| BackgroundFungal keratitis is a corneal infection caused by pathogenic fungi and is one of the most prevalent potentially blinding conditions worldwide.Among the pathogenic fungi involved in fungal keratitis,Aspergillus was identified as the dominant causative agent of infection,accounting for:26%of cases in the UK,30%in northern USA,41%in the Middle East,and 18%-60%in Asia.It is reported that 42.60%of Aspergillus keratitis patients need keratoplasty.In both developing and developed countries,the main causative pathogen of Aspergillus keratitis is Aspergillus fumigatus.Compared with Aspergillus flavus,A.fumigatus keratitis has a worse prognosis,higher recurrence rate,and greater clinical significance.Fungal infection remains a serious vision-threatening disease due to its rapid progress,poor prognosis,and absence of effective symptomatic treatment.The natural immune barrier of the ocular surface is divided into three layers,a lipid layer and a water layer of the tear film,and corneal epithelial cells.Therefore,the corneal epithelium becomes the first cell barrier of the cornea to resist fungi.The cells recognize the pathogen associated molecular patterns(PAMPs)on the surface of pathogenic microorganisms through surface-distributed pattern recognition receptors(PRRs).Over the years,our research team has conducted systematic and in-depth research on the function and regulation of innate immunity of corneal antifungal infection,confirming Toll like receptors(TLRs)located on the membrane of corneal epithelial cells and NOD-like receptors(NLRs)located intracellularly are the two most important types of PRRs that recognize fungi.They synergistically activate downstream molecular pathways such as the NF-?B pathway to induce secretion of inflammatory and antimicrobial peptides against fungal invasion.There are 11 Toll-like receptors found in the corneal epithelium.TLR1,TLR2,TLR3,TLR5,TLR6 and TLR9 are strongly expressed at the gene level,but the expression of TLR2,TLR4 and TLR5 can be detected at the protein level.Our previous study showed for the first time that pretreatment of transformed human corneal stroma fibroblast(THSFs)with the Toll-like receptor 2(TLR2)-specific ligand zymosan resulted in a state of A.fumigatus hyphae tolerance.Co-pretreatment with TLR2 and TLR4 ligands(zymosan and lipopolysaccharide)led to a stronger state of A.fumigatus hyphae tolerance,and suppressed the lethal effect of A.fumigatus.Transformed human corneal epithelial cells(HCECs)responded to challenge with TLR2 and TLR4 ligands by secreting IL-1? and IL-6.Furthermore,they recognized A.fumigatus hyphae via TLR2 and TLR4 receptors and initiated an innate immune response.This demonstrated that TLR2 and TLR4 play an important role in the regulation of innate immunity induced by A.fumigatus.Vu et al.found that the diacylated lipopeptide(TLR2-TLR6 ligand)stimulated primary cultured human keratinocytes to express thymic stromal lymphopoitein(TSLP).TSLP is a novel IL-7-like cytokine,known as a pro-inflammatory cytokine,which is induced in the innate phase of an allergic immune response.TSLP can also be released by human epithelial cells in response to microbes,trauma,or inflammation.Some recent findings suggested that TSLP might be involved in infection.Previously,we proved that TSLP and TSLP receptor(TLSPR)were expressed in transformed HCECs after A.fumigatus stimulation.TSLP could stimulate the expression of TLR2 downstream signaling molecules.Furthermore,the increase in TLR2 downstream signaling molecules induced by A.fumigatus hyphae could be hindered by RNA interference of TSLP.This inference with TSLP may exhibit cross-talk with TLR,playing an important role in Aspergillus keratitis.Previous findings from Wang et al.suggested that HCEC-derived TSLP played a key role in the adaptive immune responses of fungal keratitis via skewing Th2 differentiation and promoting humoral immunity.However,the interactions among TSLP,TLRs,anti-microbial