Expression And Function Comparison Of ARIP1 And ARIP2 In Macrophages Of Mouse

INTRODUCTIONMacrophages are important innate immune cells in the body.It is generally believed that monocytes derived from bone marrow differentiation firstly enter the blood circulation.Monocytes in the blood can be recruited into the organization,and further differentiate into mature macrophages in organization at physiological and pathological conditions.Macrophages play crucial roles in phagocytosis,sterilization,removal of body damage and senescence cells,tissue development and repair,and the role of antigen information transmission.Macrophages are closely related to the development of inflammatory response.Activin receptor-interacting proteins(ARIPs)belong to activin signal transduction proteins that interact with the C-terminus of the activin type II receptor(Act RII)through its PDZ domain and participate in signal transduction of activin.Because ARIPs are different in tissue expression and biological effects,they are considered to be key molecules in determining the role of activin.ARIP1 has a guanylate kinase domain at the N-terminus,followed by two WW regions and five PDZ-like functional regions,and ARIP1 interacts with Act RIIA through PDZ5.ARIP2 has only one PDZ domain interacting with Act RIIA,and the expression of ARIP2 increases the endocytosis of Act RII receptors and inhibits activin-induced gene transcription.There is a significant difference in histological distribution between ARIPs.Our previous study found that ARIP1 is mainly expressed in small neurons,cerebellar purkinje cells and choroidal epithelial cells in the cerebral cortex.But ARIP2 is widely distributed in tissues such as brain,heart,kidney,testis,lung,islet and adrenal gland.However,it is not clear whether ARIP1 is expressed inmacrophages and whether the expression of ARIP1 and ARIP2 in macrophages are different.Therefore,this study firstly determines the differentiation and expression of ARIP1 and ARIP2 in macrophages,and further analysis of the biological significance in macrophage.1.METHODS1.1 Expression of ARIP1 and ARIP2 in macrophagesRT-PCR and immunocytochemical staining were used to detect the expression of ARIP1 and ARIP2 in monocyte-macrophage cell line Raw264.7 and primary mouse peritoneal macrophages.1.2 Differentiation of bone marrow-derived monocyte-macrophageThe bone marrow cells of mouse femur were isolated and GM-CSF was used to induce cell differentiation and development.Gimsa staining was used to determine the morphological changes.1.3 Overexpression of ARIP1 and ARIP2 genes1.3.1 Overexpression in vitroRAW264.7 cells were transfected with pc DNA3(PC)and pc DNA3-ARIP1(ARIP1)or pc DNA3-ARIP2(ARIP2)expressing plasmids respectively.The expression efficiency was identified by RT-PCR and immunocytochemical staining.1.3.2 Overexpression in vivoBalb/c homozygous mice were randomly divided into PC,ARIP1 and ARIP2 groups.Each mouse was injected with PBS 2ml intraperitoneally.After 2 hours,the mice were injected with 3?g plasmid and transfection reagent.After 12 hours,mouse peritoneal macrophages were isolated and cultured.RT-PCR and immunocytochemical staining were used to identify the expression efficiency.2.RESULTS2.1 Expression of ARIP1 and ARIP2 in monocyte-macrophagesRT-PCR and immunocytochemical staining showed that ARIP1 and ARIP2 were expressed in mouse peritoneal macrophages and monocyte-macrophage line Raw264.7 cells.2.2 Expression of ARIP1 and monocyte-macrophage differentiation2.2.1 ARIP1 is expressed in monocyte-macrophages derived from hematopoietic stem cellsIn order to determine whether the expression of ARIP1 and ARIP2 in monocyte-macrophages are related to their differentiation and development,the expression of ARIP1 and ARIP2 in bone marrow hematopoietic stem cells and stromal stem cells were detected by RT-PCR.The results showed that bone marrow hematopoietic stem cells and stromal stem cells did not express ARIP1,but they expressed ARIP2,Smad3 and other molecules.Furthermore,GM-CSF was used to induce the differentiation and development of hematopoietic stem cell-derived monocyte-macrophage.The results showed that GM-CSF-induced hematopoietic stem cell-derived monocyte-macrophages not only expressed ARIP1 m RNA,but also expressed the mature ARIP1 protein by immunocytochemical staining.2.2.2 Effects of ARIP1 overexpression on the mature differentiation of Raw264.7cellsThe morphological changes of macrophages were observed by Giemsa staining.The results showed that ARIP1 overexpressed cells showed polygons compared with PC group,and promoted macrophage maturation-like differentiation.ARIP2 overexpression morphological changes were not obvious compared with the PC group.2.2.3 Effects of ARIP1 overexpression on phagocytosis of macrophagesFlow cytometry showed that the phagocytic capacity of ARIP1 overexpressed