Immunoregulation Mechanisms Of Hydrogen Sulfide And Lipoxin A4 In Different Inflammatory Models

Part Ⅰ Immunoregulation mechanism of hydrogen sulfide in low dose LPS-induced preeclampsia murine modelObjective:Preeclampsia (PE) is a multisystemic syndrome during pregnancy. Aberrant maternal immune responses and inflammation have been implicated in the pathogenesis of PE. Hydrogen sulfide (H2S), an endogenous gasotransmitter, has been shown anti-inflammatory and immunoregulary effects. In the present study, we investigated the effects and immunoregulary mechanisms of H2S in lipopolysaccharide (LPS)-induced PE mouse model.Methods:Preeclampsia mouse model was established by the administration of low dose LPS. Serum tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10) levels were detected by ELISA. The percentages of CD4+Foxp3+ Treg cells, CD4+ L-17+ Th17 cells and CD11c+ CD80+ DCs were detected by flow cytometry. The expressions of CBS and CSE mRNA in the spleen were detected by real-time PCR. The expressions of Foxp3, Tetl and Tet2 mRNA in the spleen and para-aortic lymph nodes (LNs) draining the uterus were detected by real-time PCR. The expressions of IL-17A and RORyt mRNA in para-aortic LNs were detected by real-time PCR.Results:1. Serum TNF-αand IL-6 levels in LPS group were significantly higher than in control group. Serum IL-10 level and fetal weight were significantly lower than in control group. H2S donor NaHS significantly attenuated the effects of LPS. The expression of CSE mRNA was downregulated in the spleen of LPS group mice compared with control group, but there was no significantly difference in the expression of CBS mRNA between two groups.3. The percentage of CD4+Foxp3+ Treg cells and the expressions of Foxp3 and Tetl mRNA in the spleen and para-aortic LNs in LPS group were lower than in control group, but the percentage of CD4+Foxp3+Treg cells and the expressions of Foxp3 and Tetl mRNA in the spleen and para-aortic LNs in LPS+NaHS group were significantly higher than in LPS group. The expression of Tet2 mRNA in the spleen and para-aortic LNs had no significant difference among three groups.4. The percentage of CD4+IL-17+ Th17 cells and the expressions of IL-17A and RORyt mRNA in the spleen and para-aortic LNs in LPS group were higher than in control group. The percentage of CD4+IL-17+Th17 cells in the spleen had no difference between LPS+NaHS group and LPS group, but the percentage of CD4+IL-17+Th17 cells in para-aortic LNs in LPS+NaHS group were significantly lower than in LPS group. The expressions of IL-17A and RORyt mRNA in para-aortic LNs in LPS+NaHS group were significantly lower than in LPS group.5. The percentage of CD11c+CD80+DCs in LPS group was higher than in LPS+NaHS group and control group.Conclusions:The results suggest that H2S donor NaHS effectively alleviates low dose LPS-induced systemic inflammation and fetal growth restriction by promoting the expansion of treg cells, decreasing the percentage of Th17 cells and keeping DCs in an immature state. NaHS induces the expansion of Treg cells involving to upregulation of Tet1 expression.Part Ⅱ Immunoregulation mechanism of lipoxin A4 in DSS-induced acute colitis murine modelObjective:Inflammatory bowel disease (IBD) is chronic, relapsing inflammatory disorders of the intestine intestinal inflammatory diseases, including ulcerative colitis (UC) and Crohn’s disease (CD). The etiology and pathogenesis is not clear so far. Now that abnormal immunity that lead to the inflammatory response play a critical role in the development of IBD. It has been reported that a unique IL-10-producing regulatory B cell subset (B10 cells) inhibit DSS-induced colitis. Other reports have found that during intestinal inflammation, CX3CR1+ macrophages from blood monocytes enter the gut to acquire distinct fates, thus breaching the intestinal homeostasis. Lipoxin A4 (LXA4) is a product of arachidonic acid that has anti-inflammatory and pro-resolution properties during inflammation. In this study, we established DSS-induced acute colitis murine model and systematically investigated