peptides(AMPs),and immune cells have not been thoroughly investigated.Thus,we aimed to reveal the role of TSLP in A.fumigatus-induced anti-fungal innate immunity.Our study also found that another inflammatory factor,IL-36,has a protective effect in the innate immunity of corneal antifungal infection by inhibiting IL-1?.The IL-3 6 subfamily is a newly named pro-inflammatory cytokine whose members include IL-36?,IL-36?,IL-36?,and IL-36Ra.Its structural model is similar to the classical 1L-1 family,which can be produced by epithelial cells and a variety of cell types,such as nuclear cells,B cells,and T cells.IL-36R is present on DCs and naive T cells.Studies have shown that IL-36?,IL-36? and IL-36? are agonists of IL-36R,which activate NF-?B,MAPK,Erk1/2 and JNK pathways;IL-36Ra is a natural IL-36R antagonist.Studies have shown that Aspergillus fumigatus can up-regulate the expression of IL-36?,IL-36? and IL-36Ra in PBMCs.Among them,the up-regulation of IL-36? expression is dependent on the Dectin-1/Syk pathway and the TLR4 pathway.In addition,Aspergillus fumigatus upregulates the expression of IL-17 and IL-22 by activating the IL-36R pathway,which is blocked by IL-36Ra.IL-36 regulates the IL-23/IL-17/IL-22 cytokine secretion axis in psoriasis.In natural immunity,IL-36 can induce bone marrow-derived dendritic cells(BMDC)in mice to produce inflammatory factors such as TNF-?,IL-1?,IL-6 and IL-23.6 and IL-23-based inflammatory factors can promote the differentiation of ThO to Th17.Studies in vitro have found that IL-36? and IL-36? can enhance the antibacterial effect of IL-17 A,while IL-17 A,IL-22 and TNF-? can up-regulate the expression of IL-36?,IL-36? and IL-36?.These studies suggest that IL-36 may be involved in the body's anti-fungal infection,induce DCs to produce cytokines,promote Th0 proliferation,and have some positive feedback mechanism with Thl 7-type acquired immunity.In this study,through the study of TSLP and IL-36 family factors in mouse corneal infection model,we aim to reveal the mechanism of the two types of factors regulate the immune regulation of inflammation and innate immune response after fungal infection.Part ? Interactions of thymic stromal lymphopoietin with TLR2 and TLR4 regulate anti-fungal innate immunity in Aspergillus fumigatus-induced corneal infectionPurpose:To revealed the function of TSLP in mediating TLR2 and TLR4 in anti-fungal inflammatory response and serving as a target to control tissue injury and infection in A.fumigatus keratitis.Materials and Methods1 Aspergillus fumigatus strain and preparation of the hyphae suspensionA.fumigatus strain CCTCC 93024 was incubated on Sabouraud dextrose agar for 24 h at 37? on a shaking table with a rotation speed of 200 rpm.Subsequently,to collect hyphal fragments,conidia were harvested and planted into Sabouraud fluid media.For in vivo studies,conidia and hyphae were diluted with PBS to 2×108 CFU and used at 1×106 CFU(in 5 ?l)per eye.For in vitro studies,the hyphae were used at a density of 2×106 pieces/ml.2 Cell culture,siRNA transfection,and cell treatmentTransformed human corneal epithelial cells(HCECs),a SV40 immortalized human CEC line,were maintained in Dulbecco's modified Eagle's medium(DMEM)/F 12 supplemented with 50%defined keratinocyte serum-free medium in an incubator with 5%CO2 at 37?.Cells were seeded at 1×105 cells/well in 6-well plates and cultured in normal growth medium.HCECs were treated with 2×106 pieces/ml of A.fumigatus suspension or 100 ng/well human recombinant TSLP protein for 6 h.For transient transfection with TSLP small interfering RNA(siRNA),HECEs were seeded at 1×105 cells/well in 6-well plates and transfected by 100 pmol TSLP siRNA for 48 h.The transfection efficiency was assessed by ELISA.HCECs were treated with A.fumigatus for 12 h after transfection.The culture supernatants and cells were harvested for RT-PCR,western blotting,and ELISA experiments.Dendritic cells(DCs)were extracted from the mouse leg bone