The Role Of IL-37b In Early Lesion Formation And Growth In A Murine Endometriosis Model

Objective: Endometriosis is a gynecological disease with abnormal expression of interleukin(IL)-37 which can suppress inflammation and the immune system.IL-37b is the best-studied splice variant of IL-37 coding gene.Here we investigated the role of the IL-37b splice variant in early lesion formation and growth in a murine endometriosis model.Methods:(1)Endometriosis was induced by intraperitoneally injected uterine segments(d0).In order to assess the effect of IL-37b on the development of endometriosis,recombinant human IL-37b protein(rh IL-37b)was intraperitoneally injected from 3 days before induction,with PBS serving as the control,and mice were killed at d3,d7 or d14.Endometriosis-like lesions in each mouse were counted,measured and weighed.Next,rh IL-37b or PBS was injected at the stage of early lesion formation(to d3)or lesion growth(d3 to d14)to assess the effect of IL-37b on lesion formation and growth,and lesions were counted,measured and weighed.(2)To determine whether macrophages play an role in the effect of IL-37b on the development of endometriosis,rh IL-37b or/and Clodronate Liposomes(Clo)which was used to deplete macrophages was injected intraperitoneally at the stage of early lesion formation or lesion growth,with PBS serving as the control.Lesions in each mouse were counted,measured and weighed.(3)To assess the effect of IL-37b on proliferation in ectopic lesions,immunohistochemistry was performed to detect the expression of proliferating cell nuclear antigen(PCNA).To assess the effect of IL-37b on invasion and angiogenesis in lesions,real-time PCR was applied to detect the expression of matrix metalloproteinase(Mmp)2,Mmp9 and vascular endothelial growth factor-a(Vegfa)m RNA.An immunohistochemistry analysis was performed of MMP9 and VEGF in lesions at d14.The levels of MMP9 and VEGF in peritoneal fluids were measured by ELISA.(4)To assess the effect of IL-37b on the expression of inflammatory factors in lesions,real-time PCR was applied to detect the expression of Il1 b,Il6,tumor necrosis factor(Tnf)a,Il10,transforming growth factor(Tgf)b1 and IL-37b receptors m RNA at d3 and d14.The level of IL-1β,IL-6,TNF-α,IL-10 and TGF-β1 in peritoneal fluids was measured by ELISA.(5)Murine uterine segments were cultured and then treated with rh IL-37b at different concentrations for 3 days in vitro.The gene expression of MMP2,MMP9,VEGF-A,inflammatory factors and IL-37b receptors in the uterine segments treated with rh IL-37b was compared to non-treated segments by real-time PCR analysis.The expression of Akt,p-Akt,Erk1/2 and p-Erk1/2protein in uterine segments was detected by Western Blot.(6)Endometrial stromal cells(ESCs)were isolated and cultured in vitro,and the expression of ESCs marker vimentin and endometrial epithelial cells marker cytokeratin was assessed using immunohistochemistry.After treatment with rh IL-37b at different concentrations,CCK-8 and transwell assay were used to assess cell proliferation and invasion ability;real-time PCR was used to assess the effect of rh IL-37b on the expression of MMP2,MMP9,VEGF-A,inflammatory factors and IL-37b receptors.(7)The expression of IL-37b in the ESCs transfected with p IL-37b or pc DNA3.1 plasmid was identified by realtime PCR and Western Blot.CCK-8 and transwell assay were used to assess the effect of IL-37b overexpression on cells proliferation and invasion.Gene expression of MMP2,MMP9,VEGF-A,inflammatory factors and IL-37b receptors in transfected ESCs was measured by real-time PCR.Western Blot was applied to detected the expression of Akt,p-Akt,Erk1/2 and p-Erk1/2 protein in transfected ESCs.(8)RL95-2 cells were cultured and then treated with rh IL-37b for 24 hours at different concentrations in vitro.Cell proliferation was assessed by CCK-8 assay,and gene expression of inflammatory factors,MMP9,IL-8,VEGF-A and IL-37b receptors in RL95-2 cells was detected by real-time PCR.Results:(1)Number,size and weight of lesions per mouse in rh IL-37b group were lower than those in control group at d14.At the stage of early lesion formation(to d3),rh IL-37b treatment did not have a significant effect on lesions,while at the stage of lesion growth(d3 to d14),rh IL-37b treatment significantly reduced the number,size and weight of lesions per mouse.These results suggested that rh IL-37b inhibited the development of endometriosis through reducing lesion growth,not lesion formation.