The Role Of Interleukin-26 In The Pathogenesis Of Malignant Pleural Effusion

Objectives:IL-26 is a member of the IL-10 cytokine family with multifaceted actions in inflammation and tumors.However,the relationships between IL-26 and lung cancer or malignant pleural effusion(MPE)remain unclear.This study aims to explore the role and specific mechanism of IL-26 in the occurrence and development of MPE caused by lung cancer and to find new therapeutic targets for lung cancer and MPE.Methods:The concentrations of IL-26 and other cytokines in MPE and peripheral blood(PB)were detected by enzyme-linked immunosorbent assay(ELISA),and correlation analysis was carried out between IL-26 concentrations and clinical indicators of patients.The cellular origins of IL-26 in MPE and PB was analyzed by flow cytometry.Purified CD4~+T cells were cultured in the presence of different combinations of cytokines in vitro,and the secretion of IL-26 and the distribution of various T cell subsets were detected by flow cytometry to clarify the interactions and mechanisms between IL-26 and CD4~+T cells subsets in MPE.The secretion of cytotoxic factors in CD8~+T cells were detected by flow cytometry and the function of CD8~+T cells was detected by CCK-8 assay in a tumor cells-CD8~+T cells co-culture system in vitro.MPE model mice were constructed and the effect of IL-26 on the formation of MPE was evaluated by various indicators;the effect of IL-26 on the microenvironment of MPE in mice was analyzed by flow cytometry as well.In addition,the expression of IL-26 in lung adenocarcinoma and normal tissues were analyzed at the protein and gene levels;the cellular origins of IL-26 in lung adenocarcinoma tissues was detected by immunofluorescence.Meanwhile,lung adenocarcinoma cell lines were cultured in vitro.The effects of IL-26 on the proliferation and migration activity of cell lines was analyzed by flow cytometry,CCK-8 assay,colony formation assay,Ed U assay,scratch assay and transwell assay.Subsequently,transcriptome sequencing analysis of IL-26-regulated lung adenocarcinoma genes was conducted.A gene knockdown vector was constructed to analyze whether IL-26 could regulate the biological functions of lung adenocarcinoma through the differential genes.Finally,immunocompromised mouse models were used to determine the roles of IL-26 and its regulatory genes in lung adenocarcinoma in vivo.Results:(1)The concentrations of IL-26 in MPE were higher than those in PB.(2)The concentration of IL-26 in MPE was positively correlated with IL-6 concentration,neutrophil/lymphocyte ratio,white cells count and lymphocytes count,and negatively correlated with adenosine deaminase as well as the prognosis of patients.(3)IL-26 was mainly secretd by Th17 cells in MPE while Tc17 cells and macrophages could secrete it as well.(4)IL-6 and IL-23 could promote the differentiation of Th17 cells and the expression of IL-26 in MPE by phosphorylating STAT3 pathway and subsequently up-regulating the expression of RORγt.(5)IL-26 could selectively induce the proliferation and differentiation of Th22 cells.(6)IL-26 could inhibit the cytotoxic functions of CD8~+T cells.(7)IL-26could promote the formation of MPE in mice.Meanwhile,the cytokine could increase the proportion of Th22 cells,M2 macrophages as well as myeloid-derived suppressor cells and decreased the proportion of CD8~+T cells in mice MPE microenvironment.(8)IL-26 is overexpressed in lung adenocarcinoma tissues and mainly expressed by tumor-infiltrating T cells and macrophages.(9)IL-26 could induce the proliferation and migration activities of lung adenocarcinoma cells in vitro.(10)IL-26 could increase the gene expression of EIF3C in lung adenocarcinoma cells through activating STAT3 pathway.(11)After inhibiting the expression of EIF3C gene,the promoting effect of IL-26 on the function of lung adenocarcinoma cells was significantly down-regulated.(12)IL-26 could promote the progression of lung adenocarcinoma cells in vivo.Conclusions:IL-26 was highly expressed in lung cancer and MPE;Th17-related cytokines IL-6 and IL-23 could promote the accumulation of IL-26 in the MPE microenvironment;subsequently,IL-26 could increase the secretion of IL-22 by upregulating the proliferation and differentiation of Th22 cells.Furthermore,the function of CD8~+T cells was inhibited by IL-26.Survival analysis had showen that high levels of IL-26 protein predicted poorer prognosis in patients with MPE.The study has also confirmed that IL-26 had tumor-promoting properties both in vivo and in vitro through regulating the activity of STAT3 signaling pathway and the expression of the tumor-promoting gene EIF3C.The important role of the IL-26/STAT3/EIF3C axis in the progression of lung adenocarcinoma was also validated.This study has confirmed that IL-26 is an important cytokine involved in the pathogenesis of lung adenocarcinoma as well as MPE,providing new prognostic biomarkers and treatment targets for the diseases.