104 associated with extended contact lens wear (30). P. aeruginosa is CFU of ATCC 19660 in 5 ml of PBS. a common causative agent of keratitis (31). To cause keratitis, The application of siRNA, recombinant protein, microbes must traverse the epithelial layer and cross the base- or neutralizing Ab ment membrane (BM) to reach the stroma (32, 33), a process that usually takes ∼18–24 h to accomplish because the BM serves as All the siRNAs used for this study were SMARTpool (a mixture of four siRNAs) ON-TARGETplus siRNAs designed by GE Healthcare Dharmacon a physiological barrier (34, 35). P. aeruginosa keratitis usually (Lafayette, CO). Mice were subconjunctivally injected twice with siRNA presents as a rapidly progressing condition, with distinct in- targeting to a specific gene (50 pmol in 5 ml of RNase-free water) over 2 d. flammatory epithelial edema and stromal ulceration, which can Six hours after the second siRNA injection, mouse corneas were inoculated lead to significant stromal tissue destruction and loss of vision with P. ae ru gi nos a. To apply recombinant proteins or neutralizing Ab, mice were subconjunctivally injected with 150 ng recombinant protein (IL-36g (36). These consequences are largely caused by the host’s in- and IL-36Ra) (R&D Systems, Minneapolis, MN), 150 ng anakinra, 100 ng flammatory responses, although bacterial toxins and exoproducts anti-S100A9 (kindly provided by Dr. P. Tessier, Laval University Hospital may contribute to P. aeruginosa keratitis (36). The innate im- Center, QC, Canada), CXCL10, or CXCR3 neutralizing Abs (R&D Systems) mune system is critical for host defense against infectious 4 h before the inoculation with P. ae ru gi nos a on the corneas. challenges. Recognition of pathogens by TLRs results in the Clinical examination initiation of innate immune responses, including the production/ secretion of inflammatory mediators, especially IL-1b and its Eyes were examined daily to monitor the disease progression with a dis- section microscope equipped with a digital camera. For the assessment of natural inhibitor IL-1Ra (37). In the cornea, epithelial cell– clinical scores (CSs), mice were color coded and examined in a blinded produced IL-1 was suggested to act as a master regulator of fashion by two independent observers at 1 and 3 d postinfection (dpi) to Downloaded from corneal wound healing; topical application of IL-1Ra was shown visually grade the disease severity. Ocular disease was graded in CSs to inhibit inflammatory cell infiltration of the cornea (7). Be- ranging from 0 to 12. A total score of five or less indicated mild eye disease, six to nine signaled moderate disease, and nine showed severe disease (35, cause they were only discovered recently, the role of IL-36 cy- 38, 39). tokines in P. aeruginosa keratitis and microbial infection of the corneas remains elusive. Bacteria load determination, cytokine ELISA,

In this study, we assessed the expression, contribution, and role and myeloperoxidase measurement http://www.jimmunol.org/ of IL-36R signaling pathway, in comparison with that of IL-1R We used our previously modified methods that allowed all three assays signaling, in the control (CT) of P. aeruginosa infection by us- (bacteria load, myeloperoxidase (MPO) determination, and cytokine ing small interfering RNA (siRNA) knockdown and recombinant ELISA measurement) to be performed with a single mouse cornea. Briefly, receptor antagonists. We found that the expression of IL-36a and the corneas were excised, minced, and homogenized in 100 ml of PBS with Dounce Micro Tissue Grinder. The homogenates were divided into two IL-36g was induced upon P. aeruginosa infection and augmenting parts. The first part was subjected to plate bacterium counting. Aliquots IL-36R signaling, either by downregulating IL-36Ra or by exog- (50 ml) of serial dilutions were plated onto Pseudomonas Isolation Agar enous IL-36g, greatly reduced the severity of P. aeruginosa ker- plates in triplicates, and colonies were counted the next day. The results atitis, in sharp contrast to that reported in P. aeruginosa infection were expressed as the mean number of CFU per cornea 6 SE. The second of the lung (29). The data support our hypothesis that IL-36 cy- part of each homogenate was mixed with 5 ml of 1% SDS and 10% Triton-X by guest on September 29, 2021 100. For MPO assay, 30 ml of homogenate was mixed with 270 mlof tokines initiate innate defense and tissue repair in response to hexadecyltrimethylammonium bromide (HTAB) buffer (0.5% HTAB in microbial infection by inducing epithelial cells to produce AMPs 50 mM phosphate buffer [pH 6]). The samples were then subjected to three and other cytokines/chemokine and by antagonizing the IL-1 freeze–thaw cycles, followed by centrifugation at 16,000 3 g for 20 min. subfamily by mediating the balanced expression of IL-1b and Twenty microliters of the supernatant was mixed with 180 mlof50mM phosphate buffer (pH 6) containing 16.7 mg/ml O,O-dianisidine hydro- IL-1Ra. chloride and 0.0005% hydrogen peroxide at a 1:30 ratio in a well of a 96-well plate. The change in absorbance at 460 nm was monitored con- Materials and Methods tinuously for 5 min in a Synergy 2 Microplate Reader (BioTek). The results were expressed in units of MPO activity per cornea. One unit of MPO Animals activity corresponded to ∼2.0 3 105 polymorphonuclear leukocytes Wild-type C57BL6 (B6) mice (8 wk of age; female) were purchased from (PMN). For ELISA, protein concentration was first determined, and Micro The Jackson Laboratory (Bar Harbor, ME). All animal procedures were BCA Protein Assay Kit (Pierce) and 1 mg of total protein was used performed in compliance with the Association for Research in Vision and for ELISA assay for CXCL2, IL-1b, CXCL10, and calprotectin (R&D Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Systems). Research and were approved by the Institutional Animal Care and Use Committee of Wayne State University. RNA extraction and real-time PCR Cell culture For RT-PCR, mouse corneas or CECs scraped off the cornea were extracted for RNA using RNeasy Mini Kit (Qiagen). cDNA was generated with an Primary human corneal epithelial cells (HCECs) were obtained from Oligo (dT) Primer (Invitrogen) followed by analysis using real-time PCR diseased human corneal samples. Corneal epithelial cells (CECs) were or RT-PCR. For quantitative PCR, cDNA was amplified using StepOnePlus dislodged from the underlying BM by dispase and then digested by trypsin. Real-Time PCR System (Applied Biosystems, University Park, IL) with Cells were pelleted, resuspended, and grown in defined keratinocyte the SYBR Green PCR Master Mix (Applied Biosystems). Data were an- serum-free medium (Life Technologies, Waltham, MA) with supplements alyzed by using DD threshold cycle method with b-actin as the internal CT. and antibiotics. For RT-PCR, PCR products were subjected to electrophoresis on 1% P3 or P4 of the primary HCECs were used in the experiments. Before agarose gels containing ethidium bromide. Stained gels were captured by treatment, cells were starved overnight in growth factor–free and antibiotic- using a digital camera. The primer pairs used are listed in Table I. free keratinocyte basic medium (KBM-2; Lonza, Basel, Switzerland). Subsequently, cells were challenged with heat-killed (H-K) bacteria (1:50 Western blot multiplicity of infection) for 1, 2, 4, 8, or 16 h. At the end of the incubation Mouse corneal samples were lysed with radioimmunoprecipitation assay period, cells were harvested for detecting gene expression. buffer. The lysates were centrifuged to obtain the supernatant. Protein Mouse model of P. aeruginosa keratitis concentration was determined by bicinchoninic acid assay. The protein samples were separated by SDS-PAGE and electrically transferred onto Mice were anesthetized with an i.p. injection of ketamine (90 mg/kg) and nitrocellulose membranes (Bio-Rad, Hercules, CA). The membranes were xylazine (10 mg/kg) before surgical procedures. Mouse corneas were blocked with 3% BSA and subsequently incubated with primary and scratched gently with a sterile 26-gauge needle to create three 1-mm secondary Abs. Signals were visualized using SuperSignal West Pico 690 IL-1Ra, IL-36Ra, AND BACTERIAL KERATITIS