following the standard bone marrow cell extraction protocol.DCs were cultured for 7 days before A.fumigatus hyphae treatment.The culture supernatants and cells were harvested for western blotting at 6 h post-infection(hpi).3 Animals,corneal infection protocol,and disease evaluationFor corneal infection,Wild-type C57BL/6 mice were anesthetized with 0.2 ml pentobarbital(10 mg/ml)by intraperitoneal injection,and the right corneas were scarified with three 1-mm incisions using a sterilized 26-gauge needle.For each cornea,a cover of parafilm M film cut using a 3 mm trephine was used as a contact lens on the surface of the scarifved colvea.A 5 ?l suspension containing 1×106 CFU of A.fumigatus was injected into the gap between the parafilm M film and the cornea.The eyelids were sutured together for 12-24 h before being observed at different time points(12,24,48,and 72 hpi,and 5 days post-infection,dpi).Clinical examination was performed with corneal photography and clinical scoring.The corneas were stored at-80? and used for mRNA or protein extraction.4 TSLP knockdown or recombinant TSLP protein stimulation treatment of miceMice were anesthetized as previously described.For TSLP knockdown experiments,TSLP siRNA(5 ?l,1 ?g/?l)was injected subconjunctivally twice at 24 h and 4 h before A.fumigatus infection.For TSLP stimulation,mouse recombinant TSLP protein(5 ?l,100 ng/?l)was injected subconjunctivally twice,4 h before and 3 h after A.fumigatus infection.TSLP expression and TSLP detection of downstream factors and receptors.Mice were infected with eyeballs,and frozen section immunofluorescence staining was used to observe the expression of TSLP,TSLPR and corneal pathologic changes;qPCR,WB and ELISA were detected respectively on expression changes of TSLP,TLR2,and TLR4.CXCL1,CXCL2,IL-6,CXCL8 and neutrophil infiltration were detected.Results1 Heat-killed A.fumigatus induces TSLP and TSLPR in HCECs and DCsHCECs and DCs were cultured in 6-well plates and treated with 2×106 pieces/ml of heat-killed A.fumigatus hyphae.The enhanced expression of TSLP was detected by western blot analysis of cell lysates of HCECs at 6 hpi but not of DCs.The secreted-TSLP(sTSLP)was detected in the treated-HCEC supernatants.In A.fumigatus-treated DC supernatants,sTSLP was hardly detectable.Another set of 6-well plates of HCECs were seeded in chamber slides and separately treated with 100 ng/well of recombinant human TSLP protein or A.fumigatus hyphae for 6 h,and were then subjected to immunofluorescence analyses of TSLP and TSLPR.TSLPR was expressed on normal untreated HCECs and expression was significantly enhanced after stimulation with both A.fumigatus and recombinant TSLP protein.2 The regulating role of TSLP on hBD2,hBD9,IL-6,and IL-8 levels in HCECs treated with A.fumigatusHCECs were incubated with heat-deactivated A.fumigatus hyphae suspension for 1,3,6,12,and 24 h.Cell supernatants were collected to assess the levels of TSLP,hBD2,hBD9,IL-6,and IL-8 by ELISA.TSLP increased in a time-dependent manner;hBD2 expression increased rapidly from 1 hpi,peaking at 12 hpi whereas hBD9 decreased significantly from 3 to 12 hpi(p<0.01).HCECs were pretreated with scrambled siRNA and TSLP siRNA for 48 h before incubation with A.fumigatus.At 12 hpi,hBD2 mRNA expression was enhanced substantially by fungal stimulation;in the TSLP siRNA group,the hBD2 level dropped considerably compared with the scrambled siRNA group.The level of hBD9 was found to be reduced in the A.fumigatus treatment group and this reduction was reversed in the TSLP depletion+A.fumigatus treatment group(p<0.01).Besides,the levels of IL-6 and IL-8 in the cellculture supernatants were decreased in the TSLP siRNA group with A.fumigatus treatment at 12 hpi.3 A.fumigatus induces TSLP expression in whole corneas and DCs respond to A.fumigatus stimulation by expressing TSLPRInfected corneas were examined under slit-lamp photography and clinical scoring at 0,12,24,and 48 hpi,and 3 and 5 dpi,fungal-infected