Thymosin α-1 Regulates The Secretion Of Cytokines And The Expression Of Membrane Surface Molecules Of Macrophages Clearing Apoptotic Cells And Its Mechanism

Thymosin α-1(Tα-1)is an immunomodulator of thymus hormones,which promotes the development,differentiation and maturation of T lymphocytes,enhances the antiviral effect of CTL,and participates in the production of antibodies by B cells.Thymosin α-1 also plays an important immune role in innate immune cells,and has been clinically applied to the treatment of viral hepatitis,malignant tumors,severe infections and the like.However,the molecular mechanism of thymosin to play an immunomodulatory role is still lack of systematic and in-depth research.Thymosin α-1 is folded in the proper conformation in the presence of sodium dodecyl sulfate(SDS)micelles,and its N-terminus can better interact with membrane negative regions with exposed phosphatidylserine(PS).PS is an important marker molecule on the surface of apoptotic cells.Macrophages clear apoptotic cells by recognizing PS,secrete a large number of negative immunoregulators,and promote tumor cell growth and metastasis.Objectives:This study is to investigate whether thymosin α-1 regulates the polarization of macrophages in the process of scavenging apoptotic cells;to studies the molecular mechanism by which thymosin α-1 regulates the secretion of cytokines by phagocytic apoptotic tumor cells.Methods:1.Investigation of regulateing the production of cytokines and membrane surface molecules during the process of macrophage clearance of apoptotic cells by Tα-1Establish a model of tumor cell apoptosis,stimulate MDA-MB-231 cells with different concentrations of chemotherapeutic drug bortezomib,and detect the percentage of apoptosis of MDA-MB-231 cells by flow cytometry 24 hours later.Tα-1 pre-incubated apoptotic MDA-MB-231 cells,apoptotic MDA-MB-231 cells were added to THP-1 macrophages,and THP-1 macrophages were stimulated with Tα-1 alone.After 3 hours,the apoptotic cells that were not phagocytized were discarded;the cells were harvested for 3 hours,and the difference in expression of macrophage phenotype-related molecules in RNA was detected;after 24 hours,the culture supernatant was collected to detect cytokines IL-6 and IL-1β,IL-10,TGF-β;harvested cells,and detected macrophage surface molecules CD206,CD 163,CD86,HLA-DR,HLA-ABC,PD-L1 expression differences after 48 hours.2.Study on the molecular mechanism of Tα-1 regulating macrophage clearance of apoptotic cells to produce cytokines2.1 Detection of the binding capacity of thymosin α-1 to the surface PS of apoptotic tumor cells and efficient phagocytosis by macrophagesTα-1(0,1,3,10,30,100,300 μg/mL)and apoptotic MDA-MB-231 cells were incubated for 1 hour in a 37℃,5%CO2 incubator.MDA-MB-231 cells with different apoptosis ratios were incubated with 100 μg/mL Tα-lfor 1 hour.The ability of Tα-1 binding to PS on the surface of apoptotic tumor cells was detected by flow cytometry.Tα-1 pre-incubated apoptotic MDA-MB-231 cells,apoptotic MDA-MB-231 cells labeled with CFSE,Annexin V-FITC,α Tα-1 Ab-APC,respectively were incubated with THP-1 macrophage for 3 hours at 37℃;the phagocytosis ratio and mean fluorescence intensity of THP-1 macrophages were detected by flow cytometry.2.2 Study on the molecular mechanism of Tα-1 promoting the secretion of IL-6 by macrophagesPretreatment of THP-1 macrophages with PI3K,ERK,JNK,p38,NF-κB inhibitors for 1 hour,were co-incubated with Tα-1 pre-incubated apoptotic MDA-MB-231 cells and apoptotic MDA-MB-231 cells,and the expression of IL-6 was detected by ELISA.THP1 macrophage was co-incubated with Tα-1 pre-incubated apoptotic MDA-MB-231 cells,then the expression of AKT,p-AKT,ERK,p-ERK,JNK,p-JNK,p38,p-p38,IKKa,pIKKα,IkBα,p-IkBα,p65,p-p65,GAPDH and β-Tublin was detected by Western blot.Results:1.Investigation of regulateing the production of cytokines and membrane surface molecules during the process of macrophage clearance of apoptotic cells by Tα-11.1 Tα-1 regulates the secretion of cytokines during the process of macrophage phagocytosis of apoptotic cellsTα-1 did not regulate the secretion of cytokines by THP-1 macrophages;apoptotic tumor cells stimulated macrophages to up-regulate IL-10,TGF-β secretion,and inhibit IL-6 secretion(P<0.05).Tα-1 pre-incubated apoptotic tumor cells,caused dose-dependent down-regulated macrophage secretion of IL-10,TGF-β,up-regulated IL-6,IL-1β in dosedependent manne(P<0.05).1.2 Tα-1 regulates the expression of cell membrane surface molecules during the process of macrophage phagocytosis of apoptotic cellsWhen macrophages engulfed apoptotic tumor cells,the co-stimulatory molecule CD86 was down-regulated,while the M2 markers CD206 and CD 163 were up-regulated.Interestingly,Tα-1 can increased the expression of CD86 of macrophages that engulfed apoptotic tumor cells.and M2 marker molecules such as CD206 and CD 163 were downregulated(P<0.01).Besides,apoptotic tumor cells can induced down-regulation of HLA-DR and HLA-ABC,and up-regulated PD-L1 expression of macrophages.Tα-1 can further down-regulated the expression of HLA-DR,up-regulated PD-L1,and reverse upregulated HLA-ABC.2.Study on the molecular mechanism of Tα-1 regulating macrophage clearance of apoptotic cells to produce cytokines2.1 Tα-1 dependent PS molecules binding to apoptotic tumor cells and were effectively phagocytosed by macrophagesThe binding of Tα-1 to the PS molecule on the surface of apoptotic MDA-MB-231 cells was concentration-dependent,and the binding ability was enhanced as the proportion of apoptosis increased.THP-1 macrophages phagocytose apoptotic tumor cells,Tα-1 can be accompanied by apoptotic tumor cells into macrophages;and Tα-1 can promote the phagocytosis of apoptotic cells by macrophages.2.2 Tα-1 activates macrophage JNK,p38,ERK,NF-κB signaling pathway up-regulates IL-6 secretionCompared with THP-1 macrophages that phagocytose PS+MDA/Tα-1 without pretreatment with inhibitors,JNK,ERK,p38,NF-κB inhibitor-pretreated macrophages secreted IL-6 levels significantly.decreased(P<0.05),and there was no significant change in IL-6 secretion by macrophages treated with PI3K inhibitor.Western Blot results showed that Tα-1 significantly induced the activation of JNK,p38,ERK and NF-κB signals in macrophage clearance of apoptotic cells.Conclusions:1.Tα-1 did not regulate the secretion of cytokines by THP-1 macrophages.But,Tα-1 can regulates the secretion of cytokines and the expression of cell surface molecules during the process of macrophage phagocytosis of apoptotic cells;2.Phosphatidylserine(PS)appears on the surface of apoptotic cells,and Tα-1 dependented PS molecules binds to apoptotic tumor cells and is effectively engulfed by macrophages;3.Tα-1 induces the activation of JNK,p38,ERK and NF-κB signaling pathways in macrophage that phagocytoses apoptotic cells and up-regulates the secretion of IL-6.