Chemiluminescent Substrate (Thermo Scientific, Pittsburgh, PA) using a detectable, whereas IL-36g and IL-36Ra had a basal-level of ex- Kodak Imaging Station 4000R. b-Actin was used as the loading CT. pression by RT-PCR in unchallenged cells. P. aeruginosa chal- Abs included anti-human IL-36a,IL-36g, and IL-36RA (a,1mg/ml; g, lenge resulted in the induction of IL-36a and the upregulation of 0.5 mg/ml; RA, 0.1 mg/ml; R&D Systems); anti-mouse IL-36Ra, IL-1Ra (1.0 mg/ml), or IL-1Ra (0.1 mg/ml; Thermo Fisher Scientific); rabbit IL-36g. As for IL-1 cytokines, P. aeruginosa stimulated robust anti-mouse IL-36g, which was generated in Dr. Standiford’s laboratory expression of IL-1b and sIL-1Ra (V2, secreted form) (7) as early (University of Michigan) (29); and anti–b-actin (A1978, 1:10000; response genes in cultured HCECs (Fig. 1A). The expression of Sigma-Aldrich). IL-36 cytokines was also assessed on the protein level by Western Immunohistochemistry blotting (Fig. 1B). Whereas IL-36g and IL-36Ra were readily detectable in unstimulated cells, the presence of H-K P. aeruginosa At the indicated time points, the corneas were excised from sacrificed mice and were frozen in OCT compound. Posterior parts along with the lens upregulated the expression of IL-36a and IL-36g at 4 hpi. There were removed shortly after the global parts were frozen. The corneas were was a detectable elevation of IL-36Ra at 16 hpi. The image ac- cut into 6-mm thick sections by cryostat sectioning, and the sections were quisition for IL-36a and IL-36RA required a much longer expo- mounted to poly-L-lysine–coated glass slides. After a 10-min fixation in sure time than that of IL-36g. Importantly, the immunoreactivity 4% paraformaldehyde, the sections were blocked with 10 mM PBS, containing 2% BSA, for 1 h at room temperature. Sections were then incubated with of IL-36g, but not IL-36a or IL-36RA, was also detected in the primary Abs: rat anti-mouse NIMP-1 (1:1000; BD) or F4/80 (1:50; culture media of HCECs at 8 and 16 h post stimulation, suggesting eBioscience), followed by the secondary Ab, FITC-conjugated goat anti- the cytokine was secreted in response to P. aeruginosa challenge. rabbit IgG (Jackson ImmunoResearch Laboratories). Slides were mounted with mounting media containing DAPI. CTs were treated similarly, but the IL-36a and IL-36g, like IL-b and sIL-1Ra, are early response primary Ab was replaced with nonspecific rabbit IgG. genes in P. aeruginosa–infected B6 mouse corneas Downloaded from In vitro bactericidal activity assay To assess IL-36 expression in vivo, we inoculated B6 mouse 4 Corneas, three per group, were treated with BSA (CT) or IL-36g, ho- corneas with 10 CFU of P. aeruginosa (strain ATCC 19660). mogenized, and sonicated 4 h postinfection (hpi). Each cornea in 0.1 ml of Corneas were collected at 3, 6, 9, and 18 hpi and subjected to PCR homogenate was incubated with 1000 CFU freshly prepared P. aeruginosa analysis. Real-time PCR analysis revealed a gradual and robust for 1 h at 37˚C, three 20-ml homogenates were spread onto TB Agarose upregulation of IL-1b and IL-1Ra (Fig. 2A) from 3 to 18 hpi in the plates, and the colonies formed overnight at 37˚C were counted and av-

whole cornea. IL-36a and IL-36g were rapidly elevated with no http://www.jimmunol.org/ eraged. The average numbers of colonies of three corneas were used to calculate the mean CFU for each condition. significant differences from 3 to 9 hpi (Fig. 2A). The increases for IL-36a and IL-36g were relatively moderate compared with those Statistical analyses of IL-1b and IL-1Ra. This may be related to the low-level basal Data were presented as means 6 SD. Statistical differences among three or expression of IL-36 subfamily of cytokines in naive corneas (NL), more groups were identified using one-way ANOVA. Differences were as shown by RT-PCR (Fig. 2B). The induction of IL-36g at the p , considered statistically significant at 0.05. protein level can be detected in the infected corneas at 1 dpi Results (Fig. 2C). As in HCECs, IL-36Ra remained detectable but un- changed from 0 to 18 hpi. HCECs express IL-36a and IL-36g in response to H-K by guest on September 29, 2021 P. aeruginosa challenge Downregulation of IL-1Ra and IL-36Ra had opposing effects We first assessed the induction of the IL-I family of cytokines in on the outcome of P. aeruginosa keratitis cultured HCECs challenged with H-K P. ae ru gi nos a (strain ATCC Having shown the expression pattern of IL-1 cytokines in human 19660). Among IL-36 cytokines, IL-36a and IL-36b were not and mouse CECs, we next used an siRNA approach to investigate

