Molecular Mechanism For Cellular Reprogramming In Corneal Fibroblasts Induced By Lipopolysaccharide Pretreatment Modulating Antifungal Innate Immunity

Fungal keratitis is among the most dangerous ocular infections. It can manifest fast and vicious progress and often leads to total sight loss in days if not properly controlled. The best known predisposing factors include trauma in underdeveloped countries or contact lens wear in developed countries. Hot and humid weather is a further risk factor in all countries. Fusarium spp. and Aspergillus spp. are the most common pathogens of fungal keratitis. Under normal conditions, the cornea is highly resistant to infection, and fungal keratitis always occur after the epithelial integrity of the cornea has been compromised, exposing underlying fibroblasts. The corneal fibroblasts are the second line of the host innate immunity defense and participate in immediate recognition and control of fungal invasion.Toll-like receptors (TLRs) are key players in innate immunity through their recognition of conserved pathogen-associated molecular patterns (PAMPs) in microbes and are expressed at the cell surface or in intracellular space in corneal fibroblasts. Among these receptors, TLR4is capable of sensing LPS while TLR2recognizes a diverse set of microbial lipopeptides and lipoproteins that are present on gram-positive and gram-negative bacteria. Our previous study demonstrated that TLR2and TLR4represents two of the master switches initiating innate immunity in the cornea and have determinant roles in Aspergillus fumigates (AF) keratitis. Their activation leads to the initiation of downstream signal cascades, which regulate secretion of inflammatory cytokines that recruit inflammatory cells to the corneal stroma and the production of antimicrobial molecules that kill the invading pathogens. However, this response of TLRs must be stringently regulated, as excessive inflammation can cause tissue damage and have devastating effects on the host, which can result in vision loss or perforations.TLRs signal through the adaptor proteins myeloid differentiating factor88(MyD88) and Toll-IL-1receptor domain-containing adaptor inducing interferon-beta (TRIF) to activate downstream transduction cascades. The detrimental effect of TLR signaling is associated with the MyD88-dependent classical pathway and MyD88-dependent mitogen-activated protein kinase (MAPK) pathway that lead to transcription factor nuclear factor κB (NF-κB) and activator protein-1(AP-1) activation, which is essential for the induction of many cytokines and chemokines such as interleukin (IL)-6,-8and tumor necrosis factor (TNF)-α. In contrast, the MyD88-independent TRIF signaling pathway that activates interferon regulatory factor-3(IRF-3) can induce interferon (IFN)-β and anti-inflammatory mediators. Thus, in TLR signaling there is a fine balance between pathways leading to injury or protection, with even minor alterations of these fine-tuned endogenous pathways having profound effects on cellular responses to TLR engagement.We previously reported that telomerase-immortalized human stroma fibroblasts (THSFs) pretreated with lipopolysaccharide (LPS) develop a state of cellular reprogramming that modulates corneal antifungal innate immunity. Pretreatment of THSFs with low-dose LPS resulted in diminished production of cytokines IL-8and IL-6, elevated expression of antimicrobial peptides CC chemokine-ligand20and thymosin b, and suppression of polymorphonuclear leukocyte migration upon subsequent A. fumigatus challenge. Therefore, LPS pretreatment may induce protective mechanisms during fungal keratitis that prevent an excessive inflammatory response and provide an innate defense in the cornea.The underlying molecular mechanism by which LPS pretreatment induces cellular reprogramming of THSFs challenge by A. fumigatus hyphae through innate immunity pathways remains to be elucidated. In the present study, the effect of LPS pretreatment on TLR4and TLR2messenger RNA (mRNA) induction was evaluated in order to explore the signaling mechanisms for the resolution of inflammation, restoration of homeostasis and prevention of corneal destruction. Then these TLRs were inhibited using monoclonal antibodies to investigate the role of TLR2and TLR4in LPS-induced tolerance using the THSF cell line. In addition, the downstream influence of LPS pretreatment was examined for the purpose of identifying the TLRs signaling pathways involved in THSFs, including the MyD88-dependent classical pathway, MyD88-dependent MAPK pathway and MyD88-independent TRIF signaling pathway.Part I TLR4mediates cellular reprogramming induced by lipopolysaccharide pretreatment in corneal fibroblasts[Purpose]To determine the key receptors in cellular reprogramming induced by lipopolysaccharide pretreatment in corneal fibroblasts challenged with Aspergillus fumigates.