Effects Of Caspase-1 Inhibitors On Experimental Autoimmune Myasthenia Gravis And Mechanism Research

Background:Myasthenia gravis (MG) is characterized by loss of acetylcholine receptor (AChR) on the postsynaptic membrane of neuromuscular junction, an antibody-mediated, T cell-dependent, complements involved autoimmune disease, and resulted in impaired neuromuscular transmission and muscle weakness. Immunization with torpedo acetylcholine receptor (TAChR) or a synthetic peptide corresponding to region 97-116 of the rat AChR a subunit (R97-116 peptide) can induce experimental autoimmune myasthenia gravis (EAMG) in Lewis rats. EAMG is a reliable model for human MG and is suitable for investigating the development of novel therapeutic strategies.IL-1β is a highly active proinflammatory cytokine produced by limited cells, such as dendritic cells, tissue macrophages, and blood monocytes, depending on the activation of caspase-1. Both pro-IL-1β and pro-IL-18 as inactive precursors are cleaved by the active caspase-1 into functional forms. Caspase-1 itself is cysteine protease, and pro-caspase-1 is cleaved into active caspase-1 in the inflammasome.IL-1β and IL-18 can induce the secretion of IL-17 from many cells of the innate immune system, including γδ T cells and invariant natural killer T (iNKT) cells. Mature IL-1β binds first to the type 1 IL-1 receptor (IL-1RI) on their cell surface and forms the receptor complex with IL-1R accessory protein (IL-1RAcP). The Toll-IL-1 receptor (TIR) domains of receptor complex recruits the adaptor molecule MyD88, consequently, NF-kB is phosphorylated. Phosphorylated NF-kB translocates to the nucleus and induces the transcription of pro-inflammatory cytokines. IL-1-targeted drugs, such as the IL-1 receptor antagonist anakinra and the neutralizing monoclonal anti-IL-1β antibody canakinumab, result in a rapid and sustained remission in inflammatory diseases. Daily injections of IL-1 receptor antagonist (IL-1ra) decreased the severity of clinical EAMG in C57BL/6 mice with suppressed serum IFN-y, TNF-a, IL-1β, C3, and anti-AChR IgGl.It has recently been reported that caspase-1 is critical for the induction of IL-17 production in innate immune cells. The role of caspase-1 in EAE pathogenesis has been demonstrated, and caspase-1 protein and activity were upregulated during the inflammatory stages of the disease. Caspase-1-/- mice were less susceptible to EAE, and this was associated with impaired Th1 cell development and reduced perivascular infiltrates in the CNS. Inhibition of caspase-1 in vivo suppressed the development of Th17 cells and the induction of EAE, and this was reversed by administration of IL-1β or IL-18, more dramatic with IL-1β.In addition, some studies have confirmed that the activated caspase-1 could lead to an upregulation of MHC class II, CD80, and CD86 on DC through the secretion of IL-1β. We addressed the hypothesis that caspase-1 may be an important drug target for autoimmune diseases.Objective:The aim of our present study was to investgate the effect of caspase-1 inhibitor on cellular immune response of EAMG and further explore the underlying mechanisms.Methods:1. EAMG model building and assessment of clinical score Female Lewis rats,160-180 g (aged 6-8 weeks old).200 μl inoculum containing R97-116 peptide, Mycobacterium tuberculosis in incomplete Freund’s adjuvant could induce EAMG, which was induced by subcutaneous injection into both hind footpads on day 0 and was boosted with the same dose along the back on day 11 after the first immunization. Rats of every group were weighed and scored from the beginning of the experiment every second day until day 43 post immunization (p.i.) in a blinded fashion.2. Experimental animal grouping Rats were randomly divided into respective groups (6 rats/group). From day 13 after the first immunization, rats in the Ac-YVAD-cmk treatment group (at doses of 100 μg/rat in a volume of 0.4 ml PBS) were administered intraperitoneally (i.p.) every second day. Rats in the Ac-YVAD-cmk with IL-1β group were injected i.p. with Ac-YVAD-cmk (at doses of 100μg/rat in a volume of 0.4 ml PBS) from day 13 after the first immunization and IL-1β (at doses of 0.4 μg/rat in a volum e of 0.4 ml PBS) from day 14 after the first immunization every second day. EAMG rats were administered with vehicle (PBS) at the same time points.3. DC preparation Spleen mononuclear cell (MNC) suspensions from Lewis rats were prepared by grinding spleen through a cell strainer in medium, then depleted of erythrocytes with osmotic lysis. Cells were incubated in Falcon culture flasks. After 1 h 40 min, we gently removed non-adherent cells, and added new complete medium to the flasks. Incubating 18 h, we collected non-adherent DC. In brief, bone marrow cells prepared