FIGURE 1. IL-1 family cytokine expression in HCECs challenged with H-K P. aeruginosa (ATCC 19660). Primary HCECs were cultured in 6-well plates and challenged with H-K P. aeruginosa (1:50) for the indicated times with 1 ml of culture media. At the indicated times, cells were lysed and subjected to RT-PCR or Western blotting analyses. (A) Two samples at each time point were assessed for the expression of IL-1b, 1Ra, IL-36a, IL-36b, IL- 36g, and IL-36Ra using RT-PCR with b-actin as internal CT. (B) Western blot analysis of IL-36a, IL-36g, and IL-36Ra expression in HCEC extracts (PHCECs) and conditioned media (medium). For cell lysates, 30-mg cellular proteins were used. To assess IL-36 secretion, culture media was collected, and 0.5 ml was subjected to TCA precipitation; total TCA precipitates were loaded in each lane for Western blotting. Chemiluminescence images were acquired using a Kodak Imaging Station 4000-mm Pro with multiple exposure times for each blot. The ﬁgure is representative of three independent experiments. The Journal of Immunology 691 Downloaded from http://www.jimmunol.org/

FIGURE 2. IL-1 cytokines are early responsive genes after P. aeruginosa infection in B6 mouse cornea. Mouse corneas were scratched with a needle and 3 4

inoculated with P. aeruginosa (ATCC 19660, CFU: 1.0 10 ). Whole corneas were collected at 3, 6, 9, and 18 hpi for RNA preparation or at 24 hpi for by guest on September 29, 2021 protein extraction and Western blotting with the NL as the CT. (A) qPCR analysis of IL-1Ra, IL-1b, IL-36Ra, IL-36a, and IL-36g. Results are presented as fold increase relative to those of NL, set as value 1. Stars on top of columns are p value results comparing with the CT. Data are representative of three independent experiments (mean + SEM). *p , 0.05, **p , 0.01, ANOVA. (B) RT-PCR analysis to show basal expression of IL-1 cytokines. Whole corneas collected as described for (A) were also subjected to RT-PCR to determine basal (Naive) and infection-induced expression with b-actin as the internal CT. (C) Immunoblot analysis of IL-36g, in cell lysates of whole corneas infected with P. aeruginosa for 1 d. b-actin serves as the loading CT. the role of IL-36 signaling in mediating corneal response to in- inflammation at 1 dpi, no further observation was made with the fection in comparison with that of IL-1 (Fig. 3). We first assessed group (Fig. 3B). At 3 dpi, knockdown of IL-1Ra resulted in an whether antagonist-specific siRNAs can effectively downregulate increase in the severity of keratitis with a much higher CS (8.6 6 targeted genes in the cornea. In the CT, low levels in NL of IL-1Ra 0.6 versus 11.8 6 0.5), ∼10-fold increases in bacterial burden and IL-36Ra were barely detectable. At 1 dpi, infection induced (4.13 3 106 versus 9.97 3 106 CFU), and an increase in MPO the upregulation of these receptor antagonists. Presence of specific activity (106 versus 217 U) (Fig. 3B, 3C). There was heavy in- siRNA suppressed infection-induced expression of these two filtration in the aqueous humor and clear signs of corneal melting factors to a level similar to the NL, indicating effective knock- in IL-1Ra siRNA-treated corneas (arrow). Because the IL-1Ra down of the target genes (Fig. 3A). Because our time course study siRNA-treated corneas were about to perforate, the experiments showed no detectable elevation of IL-36Ra mRNA up to 18 hpi were terminated at 3 dpi. (Table I). (Fig. 2), we compared mRNA levels of IL-36Ra at 18 and 24 hpi; the results were shown in the inset of Fig. 3A. There was an in- IL-36 signaling differentially regulates the expression of IL-1b, crease in IL-36Ra mRNA from 18 to 24 hpi 1 dpi. IL-1Ra, IL-36g, and antimicrobial genes in CECs at 6 hpi We next examined the pathology of P. aeruginosa–infected We next investigated the expression of IL-1 cytokines and innate corneas treated with different siRNAs. At 1 dpi, IL-1Ra knock- defense molecules in IL-1Ra and IL-36Ra downregulated corneas. down resulted in more severe keratitis with significantly higher CS Our previous study revealed that at 6 hpi, although invading (3.8 6 0.8 versus 6.0 6 1.0), bacterial burden (1.63 3 105 versus bacteria were mostly within the epithelium with low numbers of 4.50 3 105 CFU), and MPO levels (16.0 versus 29.17 U) than infiltrates, the expression of cytokines/chemokines and AMPs in those of CT siRNA-treated corneas. Knockdown of IL-36Ra, in epithelial cells had a profound impact of the outcome of infection contrast, protected the cornea from infection with no sign of in- (39–41). Fig. 4A shows the expression of IL-1 cytokines and two fection and inflammation (CS 0), no recoverable bacteria, and a known AMPs in the isolated CECs detected with quantitative greatly reduced PMN infiltration detected by MPO assay (3.68 U) PCR. Except for IL-36Ra, the expression of all five genes in CT in the cornea (Fig. 3B, 3C). As the invading pathogens in IL-36Ra siRNA-treated, infected corneas were significantly increased to a siRNA-treated corneas were eradicated with minimal signs of different extent than the value seen in noninfected CECs, which 692 IL-1Ra, IL-36Ra, AND BACTERIAL KERATITIS Downloaded from http://www.jimmunol.org/