[Methods]1. Aspergillus fumigatus strain and preparation of hyphae:The A. fumigatus strain CCTCC93024, purchased from the China Centre for Type Culture Collection, was grown on Sabouraud glucose agar for24h at37℃on a shaking table with a rotation speed of200rpm. Conidia were harvested and planted into Sabouraud fluid media, at a final concentration of108micro-organisms/ml. The tubes were shaken at26℃,500rpm for18h and then centrifuged at1000g for10minutes. The mycelia were washed twice, suspended in phosphate-buffered saline (PBS) and sterilized by heat treatment at56C for60min. Then, the mycelia were disrupted into20-40mm pieces in a Potter-Elvehjem Tissue Grinde and yielded1×106pieces/ml as A. fumigates hyphae antigen.2. Cell cultures:THSFs (kindly provided by Dr. Fu-Shin X. Yu and produced by Dr. Ilene K. Gipson) were cultured in Dulbecco Modified Eagle Medium (DMEM) supplemented with10%newborn bovine serum at37℃in a humidified atmosphere with5%CO2. For experiments, cells were seeded at4×105cells/well in6-well microculture plates and grown until80%confluence (-2days). To prepare cells for stimulation, the THSFs were then starved in serum-free DMEM for16h.3. Cell treatment and tolerance induction:The THSFs were preconditioned with a TLR4ligand, LPS at a density of10ng/ml for12h. Then the cells were washed twice with serum-free medium and re-stimulated with A. fumigatus hyphae (106pieces/ml) for various periods (30min,1h,3h,4h,6h). The Cell culture supernatants and cells were collected for measurement of TLR4, TLR2mRNA expression and IL-6, IL-8, TNF-α protein secretion.4. Evaluation of neutralizing efficiency of antibodies and blocking experiment of receptors:TLR blocking experiments were conducted by incubating THSFs with monoclonal Abs against TLR2or TLR4. THSF cells were incubated with antihuman TLR2(100mg/ml) or anti-human TLR4(100mg/ml), or both anti-human TLR2(100mg/ml) and anti-human TLR4(100mg/ml) monoclonal Abs for30min. They were then challenged with TLR2ligand zymosan at a density of1mg/ml or TLR4ligand LPS (1μg/ml) for12h at37℃, or pretreated with LPS for12h and re-challenged with A. fumigatus hyphae for4h. The cell culture media and cell lysates were harvested and used for IL-6, IL-8and TNF-a protein ELISA and TLR2mRNA Real-time reverse transcriptase polymerase chain reaction (RT-PCR).[Results]1. Efficient neutralization of TLR2or TLR4by monoclonal antibodies:TLR2-or TLR4-antibody had no apparent effect on the basal release of IL-8and TNF-α in THSFs. While THSFs secreted significantly high levels of IL-8and TNF-α in response to TLR2ligand zymosan or TLR4ligand LPS challenge, this elevated production was significantly inhibited in cells treated with TLR2-or TLR4-antibody, and there is no significant difference between blank control and the ligand-treated THSFs with previous TLRs antibodies inhibition. Thus, our monoclonal antibodies specific for TLR2and TLR4can efficiently neutralize the function of TLR2and TLR4, respectively.2. Effect of LPS pretreatment on TLR4expression induced by A. fumigatus in THSFs:To examine whether TLR4expression is affected by LPS pretreatment in THSFs, we pre-stimulated THSFs with10ng/ml LPS for12h and assessed the response of TLR4expression to subsequent challenge with A. fumigatus hyphae. RT-PCR revealed that LPS pretreatment significantly down-regulated A. fumigatus hyphae-induced mRNA expression of TLR4after1h and3h of re-stimulation.3. LPS pretreatment induced down-regulation of pro-inflammatory cytokines secretion in A. fumigates antigens-challenged THSFs depends on TLR4:In the previous study, we demonstrated that pretreatment of THSFs withlO ng/ml LPS resulted in impaired production of IL-6and IL-8in response to a secondary A. fumigatus hyphae challenge. We next sought to determine whether the reduction of pro-inflammatory