MANF Specifically Regulates Splenic Macrophage Differentiation In CCl₄-induced Hepatic Fibrosis

Splenic immune cells,especially splenic macrophages,play a key role in the liver fibrosis process.With a proved immunoregulatory function of mesencephalicastrocyte-derived neurotrophic factor(MANF)in retina repair,how MANF affects splenic immune cells in the pathological situation of hepatic fibrosis is still unknown.In this study,we established the hepatic fibrosis model in wild type(WT)and mono-macrophage-specific MANF knockout mice(M(?)MANF-/-)by intraperitoneally injecting carbon tetrachloride(CCl4)for 8 weeks.Comparing with WT,we found the number of M1 macrophages was increased in M(?)MANF-/-mice.However,M2 macrophages and plasma cells were significantly increased in M(?)MANF-/-mice under the hepatic fibrosis condition.We also found M(?)MANF-/-did not affect the number of splenic T and B cells under both the normal and hepatic fibrosis conditions.Our results suggest a specific regulation of MANF on the differentiation of splenic macrophages,which may exert a significant impact on the hepatic fibrosis process.ObjectiveTo study the effects of M(?)MANF-/-on splenic immune cells under normal and hepatic fibrosis conditions.MethodsWT and M(?)MANF-/-mice were injected intraperitoneally with 2 p,l/g CCl4 for 8 weeks.All mice were sacrificed in the ninth week,then the spleens were removed and weighed to calculate the spleen/liver weight ratio.Also,the spleens were embedded in paraffin for histology assays,and frozen in liquid nitrogen for cytokine detection.The structure of the spleen was examined by HE staining.The different splenic immune cells were detected by immunohistochemistry with the corresponding markers.The macrophages subtypes were examined by immunohistochemistry.Multiple cytokines were detected by real-time quantitative PCR.Results1.Hepatic fibrosis mice model was successfully established.The results from HE and Sirius red staining showed that the hepatic fibrosis model was successfully established in mice.2.Identification of MANF knockout in M(?)MANF-/-mice.The peritoneal macrophages were isolated from M(?)MANF-/-mice and cultured for 24 h.The results from Immunofluorescence and western blot showed that MANF were successfully knocked out in macrophages of M(?)MANF-/-mice.3.MANF knockout in mono-macrophages does not affect the morphology and size of spleen.The results from HE staining showed that there was no significant change of splenic morphology between WT and M(?>)MANF-/-mice.Also,the spleen/liver weight ratios,as well as the number of lymphoid follicle and the area of red pulp in M(?)MANF-/-mice were not different from that in WT mice.4.MANF knockout in mono-macrophages specifically up-regulates the number of M1 macrophages.To investigate the effects of MANF on splenic immune cells,we examined the splenic macrophages,plasma,T and B cells in WT and M(?)MANF-/-mice by immunohistochemistry with the antibodies against the corresponding markers.We found that splenic CD68-positive macrophages in M(?)MANF-/-mice were more than that in WT mice,whereas there was no significant difference in the number of plasma,T and B cells between WT and M(?)MANF-/-mice.Meanwhile,the splenic iNOS+ Ml macrophages in M(?)MANF-/-mice were significantly increased,compared with WT.Consistently,the mRNA levels of iNOS,TNF-α,and IL-1 β were also increased in M(?)MANF-/-mice.However,there is no difference in the number of the splenic CD206+M2 macrophages between two groups.The expressions of M2 macrophage-specific markers Ym-1,Fizz-1,Arg-1,and TGF-β in spleen remained unchanged.Therefore,the macrophage-specific MANF knockout only affect the number of splenic M1 macrophages,rather than M2 macrophages,plasma,T and B cells.5.MANF knockout in mono-macrophages exacerbates splenomegaly and increases the number of macrophages and plasma in hepatic fibrosis mice.To investigate the effect of liver fibrosis on the spleen of M(?)MANF-/-mice,we determine the size and weight of spleens,the number of immune cells,and the proliferation of splenocytes.The results from HE staining showed more severe splenomegaly in M(?)MANF-/-mice than that in WT mice under liver fibrosis.And,the spleen/liver weight ratios,as well as the area of red pulp were increased in M(?)MANF-/-mice,whereas the number of lymphoid follicle in M(?)MANF-/-mice were not different from that in WT mice.We also found that splenic CD68-positive macrophages and CD 13 8-positive plasma in M(?)MANF-/-mice were more than that in WT mice,whereas there was no significant difference in the number of T and B cells between WT and M(?)MANF-/-mice in hepatic fibrosis.6.MANF knockout in mono-macrophages specifically up-regulates the number of M2 macrophages under liver fibrosis.The splenic CD206+,Fizz-1+ and Ym-1+ M2 macrophages in M(?0 MANF-/-mice were significantly increased,compared with WT under liver fibrosis.Consistently,the mRNA levels of Ym-1,Fizz-1,and TGF-β were also increased in M(?)MANF-/-mice.However,there is no difference in the number of the splenic iNOS+ M1 macrophages between two groups.The expressions of M1 macrophage-specific markers iNOS,TNF-a,IL-1β,and IL-6 in spleen remained unchanged.Therefore,the macrophage-specific MANF knockout promotes splenic macrophages differentiation to M2 subtype.Conclusion1.MANF knockout in mono-macrophages increases the number of splenic macrophages,especially M1 macrophages.2.MANF knockout in mono-macrophages exacerbates splenomegaly and increases splenic M2 macrophages and plasma under hepatic fibrosis.