FIGURE 3. The opposing effects of IL-36RA and IL-1Ra downregulation on the outcome of P. aeruginosa keratitis. Mice were subconjunctivally injected with IL-1Ra, IL-36Ra-specific, or the CT nonspecific siRNA (50 pmol in 5 ml of RNase-free water) twice in 2 d. Corneas were inoculated with P. ae ru gi nos a (ATCC 19660,1.03 104 CFU), 6 h after the second siRNA injection. (A) Western blotting assessing the efficacy of siRNA mediated knockdown of IL-1RA and by guest on September 29, 2021 IL-36RA in the cornea in response to P. aeruginosa infection at 1 dpi with noninoculated corneas (Naive) as the CTs; b-actin serves as the loading CT. Inset shows RT-PCR showing an increase in IL-36Ra levels at 1 dpi, compared with 0 and 18 hpi. (B) Mice were monitored and photographed daily up to 3 dpi, showing IL-36Ra and IL-1Ra for 1 dpi and IL-1Ra for 3 dpi in siRNA-treated corneas. The numbers within each eye micrograph (original magnification 310) are the CSs assigned and presented as median plus interquartile range. Arrow shows melting point. (C) At 1 or 3 dpi, the corneas were excised and subjected to bacterial plate counting and MPO assay. The data were presented as total number of bacteria or MPO units per cornea and were representative of three independent experiments (n = 6 each) and indicated p values were generated using unpaired Student t test. **p , 0.01. was set as 1 (NL). Downregulation of IL-36Ra further significantly (5.25- versus 19.66-fold), and CXCL10 (2.66- versus 7.37-fold), augmented infection-induced expression of IL-1Ra (36.3- but not IL-1b, in infected CECs. Downregulation of IL-1Ra vastly versus 70.3-fold), IL-36g (3.17- versus 7.87-fold), S100A8 increased IL-1b expression (9.69- versus 22.52-fold), whereas it

Table I. Primer sequences used for PCR

Primers Forward (59→39) Reverse (59→39) mb-actin 59-GACGGCCAGGTCATCACTATTG-39 59-AGGAAGGCTGGAAAAGAGCC-39 mIL-1b 59-AAGGAGAACCAAGCAACGACAAAA-39 59-TGGGGAACTCTGCAGACTCAAACT-39 mIL-1RA 59-GGGACCCTACAGTCACCTAA-39 59-GGTCCTTGTAAGTACCCAGAC-39 mCXCL10 59-ATGAACCCAAGTGCTGCCGTC-39 59-TTAAGGAGCCCTTTTAGACCTTT-39 mS100a8 59-TGCGATGGTGATAAAAGTGG-39 59-GGCCAGAAGCTCTGCTACTC-39 mIL-36a 59-CCAAGAACTGGGGGAAATCT-39 59-GGAGGGCTCAGCTTTCTTTT-39 mIL-36g 59-CCCATGCAAGTACCCAGAGT-39 59-GGGAAAGCCACTGATTCAAA-39 mIL-36Ra 59-GGATGCTCCCATCACAGACT-39 59-CAGAAGCGGGAGTCCAGTAG-39 mIL-24 59-GCTTTCACCAAAGCGACTTC-39 59-GCCCAGTAAGGACAATTCCA-39 mIL-6 59-GTGGCTAAGGACCAAGACCA-39 59-TAACGCACTAGGTTTGCCGA-39 hIL-36a 59-GAAAGCCACAGACTCGAAGG-39 59-AAGACAGAGGGAACCCCATC-39 hIL-36b 59-GCACCTGAATTCCCACAGAG-39 59-AGCGTTTCACTCCTCATGCT-39 hIL-36g 59-AATTGTGGGCTGTCCAAC-39 59-GGTCAGAACCTTGTGGCAGT-39 hIL-36Ra 59-AGGTTTCAGCAGAGCCGTTA-39 59-AAGGTCATGCTGGATGAAGG-39 hIL-1b 59-ACCAAACCTCTTCGAGGCAC-39 59-AGCCATCATTTCACTGGCGA-39 hIL-1Ra 59-CTGGGGGTTCTTTCTTCCTC-39 59-TAGGGAACTTTGCACCCAAC-39 hb-actin 59-GGACTTCGAGCAAGAGATGG-39 59-AGCACTGTGTTGGCGTACAG-39 mBD3 59-GCTAGGGAGCACTTCTTTGCATTTAA-39 59-GCTTCAGTCATGAGGATCCATTACCT-39 mMMP13 59-TGATGAAACCTGGACAAGCC-39 59-TGGACCATAAAGAAACTGAA-39 The Journal of Immunology 693

FIGURE 4. Effects of IL-36Ra or IL-1Ra knockdowns on the gene expression in B6 mouse corneal epithelia at an early stage of P. aeruginosa infection, 6 hpi. Mouse corneas were treated with siRNAs and inoculated with P. aeruginosa as in Fig. 3. Corneal epithelial samples were collected at 6 hpi for qPCR Downloaded from analysis for the expression of IL-1b, IL-1Ra, IL-36g, IL-36Ra, S100A8, and CXCL10 (A) or ELISA for calprotectin and CXCL10 (B). The results of qPCR are presented as relative increase (fold) to those of CT siRNA-treated, uninfected corneas, set as 1 after normalization to the level of b-actin as the internal CT. The results of ELISA are presented as picograms per microgram total protein. Data are representative of three independent experiments with three corneas per group (mean + SEM). ANOVA for qPCR; unpaired t test for ELISA. *p , 0.05, **p , 0.01.