from femurs and tibias were cultured in RPMI 1640 medium supplemented with 1%(v/v) penicillin-streptom ycin,10%(v/v) fetal bovine serum,10 ng/ml of GM-CSF and 10 ng/ml IL-4. After a total culture time of 8 days, non-adherent DC were collected. DC were cultured with LPS (100 ng/ml), with or without the caspase-1 inhibitor Ac-YVAD-cmk (8μM) for 48 h. Then DC were collected, and CD80, CD86, MHC class Ⅱ, and IL-1β were determined by flow cytometric analysis.4. Lymph node MNC preparation We ultimately killed rats of every group and removed inguinal lymph nodes under aseptic conditions on day 43 p.i.. Then we grinded the organs through cell strainers in serum-free medium to obtain MNC suspensions and resuspended cells to 2×106/ml for the following experiments.5. Flow cytometric analysis of lymph node MNC After extracellular staining for surface with PerCP-conjugated anti-rat CD3, PE-conjugated anti-rat CD4, PE-conjugated anti-rat y8 TCR (eBioscience), and PE-conjugated anti-rat OX-62, lymph node mononuclear cells were then fixed with 2% paraformaldehyde for 20 min at 4℃ and permeabilized with 0.5%saponin and stained intracellularly with FITC-conjugated anti-rat IL-17 and FITC-conjugated anti-rat IL-1β. Treg cells were identified by extracellular staining with FITC-conjugated anti-rat CD4, PE-conjugated anti-rat CD25, after fixation and permeabilization, PE-Cy5-conjugated anti-mouse/rat Foxp3 antibodies was used for staining according to the protocol recommended. Follicular helper T (Tfh) cells were defined by extracellular staining with PE anti-rat-CD4, PE-Cy7 anit-rat-ICOS, and FITC anti-rat-CXCR5.6. Cell proliferation assay We cultured MNC suspended in 200 μl aliquots containing 4 × 105 cells in triplicates in flat-bottomed 96-well microtitre plates in the absence or presence of R97-116 peptide for incubating 72h. Then we gived the cells 10 μl CCK-8 and incubated cells for 4 h at 37℃. Finally, we detected results by the absorbance at 450 nm on a microplate reader, and expressed results as OD values ± SD.7. Determination of cytokines by ELISA We incubated MNC in the presence of R97-116 peptide for 72 h, and collected MNC supernatants. Then, we followed the manufacturer’s instructions to measure for IL-1β and IL-17 by ELISA kits.8. Detection of serum level and relative affinity of anti-R97-116 peptide IgG antibody We collected the rats serums on day 43 p.i., and detected level and relative affinity of anti-R97-116 peptide IgG antibody by ELISA.9. Statistical analysis of data We used the SPSS 17.0 computer programme for all calculations and statistical evaluations. Differences among two groups were tested by two-tailed Student t test and differences among three groups were tested by one-factor analysis of variance (ANOVA) followed by Least Significant Difference (LSD) test as a post-hoc test. Finally, we presented the results as means ± SD and considered a level of p< 0.05 significant.Results:1. Effects of caspase-1 inhibitor on the phenotype and intracellular cytokines of DCs in vitro DCs were cultured with or without Ac-YVAD-cmk, and the expression of CD80, CD86, MHC class Ⅱ, and IL-1β from DCs were analyzed by flow cytometry. The phenotypic analysis of spleen DCs showed that the expression of CD86 and MHC class Ⅱ were inhibited by caspase-1 inhibitor, and IL-1β production was decreased by caspase-1 inhibitor in vitro. The phenotypic analysis of bone marrow DCs showed that the expression of CD80 and CD86 were inhibited by caspase-1 inhibitor, and IL-1β production was also decreased.2. Caspase-1 inhibitor suppresses the development of EAMG and regulates the phenotype of DC in EAMG The rats in Ac-YVAD-cmk treatment group exhibited lower clinical scores when compared with rats in EAMG group. There was no difference for the levels of anti-R97-116 IgG between Ac-YVAD-cmk and EAMG groups. However, the affinity in Ac-YVAD-cmk group was lower than that in EAMG group. Further, the percentages of CD86 and MHC class Ⅱ positive cells among OX62+ DC were significantly decreased in rats treated with Ac-YVAD-cmk compared to that in EAMG rats in vivo.3. Effects of exogenous IL-1β on the caspase-1 inhibitor in EAMG The rats in Ac-YVAD-cmk treatment group exhibited lower clinical scores when compared with rats in Ac-YVAD-cmk with IL-1β treatment group. In addition, the clinical scores between Ac-YVAD-cmk with IL-1β group and EAMG group did not differ significantly. The results of IL-1β on humoral immune responses showed that the level of anti-R97-116 IgG in rats treated with Ac-YVAD-cmk with IL-1β was dramatically increased compared with the other two groups. Lower affinity indexes of anti-R97-116 peptide IgG in two treated groups was observed compared with that in EAMG rats. Furthermore, there was no difference for the levels of