corneas were subjected to real-time PCR and western blotting at 12,24,48,and 72 hpi.Both the mRNA and protein levels of TSLP increased significantly at 12 and 24 hpi,then began to decrease at 48 hpi(p<0.05).Immunofluorescence staining at 12,24,48,and 72 hpi,and 5 dpi showed remarkable enhancement of TSLP(p<0.05)along with a thicker corneal epithelium layer.After A.fumigatus treatment,the mRNA and protein levels of TSLPR showed a tendency to stably increase from 12 to 72 hpi(*p<0.05,***P<0.001).The frozen sections of infected cornea were subjected to immunofluorescence staining with anti-TSLPR(1:50,red)and anti-CD11c(1:50,green)antibodies.The images confirmed TSLPR expression both in the epithelium and stroma at 24 hpi.CD11c and TSLPR co-staining.4 TSLP aggravates inflammation,TLR2 and TLR4 expression is upregulated by TSLP in mice corneal epithelium treated A.fumigatusWild-type C57BL/6 mice were randomly divided into four groups(n=5).Corneas were pretreated by subconjunctival injection with a)scrambled siRNA,b)TSLP siRNA,c)BSA,or d)recombinant mouse TSLP protein,before A.fumigatus exposure.Corneas in all groups were excised after treatment.The TSLP depletion efficiency was determined by western blot.Western blot analysis showed that TSLP was depleted by 80%-90%by TSLP siRNA compared with the scrambled siRNA group(p<0.05).At 1 and 3 dpi,corneal injury was determined by clinical scoring;the results suggested that cornea damage,including keratitis,necrosis,and epithelial edema,was enhanced in the group treated with recombinant TSLP,while it was reversed in the TSLP siRNA group at 1 and 3 dpi.Additionally,polymorphonucleocyte(PMN)infiltration was detected by the MPO level in different groups by ELISA.The results showed a reduction in the MPO level in the TSLP siRNA group at both 1 and 3 dpi;by contrast,an increase was observed at 3 dpi in the group treated with recombinant TSLP(p<0.01)(n=5).The corneas were excised at 1 dpi and subjected to real-time PCR of chemokines and immunofluorescence analysis of Ly6g for neutrophils.The mRNA levels of chemokines CXCL1,CXCL2,IL-8,and CXCL8 were induced by A.fumigatus after cornea infection.CXCL2 expression was decreased in the TSLP siRNA group compared with the scrambled siRNA group(p<0.05).The mRNA levels of CXCL1 and CXCL8 were effected by TSLP siRNA but showed no statistically significant difference.IL-6 expression was not effected by TSLP RNA interference.By contrast,recombinant mouse TSLP protein showed remarkable enhancement of CXCL1,CXCL2,and IL-6(p<0.001)compared with BSA as the control.CXCL8 expression was also increased significantly in the rmTSLP-treated group(p<0.05).The neutrophil infiltration was further confirmed by immunofluorescence using Ly6g antibody:a decrease was observed in TSLP siRNA-treated corneas compared with PBS,while an increase occurred in recombinant TSLP-treated corneas at 1 dpi compared with BSA.In the model of fungal keratitis,TLR2 and TLR4 were detected by immunofluorescence staining and real-time PCR.The mRNA and protein levels of TLR2 and TLR4 significantly increased in the recombinant TSLP protein group with or without A.fumigatus treatment(p<0.01);the mRNA levels of TLR2 and TLR4 increased at both 1 and 3 dpi.However,the protein level of TLR2(p<0.05)was reduced in the TSLP siRNA group with or without A.fumigatus treatment;a similar effect was observed with respect to TLR4(p<0.01).These findings indicated that activation of TLR2 and TLR4 was increased by TSLP in mouse corneas infected by A.fumigatus.Conclusion:Corneal epithelial cells mediate TSLP after infection by corneal Aspergillus,and its receptor TSLPR is expressed on the surface of corneal epithelial cells and dendritic cells.TSLP can stimulate the up-regulation of TLR2 and TLR4 expression,increase the secretion of IL-6 and IL-8,and enhance the degree of innate immune response,but at the same time promote the recruitment of chemokines such as CXCL1,CXCL2 and CXCL10.Moreover,the