cytokines secretion induced by LPS pretreatment in THSFs is dependent on TLR4. THSFs pre-incubated with TLR4antibodies before LPS pretreatment showed increased IL-6, IL-8and TNF-α accumulation in culture medium compared to LPS-pretreated THSFs without the TLR4antibody, and there was no significant difference of IL-6, IL-8and TNF-a accumulation in culture medium compared to non-LPS-pretreated controls challenged with Aspergillus fumigates without TLR4antibody inhibition.4. Involvement of TLR2in LPS-induced cellular reprogramming to secondary A. fumigatus hyphae stimulation:Real-time PCR was used to assess expression of TLR2. It showed that LPS pretreatment had no apparent effect on TLR2expression in THSFs challenged with A. fumigatus hyphae. However, whereas the TLR4antibody had no effect on basal TLR2mRNA expression, THSFs treated with TLR4neutralizing antibody before LPS pretreatment exhibited significantly increased levels of TLR2after the secondary fungi stimulation compared with the pretreated controls. In addition, pre-incubation of THSFs with both TLR2and TLR4antibodies before LPS pre-stimulation resulted in dramatic reduction of common pro-inflammatory cytokines, including IL-6, IL-8and TNF-α expression compared with the non-pretreated THSFs.[Conclusion]1. As the key receptor in cellular reprogramming, TLR4mediates attenuated cytokine production induced by LPS pretreatment in THSFs.2. Levels of TLR2mRNA induced by A. fumigatus were not affected by LPS pretreatment. However, after TLR4blockage, pro-inflammatory cytokines secretion is mediated by TLR2in LPS-pretreated THSFs. Part II Influence of cellular reprogramming induced by LPS pretreatment on TLR4signaling pathways involved in THSFs[Purpose]To examine the downstream influence of LPS pretreatment for the purpose of identifying the TLR4signaling pathways involved in THSFs, including the MyD88-dependent classical pathway, MyD88-dependent MAPK pathway and MyD88-independent TRIF signaling pathway.[Methods]THSFs were pretreated with10ng/ml of LPS for12h and then re-challenged with A. fumigatus hyphae for various period(30min,1h,3h,4h,6h). The cell culture media and cell lysates were harvested and used for analysis of key signaling factors MyD88, IκB-α, NF-Kb-p65in MyD88-dependent classic pathway, MAPK3, AP-1, ERK1/2in MyD88-dependent MAPK pathway and TRIF, IRF-3, IFN-p in MyD88-independent TRIF pathway of gene and protein levels. Immunofluorescence staining were applied to determine the expression and location of NF-Kb-p65.[Results]1. Impaired response of MyD88-dependent classical signaling pathway:mRNA expressions of MyD88were proved to be inhibited after1h and3h of A. fumigates re-stimulation according to the PCR analysis, and an increase in gene and protein expression of inhibitory NF-κB (IκB)-α was observed1h and3h after fungus treatment. NF-icB-p65protein expression was diminished after1h and3h of stimulation with A. fumigatus hyphae, and the translocation of NF-κB-p65into cell nuclei was significantly attenuated by LPS pretreatment.2. Down-regulated expression of downstream molecules in MyD88-dependent MAPK signaling pathway:Expression of MAPK3and AP-1mRNA was lowered by LPS pretreatment1h and3h after the secondary A. fumigates stimulation, and Western blot analysis showed that protein level of phosphorylated-extracellular signal-regulated kinase (ERK)1/2was decreased after1h and3h of treatment with A. fumigatus hyphae.3. Up-regulation of downstream molecular expression and cytokine secretion of the MyD88-independent TRIF signaling pathway:THSFs pretreated with LPS were challenged with A. fumigatus hyphae, and TRIF, IRF3and IFN-β expression were measured by real-time RT-PCR and enzyme-linked immunosorbent assay (ELISA). In non-pretreated controls, THSFs express relatively high levels of TRIF, IRF3and IFN-β mRNA in response to A. fumigates hyphae challenge for3h and6h, and this elevated expression was significantly increased in cells pretreated with LPS. IFN-β secretion levels were consistent with trends observed for IFN-p mRNA.[Conclusion]1. The impaired activity of the MyD88-dependent classical pathway and MyD88-dependent MAPK pathway may contribute to the suppressed secretion of pro-inflammatory cytokines induced by LPS pretreatment in THSFs.2. TRIF-mediated immunological protection was augmented in LPS-pretreated THSFs.