macrophages was enhanced compared with PC group.2.2.4 Effects of ARIP1 overexpression on expression of CD14 on macrophagesFlow cytometry was performed to detect the expression of macrophage surface molecules.The results showed that overexpression of ARIP1 promoted the expression of CD14 on macrophages,and the cells were further differentiated into maturation-like macrophages.2.3 Effects of ARIP1 and ARIP2 on the activity of LPS activated macrophages2.3.1 LPS promotes the expression of ARIP1 and ARIP2 in macrophagesRT-PCR and immunocytochemical staining showed that ARIP1 and ARIP2 expression were increased and dose-dependent after stimulation with different concentrations of LPS 0.2ug / m L and LPS 0.5ug / m L.2.3.2 Effects of ARIP1 and ARIP2 overexpression on phagocytic activity of macrophagesIn LPS-activated macrophages,ARIP1 overexpression promotes macrophage phagocytosis of fluorescent particles;ARIP2 overexpression inhibits phagocytic activity of macrophages.2.3.3 Effects of ARIP1 and ARIP2 overexpression on the expression of IL-1?and TNF? in macrophagesLPS-activated macrophages were used to detect NO,IL-1? and TNF? in cell culture supernatant.The results showed that ARIP1 or ARIP2 overexpression group can reduce the production of IL-1? and TNF? compared with PC group,but the effect of NO secretion is not obvious.2.3.4 Effects of ARIP1 and ARIP2 overexpression in vivo on the expression of IL-1? and TNF? in macrophagesAfter overexpression of ARIP1 and ARIP2 in mouse peritoneal macrophages in vivo,IL-1? and TNF? in peritoneal fluid were detected by ELISA.The results showed that ARIP1 or ARIP2 overexpression group can reduce the IL-1? and TNF?production compared with PC group;ELISA was used to detect the secretion of NO,IL-1? and TNF? in LPS-activated ARIP1 and ARIP2 overexpressing peritoneal macrophages cultured in vitro.The results showed that ARIP1 or ARIP2 overexpression group compared with PC group,can reduce the production of IL-1?and TNF?,but the effect of NO secretion is not obvious.2.4 Effects of ARIP1 and ARIP2 overexpression on the expression of signaling molecules and surface molecules in macrophages2.4.1 Effects of ARIP1 and ARIP2 overexpression on the expression of NF-?Bp65 and Smad3Western Blotting showed that ARIP1 overexpression down-regulated the expression of NF-?Bp65 and Smad3 protein,but the results of flow cytometry showed no significant effect on TLR2,TLR4 and CD14 on macrophages.2.4.2 Effects of ARIP1 and ARIP2 overexpression on the expression of s surface moleculesFlow cytometry was used to detect the surface molecules of macrophages.The results showed that ARIP2 overexpression down-regulated the expression of CD14.The overexpression of ARIP1 had no significant effect on macrophage surface molecule CD14.3.CONCLUSIONIn summary,ARIP1 is expressed in monocyte-macrophages.ARIP1 is highly expressed in hematopoietic stem cell-derived monocyte-macrophages.The overexpression of ARIP1 promotes macrophage morphological maturation-like differentiation.ARIP1 overexpression increases macrophage phagocytosis activity.The overexpression of ARIP1 improves the expression of CD14 on the surface of macrophages.It is suggested that ARIP1 promotes the differentiation and maturation of macrophages.ARIP1 is expressed not only in neuronal cells,but also in mononcyte-macrophages.The expression of ARIP1 is related to the development of monocyte-macrophage differentiation.ARIP1 may be a new marker of differentiation and maturation of monocyte-macrophages.ARIP2 is also expressed in monocyte-macrophages,but its expression may be independent of monocyte-macrophage differentiation and maturation.LPS promotes the expression of ARIP1 and ARIP2 in macrophages.Both ARIP1 and ARIP2 can play an anti-inflammatory role in activated macrophages.ARIP1 and ARIP2 overexpression reduce the secretion of cytokines such as IL-1? and TNF? inmacrophages,but the mechanism of action is different.ARIP1 and ARIP2 overexpression have different effects on macrophage phagocytosis.ARIP1anti-inflammatory effect may be related to down-regulation of NF-?Bp65,Smad3,while ARIP2 anti-inflammatory effect may be related to down-regulation of CD14 molecules.In addition,ARIP1 is highly expressed in hematopoietic stem cell-derived monocyte-macrophages.Overexpression of ARIP1 can promote the differentiation of cells into mature macrophages and the expression of CD14 Fon macrophages.It is suggested that ARIP1 promotes macrophage maturation and can be used as an important biomarker for monocyte-macrophage differentiation and maturation.Thus,ARIP1 and ARIP2 play an anti-inflammatory role in macrophages,which provides new results for the elucidation of activin-inhibiting mechanisms of inflammatory cytokines.ARIP1 and ARIP2 are likely to be new therapeutic targets and are important in the pathogenesis and treatment of inflammation.