protective effect of the antimicrobial peptide is inhibited,thereby aggravating the inflammation and worsening the clinical symptoms of fungal keratitis.TSLP promotes innate immunity mediated by TLR2 and TLR4 and plays an important regulatory role in the balance of corneal antifungal infection immunity and inflammation.Part ? Role of IL-36/IL-36R signaling in corneal innate defense against Candida albicans keratitisPurposeWe want to figure out the role and mechanism of IL-36y participating in the regulation of innate immunity induced by Candida Albicans.Methods1 Animals model of C.Albicans Keratitis and the application of recombinant proteinWild-type(eight weeks of age;female)and IL-36R-/-(two pairs)C57BL6(B6)mice were anesthetized by intraperitoneal injection of Ketamine(90 mg/kg)and Xylazine(10 mg/kg)before surgical procedures.Mouse corneas were scratched with a sterile 26-gauge needle to create three 1-mm incisions to break the epithelial barrier and inoculated with 1.0×105 CFUs of ATCC MYA2876 in 5?l PBS.To apply recombinant proteins or neutralizing antibody,mice were subconjuntivally injected with 100 ng recombinant IL36? protein 4 hours before the inoculation with Ca on the corneas.2 Clinical examination,Fungal Load Determination,Cytokine ELISA,and MPO MeasurementEyes were examined daily to monitor the disease progression with a dissection microscope equipped with a digital camera.For the assessment of clinical scores,mice were examined at 1,and 3 days post infection(dpi)to visually grade the disease severity.Ocular disease was graded in clinical scores ranging from 0 to 12.We used our previously modified methods that allowed all three assays(bacteria load,MPO determination,and cytokine ELISA measurement)to be performed with a single mouse cornea.3 RNA extraction and Real-time PCRFor RT-PCR,mouse corneas or epithelium samples scraped off the cornea were extracted for RNA using RNeasy Mini Kit.cDNA was generated with an oligo(dT)primer followed by analysis using real-time PCR or RT-PCR.For quantitative PCR,cDNA was amplified using StepOnePlus Real-Time PCR system with the SYBR(?)Green PCR Master Mix.Data were analyzed using ??CT method with ?-actin as the internal control.For RT-PCR,PCR products were subjected to electrophoresis on 2%agarose gels containing ethidium bromide.Stained gels were captured by using a digital camera.The following primer pairs were used.4 Western Blot and mouse proteome profileMouse corneal samples were lysed with RIPA buffer.The lysates were centrifuged to obtain the supernatant.Protein concentration was determined by BCA assay and used for two experiments.For proteome profile,200ug total protein from 4 corneas in each group was detected by mouse protein array kit.Another part of protein samples were separated by SDS-PAGE and electrically transferred onto nitrocellulose membranes.The membranes were blocked with 5%BSA and subsequently incubated with primary and secondary antibodies.Signals were visualized using SuperSignal(?)West Pico Chemiluminescent Substrate using a Kodak Imaging Station 4000R.?-Actin was used as the loading control.Antibodies:anti mouse IL36?,36R,IL-33;anti mouse PTX3.Results1.Mouse cornea expresses IL-36 cytokines during Candida Albicans(CA)infection and IL-36R are expressed in corneal epithelium,DCs,macrophages,NK cells but not on PMNs.We assessed the expression pattern of IL-36 cytokines and receptor in time course,the mRNA level of IL-36?,IL-36?,IL-36?,and IL-36Ra in mouse whole cornea using regular PCR at 0,6,9,18,and 24hpi.Among them,IL-36?,IL-36?,and IL-36y genes quickly responded to CA at 6hpi.IL-36? expression reached the peak at 9hpi,persisted at 18hpi and receded at 24hpi.IL-36? showed relatively weak expression during whole time course but had peak at 9hpi.By contrast,IL-36? had vast increase at 9hpi and 18hpi,and still could be detected at 24hpi.IL-36Ra was hardly detected by regular PCR in CA infected cornea.We collected epithelium samples