exhibited no effects on other genes, compared with the CT siRNA- the NL, infiltration and an even distribution of neutrophils and mac- http://www.jimmunol.org/ treated CECs. rophages within the stroma were observed in P. aeruginosa–infected To determine whether the aforementioned factors were also corneas at 1 dpi. The downregulation of IL-1Ra greatly increased the expressed at the protein level, we selected CXCL10, a multi- numbers of infiltrated neutrophils and macrophages. Similar to the faceted function protein, and calprotectin (S100A8/A9) for cytokine expressions, the large numbers of infiltrated cells in IL-1Ra ELISA analyses of mouse CECs (Fig. 4B). At 6 hpi, there was siRNA-treated corneas may be related to strong host responses to the marked upregulation, compared with the NL, of both CXCL10 presence of the increased bacterial burden. No infiltrated neutrophils (7.52 versus 90.52 pg/mg total proteins) and calprotectin (22.75 and macrophages were detected in the corneas treated with IL-36Ra versus 57.02). IL-36Ra downregulation augmented CXCL10 siRNA, consistent with the lack of signs of keratitis in P. aeruginosa– by guest on September 29, 2021 markedly (90.52 versus 160.62) and calprotectin moderately infected corneas at 1 dpi. (57.02 versus 72.43), whereas IL-1Ra downregulation suppressed Exogenously-added IL-36Ra and IL-1Ra had opposing CXCL10 (90.52 versus 61.44) but not calprotectin expression in effects on the pathogenesis of P. aeruginosa keratitis in P. aeruginosa–infected corneas. B6 mouse corneas Downregulation of IL-1Ra and IL-36Ra had opposing effects To further explore the function of IL-36Ra and IL-1Ra, we in- on the expression of IL-1 cytokines and antimicrobial peptides vestigated the effects of exogenous receptor antagonists on the in B6 mouse corneas at 1 dpi pathogenesis of P. aeruginosa keratitis, using 150 mg/ml anakinra The expression of the aforementioned six genes were also inves- (human recombinant protein), IL-36Ra, or BSA as the CT. Fig. 7A tigated at 1 dpi in the corneas where heavy infiltrates, including and 7B showed that at 1 dpi, although mild keratitis can be seen in neutrophils, macrophages, dendritic cells, NK cells, and gdT cells, the CT, BSA-treated corneas (CS 4.0), recombinant IL-36Ra in- can be detected in the CT corneas. At this time point, all six genes, creased the severity of keratitis (CS 8.88), including greatly including IL-36Ra, were upregulated in CT siRNA-treated cor- augmented opacification of the cornea, 3-fold surge of bacterial neas at 1 dpi. Consistent with the minimal signs of infection and burden, and significantly elevated MPO activity (21.7 versus 38.6 inflammation observed, the levels of IL-1b, IL-1Ra, IL-36g, U). Presence of IL-1Ra, in contrast, inhibited the development of IL-36Ra, and S100A8 mRNA were similar to those of NL, with keratitis, resulting in little visible opacification, a very low number CXCL10 slightly elevated but significantly lower than that in of bacteria (176.4 6 167.2), and low, but measurable, MPO ac- infected and CT siRNA-treated corneas (35.4-versus 15.4-fold tivity (5.19 U), indicating residual levels of PMNs. The same cell increase over the NL). IL-1Ra downregulation, in contrast, greatly lysates used for CFU and MPO determination were also subjected upregulated IL-1b (77.3- versus 316.6-fold increase), IL-36g to ELISA measurements of IL-1b and CXCL2, also termed MIP-2. (5.88 versus 8.86), S100A8 (38.74- versus 81.18-fold), and Infection-induced IL-1b and CXCL2 expressions (160.1 and CXCL10 (35.4- versus 142.72-fold) in P. aeruginosa–infected 34.96 pg/mg total protein) were markedly augmented by IL-36Ra corneas. This elevation may contribute to the severe inflammation treatment (720.7 and 111.5 ng) but were greatly suppressed by in IL-1Ra–downregulated corneas (Fig. 5). IL-1Ra treatment (5.17 and 4.51 ng) (Fig. 7C). To understand the mechanisms underlying the IL-36Ra–augmented IL-36Ra and IL-1RA exhibit opposing effects on neutrophil and severity of P. aeruginosa keratitis, the expression of cytokines (IL-6, macrophage infiltrations in infected corneas IL-24) and the antimicrobial chemokines CXCL10 and S100A8 in Having shown differential CT of IL-36Ra and IL-1Ra on the ex- response to exogenous receptor antagonists in CECs at 6 hpi were pression of cytokines, chemokines, and AMP, we next assessed assessed using qPCR (Fig. 6D). Presence of IL-36Ra augmented their effects on the infiltration of innate immune cells in P. aeruginosa– the expression of both IL-6 (2.6- versus 6.28-fold increase over the infected corneas (Fig. 6). Although immune cells can be detected in naive CECs) and IL-24 (3.65- versus 6.70-fold increase) but 694 IL-1Ra, IL-36Ra, AND BACTERIAL KERATITIS Downloaded from

FIGURE 5. Effects of IL-36Ra or IL-1Ra knockdowns on the gene expression in B6 mouse cornea at 1 dpi. Mouse corneas were treated with siRNAs and http://www.jimmunol.org/ inoculated with P. aeruginosa as in Fig. 3. Corneas were excised and processed for real-time PCR analysis at 1 dpi. The results are presented as a relative increase (fold) to those of CT siRNA-treated, uninfected corneas, set as 1 after normalization to the level of b-actin as the internal CT. Data are representative of three independent experiments with three corneas per group (mean + SEM). *p , 0.05, **p , 0.01, ANOVA. suppressed CXCL10 (4.94- versus 1.71-fold) and S100A8 (6.61- inﬂammation. To test this hypothesis, we subconjunctivally injected versus 1.37-fold) expression. IL-1Ra exhibited opposing effects murine IL-36g and assessed the expression innate protective on the expression of these factors, except for S100A8 (2.6- versus (S100A9, b-defensin 3, and CXCL10) and IL-1 cytokines (IL-1b 1.2-fold for IL-6, 3.65- versus 2.67-fold for IL-24, and 4.94- and IL-1Ra) (35) in the treated corneas prior to P. aeruginosa by guest on September 29, 2021 versus 11.98-fold for CXCL10). infection (Fig. 8A). IL-36g treatment without infection stimulated the expression of S100A9 (12.21-fold increase over the NL), BD3 Exogenous IL-36g enhances corneal innate immunity and (1.83-fold), and CXCL10 (10.56-fold), whereas it exhibited no alleviates P. aeruginosa keratitis via induction of innate defense effects on IL-1Ra and downregulated IL-1b expression (1.0 versus genes in B6 mouse corneas 0.26) in the whole corneas (Fig. 8A). The IL-36g–stimulated ex- The aforementioned effects of IL-36Ra on the pathogenesis of pression of S100A9, BD3, and CXCL10 in the corneas was likely P. aeruginosa keratitis suggest that modulating IL-36 signaling may be due to participation in pathogen killing at the early stages of infection. used to enhance innate immunity and/or to ease infection-associated To test this hypothesis, we assessed in vitro bactericidal activity by

