The Role Of LIGHT-LTβR Pathway In NOD Mice

Type1diabetes is an organ-Specific autoimmune disorder characterized by chronicinflammation and pancreatic insulin-producing beta-cell destruction. T1DM usuallyoccurs in children or young adults and is accompanied with severe complication whichmay result in early death of children. Thus, the prevention and treatment of T1DM havebecome an important topic in medical study areas.The pathogenic factors of T1DM arecomplicated. T cells activation is an important factor for β cells destruction in T1DM. βcells death is mainly triggered through apoptosis, which is mediated byCTL,cytokines,oxygen-derived free radidicals, and so on. Although the roles of CTL andoxygen-derived free radidicals are rather clearly defined, it is far from clear howcytokines especially co-stimulatory moleculars play roles in T1DM. For developing thevalid therapy strategy in T1DM, it is significant for us to understand the molecularmechanisms of T cell activation resulting in β cells destruction.Direct evidence shows the bloekede of co-stimulatory signals inhlbits T cellactivation and decreases the incidence of T1DM. LIGHT-HVEM/LTβR is a novelco-stimulatory pathway. They belong to members of TNFSF/TNFRSF. It was reportedthat LIGHT pathway played an important role in the pathogenesis of rheumatoid arthritisand autoimmune hepatitis through inducing T cell activation and promoting cell apoptosis.Precious study showed LIGHT-LTβR pathway Recruited and activated Naive T cells inthe islets by LTβR-dependent tertiary lymphoid structure for the onset of diabetes; LTβreceptor-immunoglobulin fusion protein (LTβR–Ig) was administered to nonobesediabetic mice (NOD mice). Early treatment with LTβR–Ig prevented insulitis and IDDMand LTβR–Ig treatment at a late stage of insulitis also dramatically reversed insulitis andprevented diabetes, which suggested that LIGHT pathway played a critical role in T1DMdevelopment. However, the intracellular molecular mechanism that LIGHT pathwayaffects β cells apoptosis has not yet been studied.In the present project, the expression of LIGHT, HVEM and LTβR and cytokinessecretion at1,2,4w after NOD mice producing diabetes were studied. To further understand the effect of LIGHT pathway in β cells loss, we established LIGHT knock outNOD mice model for blockading LIGHT-HVEM/LTβR signalling. Then, we observeddiabetic pathogenetic condition in LIGHT knockout NOD mice through detecting bloodglucose, IPGTT, cytokines secretion as well as essaying pancreatic histopathology andthe incidence rate of T1DM. We studied the function about LIGHT in vitro. MIN6cellsor primary islet cells were treated with rmLIGHT and rmIFN-γ alone or in combine,respectively. The role of LIGHT on islet cells apoptosis was investigated with MTT,FCM methods.To interpret the molecular mechanism, the protein expression ofapoptosis-related genes was detected by Western blot or immunohistochemisty.Part I: The expression alloeosis of LIGHT/HVEM/LTβR during theinitiation and progress of diabetes in NOD miceAims: To investigate the expression of LIGHT/HVEM/LTβR and secretion ofcytokines during the development of diabetes in NOD mice.Methods: Female NOD mice (n=30) at4weeks of age were maintained underspecific pathogen–free conditions. Blood glucose in mice were measured weekly from5to30weeks of age. Diabete was monitored by levels of blood glucose. Animals wereconsidered diabetic after two consecutive measurements of≥13.8mmol/L of bloodglucose levels in a week. After three mice with non-diabetes or1,2,4w of diabetes wereput to death, Total RNA and protein were extracted from fresh pancreatic tissue. ThemRNA levels of LIGHT/HVEM/LTβR were determined by real-time quantitative PCRand protein levels of LIGHT/HVEM/LTβR were assayed by Western blot. The secretionof inflammatory cytokines including IFN-γ, IL-4was examined with ELISA.Results: At30weeks of age, the cumulative incidence of diabetes was81.5％(22/27). Real-time PCR and Western blot results showed the expression level of LIGHT,LTβR and HVEM in pancreatic tissue was significantly increased in4-week-diabetemice.The