from infected corneas at 6hpi to confirm the expression of IL-36?,IL-36?,and IL-36? by Real-time PCR.Compared to uninfected cornea epithelium,IL-36?expression elevated about 3 folds and IL-36? about 4 folds.The expression of IL-36?and IL-36R were also assessed on the protein level by Western blot.In naive corneas,IL-36? was hardly detectable while IL-36R had a basal-level of expression.The CA infection induced the expression of IL-36? at 6hpi and significantly increased IL-36?level at 18hpi.IL-36R was upregulated in response to CA infection at 6hpi and had an elevated trend till 24hpi.These results indicate that among the IL-36 cytokines,IL-36? plays most important role in CA keratitis and cornea epithelium is one of the main resource of IL-36 cytokines.To detect the expression and distribution of IL-36R,we perform double staining for IL-36R and immune cells markers on cornea treated with rmIL-36? protein.WT B6 mouse cornea was subconjunctivally injected with 5uL recombinant IL-36? protein(rmIL-36?,100 ng/?L).At 18h,corneas were excised and under Immuno-histochemical analysis with anti-CD 11c antibody(murine DC marker),anti-F4/80 antibody(murine macrophage marker),anti-NK1.1(murine NK cell marker),anti-Ly6G antibody(murine PMN marker)and double stained with anti-IL-36R antibody.The results showed that CD11c,F4/80,NK1.1,and Ly6G positive cells were found in rmIL-36?-treated central cornea stroma,while in BS A-treated cornea no immune cells infiltration was detectable.Among them,CD11c,F4/80,and NK1.1 were co-localized with IL-36R staining while Ly6G was not.2.IL-36R deficiency exacerbates CA keratitis in B6 mouse,regulates expression of IL-1?,IL-Ra,S100A8/A9 and exhibits different effects on immune cells infiltration in CA infected cornea.At 1dpi,IL-36R deficiency increases CA keratitis severity including elevated clinical scores,fungal burden,and MPO level which inferred more PMN infiltration in infected lesion area.At 3 dpi,knockout of IL-36R resulted in significant increase in the severity of keratitis with a much higher clinical score.There was heavy infiltration in the aqueous humor and clear signs of corneal melting in IL-36R knockout corneas.To investigate the mechanism of IL-36R in CA keratitis,we collected the cornea epithelium at 6hpi and whole cornea at 18hpi to detect the mRNA and protein levels of IL-1?,IL-Ra,S100A8,and S100A9 on WT and IL-36 R-/-B6 mice.The expression of IL-1?,IL-Ra,S100A8,and S100A9 by Real-time PCR.In WT infected corneas,all 4 genes were increase to a different extent than the value in non-infected corneas as control.Deficiency of IL-36R significantly suppressed infection-induced IL-Ra,S100A8,and S100A9 but further augmented the expression of IL-1?.To confirm the effects of IL-36R deficiency at the protein level,we selected IL-1? and calprotectin(S100A8/A9)for ELISA analyses on mouse whole cornea at 18hpi.In WT infected cornea,there was marked upregulation of both IL-1?(46.17 pg/ug total proteins)and S100A8/A9(345.5 pg/ug total proteins)compared to the naive corneas.IL-36R deficiency markedly downregulated S100A8/A9 but vastly upregulated IL-1?.To detect the changes of other immune cells recruitment caused by the absence of IL-36?/IL-36R signaling,Immuno-histochemical analysis was performed on staining of CD 11c,F4/80,NK1.1,and Ly6G.Figure 4C shows that,compared to WT,infected IL-36R-/-cornea had more severe epithelium damages penetrating into corneal stroma resulted in poor corneal integrity at 18hpi.There were less staining of CD11c and NK1.1 in IL-36R-/-cornea suggesting remarkable impaired DCs and NK cells recruitments.A detectable minor decrease of macrophage was showed in IL-36R-/-cornea,whereas PmN had more infiltration at infectious lesion spot and periphery of infection.3.Exogenous IL-36y improves outcome of CA keratitis in B6 mouse and enhances protective cytokines,antimicrobial proteins of innate defense while suppresses IL-1? expression.To see if exogenous IL-36? has opposite effects on the pathogenesis of CA keratitis,we subconjunctivally injected murine IL-36?