FIGURE 6. Effects of IL-36Ra or IL-1Ra knockdowns on PMN and macrophage inﬁltration in B6 mouse cornea at 1 dpi. Mouse corneas were treated with CT (marked at right side), IL-1Ra (marked at left side), or IL-36Ra (marked at right side) siRNAs and inoculated with P. aeruginosa as in Fig. 3. NL (marked at left side) were used as negative CT. The corneas were excised and processed for immunohistochemistry analysis at 1 dpi. The 6-mm cryostat sections of the corneas were stained with Ab NIMP-R14 for neutrophils and F4/80 for macrophages. The images of inﬁltrated cells (green) were merged with DAPI (blue nuclei) staining (M). Two independent experiments were performed; 1 representative image for each condition is presented. Scale bar, 100 mm. E, epithelium; S, stroma. The Journal of Immunology 695 Downloaded from http://www.jimmunol.org/

FIGURE 7. Effects of recombinant IL-36Ra or IL-1Ra (anakinra) on the severity of P. aeruginosa keratitis and on the gene expression in B6 mouse corneas. Mice were subconjunctivally injected with either recombinant IL-36Ra, anakinra (150 ng), or with 0.1% BSA as the CT. Mouse corneas were inoculated with P. aeruginosa 4 h after the protein injection. (A) P. aeruginosa–infected corneas were photographed (original magnification 310) at 1 dpi. The numbers within by guest on September 29, 2021 each eye micrograph are the CSs assigned and presented as median plus interquartile range. (B) The corneas at 1 dpi were excised and subjected to bacteria load, MPO unit determination, and (C) ELISA assay analysis for CXCL-2 and IL-1b. The letters a, b,andc correspond to the corneas shown in (A). (D)To determine the effects of receptor antagonists on corneal gene expression, recombinant IL-36Ra- or anakinra-injected corneas with or without (N) P. ae ru gi nos a infection were collected and subjected qPCR analysis. The letters a, b,andc correspond to the corneas shown in (A). The p values were generated by one-way ANOVA. Data are representative of three independent experiments with five mice per group. *p , 0.05, **p , 0.01. N, naive. mixing 2000 CFU P. aeruginosa with whole corneal homogenates. for the CT infected, 0 for IL-36g pretreated, 26760 for CXCL10, The homogenates derived from IL-36g–treated corneas killed and/or and 89320 for CXCR3 neutralized corneas), and PMN infiltration inhibited the growth of P. aeruginosa (1687 CFU remaining), was assessed by MPO assay (23.17 U/cornea for the CT infected, whereas in the BSA-treated corneal homogenates, P. aeruginosa 1.4 for IL-36g pretreated, 5.4 for CXCL10, and 11.75 for CXCR3 numbers increased to 2750 CFU (Fig. 8B). neutralized corneas). Neutralization of CXCR3 had more pro- The aforementioned IL-36a–treated corneas were then inocu- found effects on the positive effects of IL-36 than that of CXCL10 lated with P. aeruginosa. At 1 dpi, in the presence of exogenous (Fig. 10). IL-36g, the corneas had almost no detectable signs of infection (only one of five corneas had slight opacification, giving an av- Discussion erage CS of 0.2), no bacteria recoverable by culture, and very low In this study, we compared the expression and function of two large levels of MPO, consistent with corneal photographs showing no IL-1 cytokine subfamilies by manipulating their receptor antago- sign of opacification and inflammation (Fig. 9). Our previous nists in a mouse model of P. aeruginosa keratitis. We observed that study revealed that both S100A8 and A9 were the most highly P. aeruginosa triggers the expression of IL-1b, IL-Ra, IL-36a, induced AMPs and that neutralizing either subunit resulted in IL-36g, and IL-36Ra in HCECs and mouse CECs and in whole B6 much more severe keratitis in P. aeruginosa–infected corneas (40). mouse corneas. We also detected IL-36g secretion in cultured We therefore selected S100A9 neutralizing Ab to be applied HCECs challenged with H-K P. aeruginosa. Surprisingly, IL-36Ra concomitantly with IL-36g (Fig. 9). Depletion of S100A9 di- and IL-1Ra knockdown resulted in opposing effects on the severity minished IL-36g–stimulated protection, resulting in similar CSs, of P. ae ru gi nos a keratitis: IL-36Ra downregulation prevented cor- partially decreased bacterial burdens, and MPO activity compared neal infection, whereas IL-1Ra greatly increased the severity of with the BSA-treated, CT corneas (Fig. 9). P. ae ru gi nos a keratitis. During the early stages of infection (6 hpi), Finally, the involvement of CXCL10 as well as its chemokine IL-36Ra downregulation decreased the ratio of IL-1b to IL-1Ra and receptor CXCR3 in IL-36–stimulated innate protection was also increased the expression of IL-36g, S100A8, and CXCL10, whereas assessed using neutralizing Abs. Neutralizations of CXCL10 and IL-1Ra siRNA had opposing effects in mouse CECs. At 1 dpi, IL-1Ra CXCR3 only had limited effects on bacterial burden (229200 CFU siRNA altered the ratio of IL-1b to IL-Ra and increased expression 696 IL-1Ra, IL-36Ra, AND BACTERIAL KERATITIS Downloaded from