serumal concentration of IFN-γ in diabetic mice was obviously higher than thatin non-diabetic group，but the level of IL-4decreased.Conclusion: The expression of LIGHT, LTβR and HVEM was upregulated in NOD mice with diabetes. The production of IFN-γ/IL-4was increased/reduced in diabeticmice. These data indicated LIGHT pathway and cytokines such as IFN-γ, IL-4wereinvolved in the initiation and progress of T1DM.Part II: Effects of LIGHT-HVEM/LTβR signal deficiency on thedevelopment of T1DM in NOD miceAims: To observe the pathogenetic condition alleosis of T1DM in LIGHT-/-NODmice and identify the role of LIGHT pathway on insular β cells loss through contrastiveanalysis.Methods: LIGHT-/-mice were paired with NOD mice.Their descendants werebackcrossed with NOD mice beyond ten generations, whose offsprings were LIGHT-/-NOD mice. LIGHT-/-NOD mice at4w of age (n=25) and control NOD mice at4w of age(n=25) were maintained for26weeks. Body weight and blood glucose were regularlydetected and the incidence of diabetes was added up. We also performed intraperitonealglucose tolerance test to analyze insulin activity in vivo. Five of11-week-old mice or fiveof20-week-old mice from above-mentained two groups were executed. Then, the serumwas collected. Pancreatic histopathology was observed with microscope. Theinflammation of islets was graded. Apoptotic islet cells were observed with TUNEL insitu end-labelling method. Serum IFN-γ and IL-4were measured by ELISA.Results: At30weeks of age, compared with control group, the incidence rate ofT1DM in LIGHT-/-NOD mice group was lower (LIGHT-/-NOD group:13.3%,2/15;LIGHT+/+NOD group:73.3％,11/15). Glucose tolerance in LIGHT-/-NOD group wasnormal, while that in control NOD group obviously decreased. The absolute number ofislets in control NOD group was less than that in LIGHT-/-NOD group because of theislets being destroyed. In LIGHT-/-NOD group, the insulitis grade was most zero or firstgrade of lymphocytic inflammation in islets; while in control NOD group, the insulitisgrade was most second, third or four grade of insulitis. The percentage of apoptosisnotably decreased in LIGHT-/-NOD group. Deficiency of LIGHT-HVEM/LTβRsignalling significantly reduced the production of IFN-γ. Conclusion: Deficiency of LIGHT signalling can reduce the secretion of cytokinessuch as IFN-γ and alleviate inflammation extent of islets to prevent cytokine-mediated βcells destruction; Deficiency of LIGHT signalling can alleviate cell apoptosis, improveglucose tolerance abnormal and decrease the incidence rate of T1DM in NOD mice.Part III: Effects of LIGHT-LTβR signalling on islet cells apoptosisAims: To observe whether LIGHT pathway directly participates incytokines-mediated islet cells apoptosis.Methods: MIN6cells (SV40T-transformed insulinoma cells derived from NODmice) and primary islet cells from Bal b/c mice were grown in DMEM containing15%FBS,2mM glutamine and penicillin-streptomycin. Cells were treated with rmLIGHT(5μg/ml) and rmIFN-γ (100ng/ml) alone or in combination. Then, Cell viability wasmeasured by MTT assay. Several types of cell death are recognized, including necrosis,apoptosis. To further investigate which form of cell death was induced by LIGHT andIFN-γ in MIN6cells. Morphological changes in the nuclear chromatin of cellsundergoing apoptosis were detected by staining with2.5mg/ml DNA-bindingbis-benzimide Hoechst33258fluorochrome, followed by an examination on a confocallaser scanning microscope. Cells were double stained with Annexin-V–FITC apoptosisdectection Kit and7-AAD according to the manufacturer’s suggested protocols. Thepercentage of cells apoptosis was analyzed by flow cytometry. Genomic DNA in MIN6cells was isolated and isolated genomic DNA was electrophoresed on1.5%agarose geland stained with ethidium bromide to detect internucleosomal cleavage. After theblockade of LIGHT pathway by anti-LTβR Ab or anti-HVEM Ab, MTT assay detectedthe cell viability and FCM analyzed the cell apoptosis percentage.Results: A combination of LIGHT and IFN-γ, but neither cytokine alone, inducedcell death in time dependent manners as measured