Effects And Mechanisms Of Activin A On Regulation Of Monocytes/Macrophages Activities

Activin A, a member of transforming growth factor β (TGFβ) superfamily, is produced bya variety of immune cells and plays a regulatory role in most of them. for example,monocytes, macrophages, dendritic cells, T lymphocytes and mast cells can synthesizeactivin A following the immune stimulation or activation of Toll-like receptor (TLR)agonists, which plays an important role in maintaining the homeostasis of the local immunemicroenvironment. Activin A can affect the monocyte-macrophages activity, but whether itinduces monocyte differentiation and development and regulate the monocyte-macrophagesactivity dependent on GM-CSF or not has not been elucidated.The first part was to reveal the effect and mechanism of activin A on inducingdifferentiation and development of monocyte-like RAW264.7cells viaGM-CSF-independent parthway, the second part was to reveal cell polarization andregulatory effect of activin A in vivo and in vitro on rested mouse peritoneal macrophagesactivity, and the third part was to detect the role and mechanism of activin A on regulatingthe lipopolysaccharide LPS-activated macrophages activity.1. Activin A induced RAW264.7cells differentiationOur previous studies have discovered that activin A can induce RAW264.7cells todendritic-like cells (DC), but monocytes in the activation state have the autocrine functionof GM-CSF. It remains unclear that whether activin A induces RAW264.7cells to DC isdependent on the secretion of endogenous GM-CSF. Therefore, we will first confirm theeffect of activin A on monocytes.1.1Activin A induced differentiation of RAW264.7to DCThe RAW264.7morphological changes by Gimsa staining was observed, as well as theexpression of RAW264.7cells surface markers, including CD40, CD11c, CD80, CD86andMHC-class II molecule by flow cytometry. The results showed that activin A can stimulate RAW264.7cells to DC.1.2Activin A promoted the differentiation of RAW264.7cells via GM-CSF-independentpathwayWith the treatment of both activin A and GM-CSF, the results suggested that activin Aand GM-CSF synergistically promote RAW264.7cells differentiation to DC. After theRAW264.7cells were blocked beforehand with anti-GM-CSF antibody, the flow cytometryresults showed that activin A was also able to induce DC surface markers increasing,suggesting that activin A-induced differentiation of RAW264.7cells does not depend on theGM-CSF pathway and the role of Activin A and GM-CSF have a synergistic effect.1.3Relationship between activin A-induced RAW264.7cell differentiation and SmadspathwaySmad2, Smad3and Smad4compose the activin classic signal transmission pathways.Studies have shown that treated with activin A, the expression of Smad2, Smad3and Smad4mRNA of RAW264.7cells all increased. Overexpression of Smad3could upregulate theexpression of surface markers of CD40, CD11c, CD80and MHCII of Activin A-inducedRAW264.7cells, but overexpression of Smad3shRNA in RAW264.7cell lines did not affectthe role of activin A to induce RAW264.7cells differentiation to DC, suggesting that activinA inducing RAW264.7cells differentiation to DC was not totally dependent on the Smadpathway.1.4.Activin A inhibited LPS-induced RAW264.7cells differentiation to DCOur previous studies found that activin A could inhibit LPS-activated macrophages. Otherstudies have reported that both GM-CSF and LPS induced RAW264.7cells differentiation toDC-like cells. Therefore, in our studies, the relationship of activin A and LPS inducingRAW264.7cells to DC-like cells was further analyzed. RAW264.7cells morphologicalchanges by Gimsa staining and the expression of MHC-II molecules CD80, CD40, TLR4and CD11c molecules by flow cytometry showed that activin A can significantly inhibitLPS-induced RAW264.7cells differentiation to DC like cells.Due to LPS activating signal by monocyte-macrophages surface TLR4and CD14, RT-PCR and flow cytometry were used to detect the expression of TLR4and CD14. Theresults showed that activin A can inhibit the TLR4and CD14expression in LPS-inducedRAW264.7cells and Western Blotting results showed that activin A also inhibitedLPS-induced NF-κB expression. Suggesting that activin A can inhibit LPS inducedRAW264.7cells differentiation by inhibiting the expression of TLR4and CD14and also bydown-regulating the TLR4signaling.1.5The effect of activin A on RAW264.7cells tumorigenicity in vivoInduction of differentiation and apoptosis are the primary anti-tumor treatments. Ourresults