(100ng/eye)or BSA as control at 4h before and every other day after CA infection.At 1,3,5,and 7dpi,the keratitis severity were assessed by clinical scoring.At 1dpi,corneas were excised and subjected to fungal burden and MPO level detection.IL-36? pretreatment resulted in tiny opacification with much lower clinical score,very low number of fungi at 1dpi,and low but measurable MPO activity.At 7dpi,rmIL-36?-pretreated corneas were totally healed and had pretty clear ocular surface almost like normal naive corneas.To further determine the protective effect of IL-36?,we detected the level of several antimicrobial proteins,chemokines,and pro-inflammatory factors between rmIL-36?and BSA-pretreated groups,with or without CA infection.At 6hpi,epithelium samples were scraped from infected corneas and subjected to real-time PCR.Among the target genes,CA stimulation induced S100A8,S100A9,CCL3,TNFaIP3,IL-1?,and IL-1Ra(secreted form)at early stage of infection.Compared to BSA,the application of rmIL-36? protein without infection could significantly stimulate the expressions of S100A8,S100A9,CCL3,IL-1Ra,and moderately increase TNFaIP3 whereas it showed no effects on IL-1?.In the infected corneas,compared to BSA,rmIL-36? protein downregulated IL-1? expression while elevated the expression of S100A8,S100A9,CCL3,TNFaIP3,and IL-IRa.We also detected mBD3,mBD4,and CRAMP by real-time PCR and found rmIL-36? protein had not much significant effects on these AMPs.We further confirmed the effects in 18hpi whole corneas on protein level of IL-1?,S100A8/9,and CCL3 by ELISA.The rmIL-36? protein without infection enhanced S100A8/9 vastly whereas had no stimulation to IL-1?.The rmIL-36?-pretreatment upregulated CCL3 expression in uninfected corneas.However,after infection,the protein level of CCL3 in rmIL-36?-pretreated corneas decreased compare to normal infection ones,suggesting rmIL-36? lead to milder keratitis and less inflammatory cells infiltration.IL-36?/IL-36R signaling regulates many other anti-microbial factors revealed by proteome profile assayTo comprehensively understand the function of IL-36? in infection and IL-36?/IL-36R signaling downstream molecules interactions,we investigated the relative expression levels of 111 soluble mouse proteins including cytokines,chemokines and growth factors by proteome profile assay.WT and IL-36R-/-mouse were divided in 4 groups,a)WT naive,b)WT normal infection,c)WT+rmIL-36?-pretreated,and d)IL-36R-/-;corneas in b)c)d)were infected with CA for 18hpi;rmIL-36?(100ng/eye)were subconjunctivally injected 4h before CA infection.Total protein of whole corneas was extracted with PBS and subjected to protein array.Among 111 soluble mouse proteins,many factors related to infection and inflammation had huge differences and 3 proteins which exhibited the biggest differential significance.The protein level of IL-33 decreased in rmIL-36?-pretreated group,indicating that IL-36? play a protective role in ease infection-associated inflammation.Interestingly,we found that two antimicrobial protein PTX3 and Reg3g may regulated by IL-36?/IL-36R signaling.PTX3,a member of the pentraxin superfamily,could be enhanced by rmIL-36? and suppressed by IL-36R deficiency.Reg3g,a bactericidal C-type lectin with potential angiogenesis effect,was hardly detected in rmIL-36?-pretreated group,and found to be upregulated vastly in IL-36R-/-corneas with the most severe keratitis among 4 groups.We further performed western blotting to confirm these findings on IL-33,PTX3,and Reg3g.ConclusionsIL-36? can induce anti-fungal immunity in Candida keratitis as a pro-inflammatory factor to recruit vital immune cells like DCs,macrophage,??T cells,and NK cells.At the same time IL-36? enhance protective cytokines,antimicrobial peptide and chemokines to protect the cornea from fungal infection. |
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