| Objective:Pregnancy is a complex physiological process,depending on the balance of immune rejection and immune tolerance between the mother and the fetus.However,the corresponding mechanism of immune regulation is still unclear.CaMK4 is a multifunctional serine/threonine kinase expressed in T cells and can activate transcription factors that play important roles in T cell differentiation.More researches evaluated that CaMK4 is an important signaling molecule for the differentiation of Th17 cells.Th17 cells are a subset of cell types in CD4~+T cells that secrete inflammatory cytokines such as IL-17 to participate in the inflammatory response and autoimmune diseases.The excessive activity of Th17 cells may lead to pathological pregnancy such as spontaneous abortion.However,whether CaMK4 can participate in maternal-fetal immune regulation during pregnancy has not been reported,and whether it can affect the number and function of Th17 cells at the maternal-fetal interface to improve pregnancy outcomes is unknown.In this study,we investigated the role of CaMK4 at the maternal-fetal interface,whether it is involved in immune regulation and its related mechanisms.Methods:Part ? Influences of CaMK4 on pregnancy were evaluated through intraperitoneal injection of its inhibitor(KN-93)into C57BL/6 female mice mated with BALB/c male mice,administration of LPS was performed simultaneously to induce abortion.In detail,BALB/c mated C57BL/6 female mice randomly,and the pregnant mice were divided into four groups:group 1=control group(corresponding vehicle injected at E7.5,E8.5,E9.5);group 2=abortion group(200?l vehicle injected at E7.5,E8.5,E9.5+2.0?g/20g LPS injected at E7.5);group 3=LPS+KN-93 group(8?g/g KN-93injected at E7.5,E8.5,E9.5+2.5?g/20g LPS injected at E7.5);group 4=KN-93group(8?g/g KN-93 injected at E7.5,E8.5,E9.5+200?l vehicle injected at E8.5).At E11.5,the morphology of uteruses,fetuses and spleens of the four groups were observed,and rates of fetal absorption were calculated.Peripheral blood,uteroplacental complexes and spleens of four groups were subsequently obtained.HE staining of uteroplacental complexes was obtained to observe the inflammatory response at the maternal-fetal interface.Part ? After the peripheral blood,uteroplacental complexes and spleens of the four groups were obtained on E11.5,the contents of Th17 cells in the peripheral blood and spleen tissues of the mice in each group were detected by flow cytometry and those at the maternal-fetal interface by immunohistochemistry.Part ? After the uteroplacental complexes of the four groups were obtained on E11.5,Th17 cell-related factors IL-17 and ROR-?t and CD4~+T cell-related factors IFN-?,IL-6,IL-21,IL-22 on m RNA level were examined.Part ? After the uteroplacental complexes and spleens of the four groups were obtained on E11.5,the expression levels of CaMK4 were compared by Western Blot.Furthermore,AKT,S6K and their phosphorylated proteins,which are key proteins of AKT/m TOR/S6K,were detected.Results:Part ? Exposure of LPS led to abortion,and rate of fetal abortion of group 2 was100%,which was significantly higher than that of the normal pregnancy group.KN-93 could effectively reverse the abortion induced by LPS.There was no significant difference in the rate of fetal absorption between group 1 and group 4.Comparing the spleen length,LPS induced spleen enlargement while inducing abortion,and KN-93 inhibited spleen enlargement caused by LPS application.There was no difference in spleen lengths between group 4 and normal pregnancy group.The HE section showed that the maternal-fetal interface had severe inflammation,incomplete tissue structure,and a large number of inflammatory cells infiltrated caused by LPS.The uteroplacental complexes tissues of groups 1,3,and 4 mice were intact,and no obvious inflammatory cell infiltration was observed.Part ? The flow cytometry presented that LPS caused a significant increase in Th17 cells in peripheral blood and spleens compared with the normal pregnancy group.KN-93 could effectively inhibit the level of Th17 cells in peripheral blood and spleens of mice.Analysis of the results of immunohistochemical staining of uteroplacental complexes tissue showed that the content of Th17 cells at the maternal-fetal interface of group 2 was significantly increased compared to the normal pregnancy group.KN-93 can reduce the infiltration of Th17 cells at the maternal-fetal interface.Part ? The results of real-time PCR showed that the m RNA levels of the characteristic cytokines and the transcription factor of Th17 cells(IL-17 and ROR-?t)at the maternal-fetal interface were significantly increased by LPS.KN-93 has no reversal effect on the expression of IFN-?,IL-6,IL-21,and IL-22,which were related to immune regulation.Part ? Western Blot results showed that the expressions of CaMK4 in uteroplacental complexes and spleens of mice were increased after LPS injected,and KN-93 could inhibit the expression of CaMK4.After KN-93 treated,the expressions of AKT/p-AKT and S6K/p-S6K at the maternal-fetal interface and spleens were significantly lower than those of group 1 and group 2.Conclusions:1.KN-93(CaMK4 inhibitor)can effectively reverse LPS-mediated mice abortion and imbalance of the immune microenvironment at the maternal-fetal interface,suggesting that high expression of CaMK4 may be involved in the mechanism of abortion,which is not conducive to the stability of the immune microenvironment at the maternal-fetal interface;2.Changes in the expression of CaMK4 not only cause changes in the levels of Th17 cells in peripheral blood,spleens and placentas,but also affect the expression of characteristic cytokines and transcription factors which are IL-17 and ROR-?t,suggesting that CaMK4 is involved in regulating the peripheral environment and the proliferation,differentiation and functions of Th17 cells at maternal-fetal interface.3.KN-93 reverses LPS-induced abortion in mice while inhibiting AKT/m TOR signaling pathway activity at the spleen and maternal-fetal interface,suggesting that AKT/m TOR/S6K pathway is one of the mechanisms by which CaMK4 participates in the pathogenesis of abortion.In summary,CaMK4 can participate in the regulation of maternal-fetal immune microenvironment and the occurrence of abortion by affecting the proliferation,differentiation and function of Th17 cells.AKT/m TOR/S6K signaling pathway might be one of the mechanisms.Our findings will be beneficial for the development of treatment strategies for miscarriage. |
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