anti-R97-116 IgG1, IgG2a, and IgG2b between EAMG group and Ac-YVAD-cmk group. The level of anti-R97-116 IgG1 and G2a in rats treated with Ac-YVAD-cmk with IL-1β was dramatically increased compared with the other two groups.4. Caspase-1 inhibitor attenuates EAMG via suppressing DC IL-1β, CD4+T and γδT cells IL-17 pathways The percentage of IL-1β positive cells among OX62+DC was reduced in rats treated with Ac- YVAD-cmk when compared with that in EAMG rats, which was partly reversed by IL-1β but not statistically significant. There was no difference between EAMG group and Ac-YVAD-cmk with IL-1β group. The percentage of CD4+IL-17+cells was reduced in rats treated with Ac-YVAD-cmk when compared with that in EAMG rats, which was partly reversed by exog enous IL-1β but not statistically significant. There were no differences among the three groups in terms of the numbers of βδ T cells in MNC from lymph nodes. However, the percentage of IL-17-expressing βδ T cells was reduced in rats treated with Ac-YVAD-cmk when compared with that in EAMG rats, and there were no statistical difference between two treatment groups. The production of IL-1β in the culture supernatants of Ac-YVAD-cmk treated group was reduced when compared with that in EAMG group. The production of IL-1β in the culture supernatants in rats treated with Ac-YVAD-cmk and IL-1β was increased when compared with that in Ac-YVAD-cmk rats, but this difference did not reach to a statistically significant value. There was no difference between EAMG group and Ac-YVAD-cmk with IL-1β group. There were no differences for the levels of IL-17 among the three groups.5. Effects of caspase-1 inhibitor on lymphocyte proliferation and CD4+CD25+Foxp3+T, CD4 +CXCR5+ICOS+T cell subset differentiation The results showed that there was no difference for the percentage of CD4+CD25+Foxp3+ T cells among three groups. The percentage of CD4+CXCR5+ICOS+Tfh cells in rats treated with Ac-YVAD-cmk decreased compared with that in EAMG rats. The percentage of CD4+CXCR5+ICOS+ Tf h cells in rats treated with Ac-YVAD-cmk with IL-1β exhibited a decreased trend but did not reach the statistically significant value compared with that in EA MG group. In the absence or presence of R97-116 antigen, lymphocyte proliferation in Ac-YVAD-cmk group was decreased compared with that in EAMG group. In the meantime, statistical difference between two treatment groups was not found.Conclusions:1. Caspase-1 inhibitor can inhibit DC maturation and IL-1β production from DC.2. Caspase-1 inhibitor decreased IL-17 production by γδ T cells and CD4+T cells, and decreased the affinity of anti-R97-116 peptide IgG, thereby suppressing EAMG progression.3. Caspase-1 inhibitor decreased the number of Tfh cell.4. Administration of exogenous IL-1β in vivo could not reverse the suppressive effects of caspase-1 inhibitor.Background:Experimental autoimmune myasthenia gravis (EAMG), an animal model for human myasthenia gravis (MG), is an antibody-mediated, T cell-dependent, complements involved autoimmune disease. EAMG can be induced in Lewis rats by immunization with torpedo acetylcholine receptor (TAChR) or a synthetic peptide corresponding to region 97-116 of the rat AChR α subunit (R97-116 peptide), which serves as a reliable animal model used to investigate the immunopathological mechanisms and novel therapeutic strategies.Antibody-secreting B cells are generally considered to positively regulate immune responses, and they play a central role in the development of MG. The regulatory role of B cells in autoimmune diseases was first reported in experimental autoimmune encephalomyelitis (EAE). Regulatory B cells (Breg), a specific and functionally unique subset of B cells, have been shown to negatively control the immune system. Germinal centers (GCs) develop within B cell follicles of secondary lymphoid organs where B cells undergo somatic hypermutation, high affinity maturation, and result in plasma and memory B cell development. The Breg subtypes may be similar to the regulatory T cells (Treg) subtypes, including B cells producing IL-10 (B10) or TGF-β, and B cells expressing Foxp3 (B-Foxp3). Follicular Th (Tfh) cells, a subset of CD4+ Th cells that migrate into GCs, are indicated to promote B cell proliferation and affinity maturation, particularly during the GC reaction. Distinguishing features of Tfh cells are the expression of surface molecules including CXC chemokine receptor 5 (CXCR5), inducible T-cell costimulator (ICOS), and programmed death protein-1 (PD-1), and the transcription factor B cell lymphoma 6 (Bcl-6) is the master regulator needed for Tfh development and function. Recent studies have shown that a proportion of follicular T cells, with phenotypic characteristics of Tfh cells and expressing Foxp3, are