FIGURE 8. Recombinant IL-36g induces the expression of innate immune defense molecules in B6 mouse corneas. (A) B6 mice were either treated with 0.1% BSA as the CT or IL-36g for 4 h. The treated corneas were excised and processed for real-time PCR analysis. The results are presented as relative increase (fold) to those of BSA-treated, uninfected corneas, set as 1 after normalization to the level of b-actin as the internal CT. Data are representative of three independent experiments with three corneas per group (mean + SEM). *p , 0.05, **p , 0.01, t test. (B) The 0.1% BSA– or IL-36g–treated corneal http://www.jimmunol.org/ homogenates were incubated with 1000 CFU P. aeruginosa for 1 h at 37˚C. The homogenates were then subjected to plate bacterial counting. The results were presented as total CFU per cornea, n =3.*p , 0.05. of IL-36g, S100A8, and CXCL10; these factors were minimally on the pathogenesis of P. aeruginosa keratitis: IL-1Ra (anakinra) detected in IL-36 siRNA-treated corneas. Furthermore, exogenously- supported whereas IL-36Ra suppressed corneal innate defense added IL-1Ra and IL-36g prevented, whereas IL-36Ra augmented, against P. aeruginosa. In these treated corneas, the severities of the pathogenesis of P. aeruginosa keratitis. IL-36g induced or keratitis were assessed by opacification, quantitated as CSs, and augmented the expression of innate defense molecules S100A9, by pathogen burdens. Zhou et al. (42) have shown that recruitment by guest on September 29, 2021 mouse b-defensin 3, and ISG15 in NL and enhanced bactericidal ac- of PMN and activation of IL-1R signaling were associated with tivity of corneal homogenates. IL-36b–induced protection was abol- stromal thickness and haze in Serratia marcescens–infected cor- ished in the presence of S100A9 neutralizing Ab. Thus, IL-36/IL-36R neas. Our data also showed a strong association of elevated IL-1b and IL-1/IL-1R signaling pathways had opposing effects on the ex- expression and/or altered balance of IL-1b/sIL-1Ra with neutrophil pression of immunoregulatory and effector genes such as AMPs and, as infiltration, which in turn causedcornealopacificationinanIL-36– a consequence, on the outcome of P. aeruginosa keratitis. related manner. To our knowledge, this is the first study that directly The expression, secretion, and activation of the IL-1 subfamily of compared the effects of two receptor antagonists on bacterial infection. cytokines have been studied extensively. In this study, we compared The striking differences in the outcomes of keratitis between the the expression patterns of IL-1 and IL-36 cytokines in CECs and in the manipulations of IL-1R and IL-36R mediated signaling pathways corneas in response to infection. Our study revealed that unlike IL-1b indicate that although the activation of IL-1 signaling may be detri- and sIL-1RN, IL-36g and IL-36RN mRNA were present in these mental, the IL-36/IL-36R axis plays a protective role in controlling naive cells, suggesting a potential role of IL-36/IL-36R signaling P. aeruginosa keratitis in B6 mouse corneas. in corneal homeostasis. Infection induced the expression of IL-1b, Like IL-1 cytokines, IL-36 agonists are generally considered as sIL-1Ra, IL-36a, and IL-36g as early as 2 hpi and induced the ex- proinflammatory (43, 44). The IL-1 and IL-36 subfamilies share pression of IL-36Ra at 1 dpi. Moreover, to our knowledge, our study IL-1RAcP as a coreceptor and activate NF-kB and p38 signaling for the first time showed the secretion of IL-36g by P. aeruginosa– pathways through MyD88 (3, 43). It is now evident that IL-36 challenged HCECs. The mechanisms underlying IL-36 cytokine cytokines play an important role in inflammatory disorders in secretion, including IL-36Ra, remain largely undetermined. The ex- different organs such as the gastrointestinal tract (45) and the lung pression pattern demonstrated in the current study suggests that IL-36 (25, 46). In the skin, all three IL-36R agonists were found to be cytokines are a part of the network of cytokines/chemokines involved highly expressed and were involved in the pathogenesis of pso- intissueinnateimmuneresponsetomicrobial infection, similar to riasis, a chronic inflammatory, multisystem disease (47–49). that shown in the lung (25, 28, 29). Mutations of IL-36RN, which encode IL-36Ra, cause generalized Taking advantage of the subconjunctival injection procedure and pustular psoriasis (19), providing support for the proinflammatory siRNA technology, we observed striking differences in the patho- nature of IL-36/IL-36R signaling. Interestingly, generalized pus- genesis of P. aeruginosa keratitis: IL-1Ra downregulation greatly tular psoriasis carrying IL-36Ra mutations can be successfully exacerbated keratitis, whereas IL-36Ra siRNA protected B6 mouse treated with anakinra, a human recombinant IL-1Ra, suggesting corneas from P. aeruginosa. The opposing effects of IL-1Ra and that the culprit for the diseases was excessive activation of IL-1 IL-36Ra on the outcomes of P. ae ru gi nos a keratitis were further signaling pathway. Hence, how does anakinra abrogate the detri- corroborated by the use of recombinant human IL-1Ra (anakinra) mental effects of IL-36Ra insufficiency? One possibility is that IL-1 and mouse IL-36Ra. These antagonists exhibited opposing effects cytokines are the downstream targets of IL-36. Indeed, our study The Journal of Immunology 697 Downloaded from http://www.jimmunol.org/

FIGURE 9. Recombinant IL-36g protects B6 mouse corneas from P. ae ru gi nos a infection in a S100A9-dependent manner. Mice were subconjunctivally injected with BSA as the CT, 150 ng of IL-36g + 100 ng of IgG, or 150 ng of IL-36g + 100 ng of S100A9 neutralizing Ab in 5 ml PBS. Mouse corneas were inoculated with P. aeruginosa 4 h after the injection. (A) P. aeruginosa–infected corneas were photographed (original magniﬁcation 310) 1 dpi. The numbers

1, 2,and3 within each eye micrograph are the CSs assigned and presented as median + interquartile range. At 1 dpi, the corneas were excised and subjected to by guest on September 29, 2021 bacteria load (B) and MPO unit determination. Data are representative of three independent experiments with ﬁve mice per group. **p , 0.01.