the regulatory role of LXA4 on B cells and CX3CR1+ macrophages during protecting colitis. These findings may provide new theoretical basis for LXA4 treatment of IBD.Methods:1. Mice were administered 3.5% DSS in normal drinking water for 7 days to establish DSS-induced acute colitis, meanwhile LXA4 (10μg/kg) was injected i.p. daily. We recorded weight, DAI score and pathological grade. Colonic LXA4, IL-6, TNF-α, IL-1β and IFN-γ levels were measured using enzyme-linked immunosorbent assay (ELISA) kit. The expression of Colonic 5-LOX, IL-6, TNF-α, IL-1β, IFN-γ and iNOS mRNA were detected by real-time PCR.2. Splenic CD19+ B cells were purified by magnetic cell separation (MACS). The expression of ALX/FPR2 mRNA in spleen and purified B cells was detected by RT-PCR.3. Splenic cells were stimulated with LPS (10μg/ml), PMA (50ng/ml), ionomycin (750ng/ml) and monensin (2μM) for 5 hours. The percentage of B10 cells were detected on day 3,5 and 7 by flow cytometry. The expressions of IL-10 and GM-CSF mRNA in purified CD19+ B cells of the spleen were detected on day 8 by real-time PCR.4. Mice were administered 3.5% DSS in normal drinking water for 7 days after splenectomy, meanwhile LXA4 (10μg/kg) treatment daily. DAI score was recorded. The expressions of colonic IL-6, TNF-α, IL-1β and iNOS mRNA were detected by real-time PCR.5. Phagocytic ability of peritoneal macrophages was examined by FITC-dextran.6. The percentage of blood Ly6C+ cells, CX3CR1+ cells in colonic lamina propria and CX3CR1+ cells, CD4+IFN-γ+ T cells and CD4+IL-17+ T cells in mesenteric lymph nodes were detected by flow cytometry. The expression of CD68 positive cells in colonic lamina propria was detected by immunohistochemistry. The expression of CCR7 mRNA in colonic lamina propria and IL-6, IFN-y and IL-17 mRNA in mesenteric lymph nodes were detected by real-time PCR.6. The expression of NF-κB p65, ERK1/2 and STAT3 proteins and their phosphorylation proteins in colonic tissue were detected by Western blot.Results:1. DAI score and pathological grade in DSS+LXA4 group were significantly lower than in DSS group. Colonic LXA4, IL-6, TNF-α, IL-1β and IFN-γ levels in DSS+LXA4 group were substantially decreased compared with DSS group. The expression of Colonic 5-LOX, IL-6, TNF-α, IL-1β, IFN-γ and iNOS mRNA were also decreased.2. Splenic Purified CD19+ B cells expressed ALX/FPR2.3. The percentage of splenic B10 cells in DSS group and DSS+LXA4 group were significantly lower than Control group, but there was no significant difference in DSS+LXA4 group and DSS group.4. After splenectomy, LXA4 still alleviated DSS-induced acute colitis. DAI score in DSS+LXA4 group was significantly lower than in DSS group. Colonic LXA4, IL-6, TNF-α, IL-1β and IFN-γ levels in DSS+LXA4 group were substantially decreased compared with DSS group.5. Phagocytic ability of peritoneal macrophages in DSS+LXA4 group was significantly lower than in DSS group, but the percentage of peritoneal macrophages had no significant difference in between DSS+LXA4 group and DSS group.6. The percentage of blood Ly6C+ cells had no significant difference in between DSS+LXA4 and DSS groups. The percentage of CX3CR1+ cells in colonic lamina propria and mesenteric lymph nodes in DSS+LXA4 group were substantially decreased compared with DSS group. The percentage of CD4+IFN-γ+ T cells and CD4+IL-17+ T cells in mesenteric lymph nodes were also significantly decreased in DSS+LXA4 group. The expressions of CCR7 mRNA in colonic lamina propria and IL-6, IFN-γ and IL-17 mRNA in mesenteric lymph nodes in DSS+LXA4 group were also significantly decreased compared with DSS group. The expression of CD68 positive cells in colonic lamina propria was higher in DSS group than DSS+LXA4 group.7. The expression of NF-κB p65, ERK1/2 and STAT3 proteins in colonic tissue had no significant difference in DSS+LXA4 group and DSS group, but the expression of their phosphorylation proteins in DSS+LXA4 group were significantly lower than in DSS group.Conclusions:The results suggest that LXA4 alleviates DSS-induced acute colitis, independent of the regulation