confirmed that activin A can induced RAW264.7cells differentiation, but whetheractivin A can play an anti-tumor effect by inducing monocytes differentiation is still unclear.Therefore, a mouse tumor model with mouse monocytes/macrophages cell line RAW264.7cells was further established. The studies found that the mean tumor volume, tumor indexand tumor formation rate in the mice grafted with activin A-pretreated RAW264.7cellsgroup were significantly lower than those of control group. An exogenous source ofintratumoral injection of activin A could decrease mice tumor growth. The tumor tissueapoptosis was detected by flow cytometry with Annexin V-PI double staining The resultsshowed that there was no significant difference in activin A-treated group and the controlgroup which illustrated that activin A has no effect on RAW264.7cell proliferation andapoptosis. These results further confirmed that activin A inhibited the tumor formation ofRAW264.7cells in vivo by inducing differentiation of RAW264.7cells to DC, but not cellapoptosis.2. The effect of activin A on rested mouse peritoneal macrophages activity2.1The effect of activin A on the phagocytosis of mouse peritoneal macrophages detected byflow cytometryFlow cytometry results showed that activin A-pretreated mouse peritoneal macrophageshave a higher ability of phagocytic fluorescent spheres both in vitro and in vivo.2.2The effect of activin A on the secretion of cytokines of macrophagesIL-10, TNF-α and NO in the cell culture supernatant of mouse peritoneal macrophages and peritoneal fluid of mouse peritoneal macrophages was examined, both of which werestimulated by activin A or not. The results showed that activin A promoted the secretion ofIL-10, but it could not promote the secretion of TNF-α. and activin A could also stimulatedthe secretion of NO, suggesting that activin A may induce polarization of mouse peritonealmacrophages to the M2both in vitro and in vivo.2.3The expression of macrophages CD206, Arg-1detected by flow cytometryThe expression of CD206and Arg-1of activin A-stimulated mouse peritonealmacrophages was detected,by flow cytometry.The results showed that activin A can increasethe expression of the M2macrophage cell markers CD206and Arg-1of mouse peritonealmacrophages. This study further proved that activin A can induce polarization of mouseperitoneal macrophages to M2both in vitro and in vivo.3. The mechanism of activin A inhibiting LPS activated macrophages3.1Activin A inhibited the phagocytic activity of LPS-activated mouse peritonealmacrophagesThe flow cytometry results showed that the phagocytic activity of mice peritonealmacrophages co-stimulated by LPS and Activin A were significantly lower than that ofperitoneal macrophages stimulated by LPS alone both in vitro and in vivo.3.2Activin A inhibited the secretion of cytokines of LPS-induced mouse peritonealmacrophagesThe experiment further detected the secretion of cytokines of activin A-treatedmacrophages. The results showed that the secretion of TNF-α, NO of LPS stimulated-groupwere significantly higher than those of LPS and activin A-co-stimulated group, which furtherconfirmed that activin A can inhibit the function of LPS-activated mouse peritonealmacrophage.3.3Activin A inhibited the expression of TLR4of LPS-activated mouse peritonealmacrophagesFlow cytometry results showed that the expression of TLR4of LPS and activin A-co-stimulated mouse peritoneal macrophages were significantly lower than that of LPS-stimulated alone group both in vitro and in vivo.3.4Smad3knock-down attenuated the inhibitory effect of activin A on TLR4Furthermore, to confirm the relationship between the inhibitory effect of activin A on LPSactivated macrophages and TLR4, Smad3knock-down RAW264.7cell line was establishedby stably transfected with pGCsi-U6/Neo-Smad3shRNA. The the results showed thatinhibitory effect of Activin A on the expression of MHC-II,CD80, CD40, CD11c and TLR4induced by LPS were significantly attenuated in Smad3knock-down RAW264.7cells. Thesedata further demonstrated that activin A inhibited the TLR4expression of macrophages.The study not only revealed activin A could induce RAW264.7cells to DC-like cells viaGM-CSF-independent mode, but also revealed that activin A might enhance the bodydefense by promoting the phagocytic activity of macrophages in early inflammation, anddown-regulating LPS-TLR4pathway to prevent excessive activation of macrophages in thelate stage of inflammation and the occurrence of inflammatory injury.