(2)After rh IL-37b or Clo or rh IL-37b + Clo treatment at the stage of early lesion formation,the number,weight and size of lesions per mouse in Clo and rh IL-37b + Clo group,not rh IL-37b group,was remarkably reduced when compared with control group,but there was no difference between Clo group and rh IL-37b + Clo group.These suggested that macrophages were involved in the early formation of lesions,but rh IL-37b had no effect not only on lesion formation but also on the macrophages involved in lesion formation.After treatment at the stage of lesion growth,the number,weight and size of lesions per mouse was significantly decreased in rh IL-37b group and rh IL-37b + Clo group,not Clo group,when compared with control group.Moreover,there was no difference between rh IL-37b group and rh IL-37b + Clo group.These suggested that the effect of rh IL-37b on lesion growth was not through macrophages.(3)The expression of Mmp2,Mmp9 and Vegfa m RNA in lesions was decreased in rh IL-37b group at d3 and d14 when compared with control group.The protein expression of MMP9 and VEGF in lesions detected by immunohistochemistry was also reduced in rh IL-37b group.In addition,a significant reduction in rh IL-37b group was observed for MMP9 protein by ELISA,compared with control group.These results suggested that rh IL-37b suppressed the proliferation,invasion and angiogenesis in lesions.(4)The expression of Tgfb1 m RNA was notedly increased in ectopic lesions in rh IL-37b group at d3,while the expression of Il1 b,Il6,Il10,Tnfa and Il18r1 m RNA was decreased,compared with control group.At d14,rh IL-37b treatment decreased the expression of Il1 b,Il6,Tnfa,Il10,Tgfb1 and Il-18r1 m RNA,and increased the expression of Sigirr m RNA in lesions.ELISA results showed that the level of TNF-α protein in peritoneal fluids was significantly reduced in rh IL-37b group when compared with control group.These results suggested that rh IL-37b suppressed the expression of inflammatory factors in ectopic lesions.(5)Immunohistochemistry results showed that the uterine tissue cultured in vitro for 4 days still expressed the vimentin and cytokeratin.Treatment with rh IL-37b significantly increased the m RNA expression of Tgfb1 and decreased the m RNA expression of Mmp9,Vegfa,Il18r1 and Il1 b which suggested that rh IL-37b directly suppress the expression of invasion,angiogenesis and pro-inflammatory factors in uterine tissue.In addition,treatment with rh IL-37b reduced the phosphorylation of Akt and Erk1/2.(6)Isolated ESCs mostly expressed vimentin and showed little expression of cytokeratin.ESCs treated with rh IL-37b showed significantly suppressed proliferation,invasion and decreased the expression of Mmp9,Vegfa,Il1 b,Il6,Tnfa,Il10,Il18r1 m RNA and increased the expression of Tgfb1 and Sigirr m RNA.(7)Real-time PCR and Western Blot results confirmed that the ESCs transfected with p IL-37b plasmid expressed IL-37b,while those transfected with pc DNA3.1 plasmid not.Transfected with p IL-37b plasmid also suppressed cells proliferation and invasion,decreased the expression of Mmp2,Mmp9,Vegfa,Il1 b,Il6,Tnfa,Il10,Il18r1 m RNA and increased the expression of Tgfb1 and Sigirr m RNA,compared with pc DNA3.1 plasmid.Western Blot results showed that transfected with p IL-37b plasmid downregulated the phosphorylation of Akt and Erk1/2,compared with pc DNA3.1 plasmid.(8)Treatment of RL95-2 cells with rh IL-37b inhibited cell proliferation,decreased the m RNA expression of MMP9,VEGFA,CXCL8,IL1 B,TNFA,IL10 and IL18R1 and increased the expression of TGFB1.Conclusion: These findings suggest that IL-37b inhibited the development of endometriosis through reducing lesion growth,not lesion formation,which was not affected by macrophage depletion.IL-37b may inhibit the growth of lesions by regulating proliferation,invasion,angiogenesis and inflammation through Akt and Erk1/2 signaling pathway.