by MTT assay. LIGHT or IFN-γ hadjust slight cytotoxic action on cells, but LIGHT/IFN-γ synergism was responsible for cellapoptosis in vitro. Morphological changes of MIN6cells were further detected by thephase contrast microscope. Cells shape changed gradually from tile to round, evenfloating and cell density decreased after LIGHT and IFN-γ treatment. Cytokines-treated cells displayed apoptotic nuclear morphological changes, including formation ofapoptotic bodies, chromatin condensation and thickly staining. Genomic DNA in MIN6cells presented DNA fragmentation. The percentage of early apoptosis notably augmented,while the percentage of advanced apoptosis or necrosis had no obvious change. Thesedata suggested islet cell death did not result from necrosis but cell apoptosis. Theblockade of LIGHT pathway through LTβR Abs could obviously increase the cellviability and reduce the cell apoptosis percentage.Conclusion: LIGHT may directly participate in cytokines-mediated islet cellsapoptosis by interacting with LTβR; Synergetic effect on islet cells apoptosis may lie inbetween LIGHT and IFN-γ.Part Ⅳ: Molecular mechanism of LIGHT-LTβR pathway on isletcell apoptosisAims: To investigate the protein expression of downstream genes involved in isletcell apoptosis induced by LIGHT-LTβR pathway and further study the molecularmechanism of LIGHT-LTβR pathway on islet cell apoptosis in NOD mice.Methods: MIN6Cells were seeded in6-well plates and treated with rmLIGHT(5μg/ml) and rmIFN-γ (100ng/ml) combination at different time point. In someexperiments, cells were pre-incubated with or without50μM NF-κB inhibitor PDTC or50μM STAT1inhibitor Flud for1h and then treated with or without LIGHT and IFN-γcombination for12h. At the end of culture, Total protein was extracted from cellsincluding the floating and attached cells. The protein concentration in samples wasmeasured using NanoDrop ND-1000spectrophotometer. Then, the protein expression oftranscription factor NF-κB (p65) and STAT1, Caspase-3, Cleaved Caspase-3, Caspase-8,Caspase-9, CytoC, Bcl-2, Bcl-XL, Bak, Bax, Bid and PARP was detected by Western blot.Relative protein levels were calculated in comparison to β-actin as standard. Cells wereseeded on coverslips of flat bottom6-well microtiter plates and treated with LIGHT andIFN-γ in combination. NF-κB activation was measured by immunofluorescence Staining.Cleaved Caspase-3and Bax were measured by immunocytochemistry Staining. To further identify the effect of NF-κB,STAT1,Caspase in cell apoptosis induced by LIGHT andIFN-γ, MIN6Cells were pretreated with or without50μM NF-κB inhibitor PDTC,50μMSTAT1inhibitor Flud or Caspase inhibitor Z-VAD-FMK for1h and then treated withLIGHT and IFN-γ. Cell viability was detected with MTT. The percentage of cellsapoptosis was analyzed by FCM. Western blot detected Bcl-XL, Bak and Bax expression.Caspase-3activity was measured with a commercial Caspase-3Colorimetric assay kitaccording to the manufacturer’s suggested protocols. Immunohistochemical methodobserved the expression of Bax and Caspase-3in LIGHT knockout NOD mice.Results: LIGHT/IFN-γ synergism could obviously activate cytoplasmictranscription factor NF-κB (p65) and STAT1. Cyto C was released because ofmitochondrial membrane permeation. Caspase-3,-8,-9were activated in LIGHT andIFN-γ-induced apoptosis of MIN6cells. Caspase specific substrate PARP was cleavedinto inactive pattern. Bcl-2family is involved in the regulation of apoptosis. It was foundthat Bcl-XLexpression was downregulated with a time-dependent manner after theLIGHT and IFN-γ treatment, while the protein expression of proapoptosic genesincluding Bak and Bax was upregulated. Cytokines-induced Bcl-XLdownregulation/Bakand Bax upregulation were reversed by PDTC/Flud. Cell viability was significantlyincreased in the PDTC/Flud pretreating cells. Cleaved Caspase-3and Bax wereobviously downregulated in LIGHT knockout NOD mice.Conclusion:①Molecular mechanism of LIGHT and IFN-γ-induced islet cellapoptosis may be relevant to transcription factor NF-κB and STAT1activation.②Mitochondrial pathway may be involved in cell apoptosis.