of splenic B cells. LXA4 plays a protective role by reducing the colonic lamina propria CX3CR1+ cells and inhibiting their migration to the lymph nodes decreased T cell immune response. LXA4 alleviates DSS-induced acute colitis by inhibiting phosphorylation levels of NF-κB, ERK1/2 and STAT3 proteins in colonic tissue.PartⅢ Immunoregulation mechanism of lipoxin A4 on B cells in LPS-induced sepsis murine modelObjective:Sepsis is a severe life-threatening disease, with severe inflammation, multiple organ failure and death. Recently it has been reported that an effector B cell population produces granulocyte-macrophage colony stimulating factor (GM-CSF) and protects against sepsis. Lipoxin A4 (LXA4) is an arachidonic acid product with specific anti-inflammatory and pro-resolution properties. LXs can play a regulatory role on a variety of immune cells. Recently it is reported that LXA4 can inhibit human memory B-cell antibody production and proliferation. However, little is known about the regulation action of LXA4 on GM-CSF-producing B cell subpopulation. In this study, we established LPS-induced sepsis mouse model and investigated the regulatory role of LXA4 on B cells which produces GM-CSF. These findings may provide new insights for immune regulatory function of LXA4.Methods:1. Mice were administered LPS (10μg/mouse) daily by i.p. injections in PBS over the course of 4 days to establish LPS-induced sepsis model, meanwhile LXA4 (10μg/kg) was injected i.p. daily. On day 5, serum and peritoneal lavage fluids IL-6, TNF-α and IL-1β levels were measured using enzyme-linked immunosorbent assay (ELISA) kit.2. The expressions of GM-CSF mRNA in mice spleen and purified CD19+ B cells were detected by real-time PCR. The percentage and mean fluorescence intensity (MFI) of splenic GM-CSF+CD19+ B cells were detected by flow cytometry.3. The expressions of VLA-4 and LFA-1 mRNA in purified CD19+ B cells of the spleen were detected by real-time PCR. The percentage of peritoneal CD19+ B cells was detected by flow cytometry. Mice were injected peritoneal CFSE-labeled CD19+ B cells by i.p. and next administered LPS (10μg/mouse) and LXA4 (10μg/kg) daily by i.p. injections in PBS over the course of 4 days, the percentage of splenic CFSE-labeled CD19+ B cells was detected by flow cytometry after 48 hours.4. The expression of ALX/FPR2 mRNA in purified peritoneal macrophages and B cells was detected by real-time PCR. After Clodronate-liposomes depleted peritoneal macrophages, mice were injected i.p. LPS (10μg/mouse) daily for four consecutive days, while treatment of LXA4 (10μg/kg) daily, the percentage of splenic GM-CSF+CD19+B cells was measured by flow cytometry on day 5.Results:1. Serum and peritoneal lavage fluids IL-6, TNF-α and IL-1β levels in LPS+LXA4 group were substantially decreased compared with LPS group.2. The expression of GM-CSF mRNA in mice spleen and purified CD19+ B cells in LPS+LXA4 group were both significantly higher than LPS group. The percentage of splenic GM-CSF+CD19+ B cells in LPS+LXA4 group was increased significantly than LPS group, but there was no significant difference in mean fluorescence intensity of LPS+LXA4 and LPS groups.3. The expressions of VLA-4 and LFA-1 mRNA in splenic purified CD19+ B cells in LPS+LXA4 group were both significantly higher than that in LPS group. After 48 hour mice were injected i.p peritoneal CFSE-labeled CD19+B cells, the percentage of splenic CFSE-labeled CD19+B cells in LPS+LXA4 group was increased significantly than in LPS group.4. The percentage of splenic GM-CSF+CD19+B cells in LPS+LXA4 group was significantly increased than in LPS group after peritoneal macrophages were depleted. 5. The phosphorylation levels of signal transducer and activator of transcription 5 (STAT5) of splenocytes and purified CD19+ B cells in LPS+LXA4 group was significantly increased than in LPS group.Conclusion:These results suggest that LXA4 protects against LPS-induced sepsis by promoting the generation and migration of splenic IRA B cells, and the underlying molecular mechanism may be related to STAT5 activation.