defined as follicular regulatory T (Tfr) cells, which inhibited Tfh function and limited GC reactions.We previously demonstrated that caspase-1 inhibitor ameliorates EAMG via suppressing DC IL-1β, CD4+ T and γδT cells IL-17 pathways. The previous study focused on the effect of caspase-1 inhibitor on cellular immunity, and this study further investigated the effect of caspase-1 inhibitor on humoral immunity.Objective:The aim of our present study was to investgate the effect of caspase-1 inhibitor on humoral immune response of EAMG and further explore the underlying mechanisms.Methods:1. EAMG model buildingFemale Lewis rats,160-180 g (aged 6-8 weeks old).200 μl inoculum containing R97-116 peptide, Mycobacterium tuberculosis in incomplete Freund’s adjuvant could induce EAMG, which was induced by subcutaneous injection into both hind footpads on day 0 and was boosted with the same dose along the back on day 11 after the first immunization.2. Experimental animal groupingRats were randomly divided into respective groups (6 rats/group). From day 13 after the first immunization, rats in the Ac-YVAD-cmk treatment group (at doses of 100 μg/rat in a volume of 0.4 ml PBS) were administered intraperitoneally (i.p.) every second day. EAMG rats were administered with vehicle (PBS) at the same time points.3. Detection of serum level and relative affinity of anti-R97-116 peptide IgG antibodyWe collected the rats serums on day 43 p.i., and detected level and relative affinity of anti-R97-116 peptide IgG antibody by ELISA.4. Lymph node MNC preparationWe ultimately killed rats of every group and removed inguinal lymph nodes under aseptic conditions on day 43 p.i., Then we grinded the organs through cell strainers in serum-free medium to obtain MNC suspensions and resuspended cells to 2×106/ml for the following experiments.5. Flow cytometric analysis of lymph node MNCTfh or Tfr cells were identified by determining the staining profile of cells following incubation with FITC anti-rat-CD4, PE-Cy7 anit-rat-ICOS, CXCR5 and second antibody PE-conjugated goat anti-rabbit IgG, in the presence or absence of PE-Cy5-conjugated anti-mouse/rat Foxp3 antibodies. B-Foxp3 or B10 cells were defined by staining of FITC-conjugated anti-CD 19 and PE-conjugated anti-Foxp3 or PE-conjugated anti-IL-10.6. Determination of cytokines by ELISAWe incubated MNC in the presence of R97-116 peptide for 72 h, and collected MNC supernatants. Then, we followed the manufacturer’s instructions to measure for IL-10 by ELISA kits.7. Detection of GCs by immunofluorescenceWe prepared 10 μM-thick frozen sections from rat popliteal lymph nodes, and then air-dried, fixed in cold acetone for 10 min at 4 ℃. After blocked by 0.5% bovine serum albumin for 30 min, we incubated sections with Fluorescein-labeled Peanut agglutinin (PNA) and DyLight(?) 594-labeled goat anti-rat IgM at 4℃ overnight. Finally, we washed sections and examined under an immunofluorescence microscopy.8. Statistical analysis of dataWe used the SPSS 17.0 computer programme for all calculations and statistical evaluations. Differences among two groups were tested by two-tailed Student t test. Finally, we presented the results as means ± SD and considered a level ofp< 0.05 significant.Results:1. Caspase-1 inhibitor inhibits anti-R97-116 IgG and germinal center response in EAMG rats. Serum were collected from rats in the EAMG and caspase-1 inhibitor Ac-YVAD-cmk groups on day 43 p.i. and antibody responses assessed by ELISA. There was no difference for the levels of anti-R97-116 IgG between EAMG and Ac-YVAD-cmk groups. However, the affinity in Ac-YVAD-cmk group was lower than that in EAMG group. PNA+GC-B cells were significantly decreased in the Ac-YVAD-cmk group than that in EAMG group.2. Caspase-1 inhibitor balances the Tfh and Tfr cell populations in EAMG rats.The results showed a decrease in Tfh and an increase in Tfr levels in EAMG rats treated with caspase-1 inhibitor Ac-YVAD-cmk compared to untreated EAMG rats.3. Caspase-1 inhibitor increases Breg cells in EAMG rats.The percentage of B-Foxp3 cells was increased in the Ac-YVAD-cmk treatment group compared with that in EAMG group. B10 cells were aslo increased in the Ac-YVAD-cmk group compared with the EAMG group but not statistically significant. We further examined the levels of IL-10 in culture supernatants of lymphocytes stimulated with R97-116 peptide. The production of IL-10 in the culture supernatants of Ac-YVAD-cmk treated group was increased when compared with that in EAMG group.Conclusions:1. Caspase-1 inhibitor reduced the relative affinity of anti-R97-116 IgG.2. Caspase-1 inhibitor suppressed germinal center response.3. Caspase-1 inhibitor decreased follicular helper T cells, and increased follicular regulatory T cells and regulatory B cells.