revealed that downregulating IL-36Ra or exogenous IL-1Ra, in elevated, in contrast to those observed in CECS at 6 dpi. This, contrast to knockdown of IL-1Ra or exogenous IL-1Ra, attenuated however, may be related to the strong response of the host to the IL-1b but augmented the expression of sIL-1Ra at an early stage increased pathogen burden in the treated corneas. Furthermore, in (6 hpi) of infection. At this time point, most invading bacteria are IL-36g–treated corneas, three innate defense molecules, S100A9, restricted within the epithelial layers (35). Hence, IL-36 signaling mouse b-defensin 3 (homolog of human b-defensin 2), and CXCL10, may alter the IL-1b/IL-1Ra balance in favor of anti-inflammatory were induced in NL while IL-1b was suppressed. Hence, IL-36 effects, and IL-36/IL-36R signaling is anti-inflammatory in cytokines may control the outcome of bacterial keratitis by P. aeruginosa keratitis. suppressing proinflammatory IL-1R signaling and by stimu- How might IL-36R activation by siRNA knockdown of IL-36Ra or lating the expression of AMPs in the epithelium at the initial exogenous IL-36g mediate the innate killing of invading pathogens, stage of infection, resulting in the eradication of invading pathogens resulting in total eradication of inoculated P. aeruginosa in B6 mouse andtherapidresolutionofinflammation associated with infection. Our corneas? Our data showed that the downregulation of IL-36Ra, in in vitro P. aeruginosa killing assay results support a role of IL-36R contrast to that of IL-1RA, augmented the infection-induced expres- signaling in promoting innate immune killing of invading pathogens in sion of S100A8 and CXCL10 at both the mRNA and protein levels, the cornea. Moreover, neutralizing Ab of S100A9 abolished IL-36g– whereas exogenous IL-36Ra stimulation greatly downregulated their induced protection, resulting in P. aeruginosa keratitis with smaller expression in CECs at 6 hpi. Hence, we propose that IL-36R activa- pathogen burden and fewer infiltrated PMNs when compared with the tion–stimulated CXCL10 and/or calprotectin are the effectors that play CT, P. aeruginosa–infected corneas. In contrast, neutralizing CXCL10 an important role in innate defense during early time of P. aeruginosa had significant but limited effects on bacterial burden and on PMN infection of the cornea. However, at 1 dpi, the expression of afore- infiltration, whereas the effects of neutralizing its receptor CXCR3 mentioned factors, except CXCL10, were either undetectable were significantly higher. Our previous studies revealed that CXCL10 or similar to the naive CTs in IL-36Ra siRNA-treated corneas, neutralization had no effect on the outcome of P. aeruginosa keratitis consistent with the eradication of invading pathogens and the reso- (50), whereas the neutralizations of CXCL10 and CXCR3 resulted in lution of inflammation, as evidenced by the lack of infiltrated neu- much more severe keratitis in Candida albicans–infected corneas trophils and macrophages in the corneas at this time point. In IL-1Ra (38, 41). Cells expressing CXCR3 at early stage of infection are NK siRNA-treated corneas, although the balance of IL-1b/IL-1Ra cells that are necessary for innate defense against both P. aeruginosa remained in favor of a proinflammatory state, the infection- and C. albicans infection of the cornea (38, 41, 50). Thus, CXCL10, induced expressions of IL-36g, S100A8, and CXCL10 were all likely functioning through CXCR3 as a chemokine, plays a minor 698 IL-1Ra, IL-36Ra, AND BACTERIAL KERATITIS Downloaded from http://www.jimmunol.org/

FIGURE 10. CXC10/CXCR3 signaling plays a minor role in IL-36g–stimulated innate protection against P. aeruginosa infection. Mice were subconjunctivally injected with 0.1% BSA as the CT, 150 ng IL-36g + 100 ng IgG, or 150 ng IL-36g + 100 ng CXCL10 or CXCR3 neutralizing Ab in 5 ml PBS. Mouse corneas were by guest on September 29, 2021 inoculated with P. aeruginosa 4 h after the injection. (A) P. aeruginosa–infected corneas were photographed (original magnification 310) 1 dpi. The numbers within each eye micrograph are the CSs assigned and presented as median plus interquartile range. At 1 dpi, the corneas were excised and subjected to bacteria load (B)and MPO unit determination (C). Data are representative of three independent experiments with five mice per group. *p , 0.05, **p , 0.01. role in IL-36g–stimulated innate protection against P. aeruginosa bouts of infection with Gram-positive and Gram-negative bac- infection, and other CXCR3 ligands, CXCL9 and/or CXCL11, teria as well as fungi, anakinra treatment reduces the severity of might also be involved in B6 mouse corneas. We proposed that inflammatory bowel disease and disease-associated granulomas upregulation of AMPs, such as calprotectin and/or b-defensins, without increased infection (56–58). Hence, local application are an underlying mechanism for the IL-36/IL-36R axis to of anakinra, an IL-36 agonist, alone or in combination, may be enhance cornea innate immunity against infectious keratitis. used as an adjuvant treatment with antibiotics for infectious WefurtherproposethenovelconceptthattheIL-36subfamily keratitis. or IL-36R signaling may antagonize that of the IL-1 subfamily in mediating innate immunity early during P. aeruginosa infection Acknowledgments of B6 mouse corneas. We thank all the members of the Yu laboratories for assistance and Finally, our study suggests that anakinra may be used as an comments on the work. We thank Patrick Lee for critical reading of adjuvant treatment for bacterial keratitis and that IL-36 agonist the manuscript. may be used as an alternative therapeutic of IL-1 neutralization medications in treating a host of inflammation and infection Disclosures diseases. This seems paradoxical, given that IL-1 is a master The authors have no financial conflicts of interest. cytokine of local and/or systemic inflammation, which is required for immune cell infiltration and for the effective eradication of invading pathogens. For example, mice deficient in IL-1R had References higher P. aeruginosa burden in the lungs (51, 52) and suffered 1. Dinarello, C. A. 2009. Immunological and inflammatory functions of the interleukin-1 family. Annu. Rev. Immunol. 27: 519–550. lethal necrotic pneumonia following Mycobacterium tuberculosis 2. Gabay, C., C. Lamacchia, and G. Palmer. 2010. IL-1 pathways in inflammation infection (53). This paradox can perhaps be resolved by the and human diseases. Nat. Rev. Rheumatol. 6: 232–241. findings regarding long-term use of anakinra; anakinra has been 3. Garlanda, C., C. A. Dinarello, and A. Mantovani. 2013. The interleukin-1 family: back to the future. Immunity 39: 1003–1018. used to treat rheumatoid arthritis and related diseases in over 4. Hahn, M., S. Frey, and A. J. Hueber. 2017. The novel interleukin-1 cytokine 100,000 patients, some for over 15 y, without significant increases family members in inflammatory diseases. Curr. Opin. Rheumatol. 29: 208– 213. in the rate of infection for the patients (54, 55). In patients with 5. Latz, E., T. S. Xiao, and A. Stutz. 2013. Activation and regulation of the chronic granulomatous disease, an inherited condition with multiple inflammasomes. Nat. Rev. Immunol. 13: 397–411. The Journal of Immunology 699

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