Erythropoietin Signalling In Macrophage Promotes Apoptotic Cell Phagocytosis And Immunotolerance

Since the human cell differentiation, development, infection, aging and other reasons, the body produces billions of apoptotic cells every day(1). Apoptosis is an energy-consuming biological process and if apoptotic cells are not timely removed, cells occur secondary necrosis(2). The secondary necrotic cells can not maintain the integrity of the cell membrane, leading to cell membrane rupture and releasing of cell contents including autoantigens. Under normal circumstances, apoptotic cells were promotely uptaken by professional phagocytes like macrophages and dendritic cells(DC) or non-professional phagocytes like endothelial cells(3). Thus clearance of apoptotic cells by phagocytes is critical to maintain homeostasis of internal environment. Systemic lupus erythematosus(SLE) is a chronic autoimmune diseases involving skin, kidney, cardiovascular, nervous and other tissues(4). The autoantibodies against self-antigens such as anti-nuclear antibodies(ANA) and anti-DNA antibodies(ADA) were produced in a large number of patients with SLE. Studies have shown that inefficient phagocytosis of apoptotic cells is one of the important reasons for the incidence of SLE(5). The phagocytosis of apoptotic cells by macrophages from SLE patients was significantly reduced compared with healthy controls, while accumulations of apoptotic cells were observed in SLE tissues(4, 6-9).Apoptotic cells can release the "find me" signal molecule in order to effectively recruit the phagocytic cells(10). Studies have shown that the sphingosine kinase(Sphk), a S1 P synthesizing enzyme, was activated in apoptotic cells by a caspase dependent pathway. Apoptotic cells releases S1 P as a "find me" signal for phagocytes(11). S1 P induces macrophage chemotaxis to apoptotic cells through S1PR1 on macrophage surface(12). Research shows that in samples of peripheral blood mononuclear cells(PBMC) from SLE patients and spleen of Faslpr/lpr mice, m RNA and protein expression of S1PR1 was significantly lower than those in normal control(13). Therefore "find me" molecule S1 P plays an important role in the clearance of apoptotic cells and the pathogenesis of SLE. In addition to the secretion and release of the "find me" molecules, apoptotic cell surface also provides "eat me" signal to macrophages, thus macrophages are able to identify and uptake apoptotic cells but not normal cells(10). At present, the most widely studied "eat me" signal molecule is phosphatidylserine(PS)(14). PS is a component of the cell membrane, in normal cells PS is located in inner surface of cell membrane. When the cell becomes apoptosis, PS valgus to the cell membrane outer surface. Blocking PS on the outer surface of apoptotic cells can block the phagocytosis of apoptotic cells by phagocytes. In addition, macrophages can secrete some proteins to recognize and bind PS, such as Milk fat globule EGF factor 8(MFG-E8) and Gas6(15). MFG-E8 can combine with PS and the integrin of macrophage. Plasma Gas6(growth arrest specific 6) can be coupled with PS and macrophages tyrosine-kinase receptors such as Mer, mediating recognition of PS by macrophages(16). Studies have shown that Mfge8 deficient mice occurres age dependent SLE like autoimmune phenotype, the mice produces high concentrations of ANA and ADA. The Mer-deficient mice also occurres SLE like autoimmune symptoms(17-20). All of these results indicate that the phagocytic clearance of apoptotic cells is one of the important causes of autoimmune diseases.The glycoprotein hormone erythropoietin(EPO) is an important regulatory factor for hematopoiesis. EPO down regulates expression of Fas L/Fas on red blood cell precursors and inhibites apoptosis of immature red blood cells. Therefore EPO increases the number of peripheral blood mature red blood cells and promotes hematopoiesis function. Recent studies show that EPO possesses non-hematopoietic functions including regulation of inflammation(21, 22). For example in our previous report we found that EPO could inhibit the inflammatory cytokines and inflammatory cell infiltration in the rat experimental autoimmune neuritis(EAN)(23). Studies found the expression of erythropoietin receptor(EPOR) on macrophages and macrophage is an important target cell for EPO regulation of inflammation. EPO can inhibit proinflammatory cytokines such as TNF-α, IL-6 and IL-12 expression by macrophage after LPS or IFN-γ stimulation(24). It is found that EPO can promote human peripheral blood mononuclear macrophage phagocytosis of bacteria and particles(25). In the mouse model, intraperitoneal injection of EPO significantly increased the phagocytic rate of bacteria and particles by macrophages(26). Studies also found that the macrophage phagocytosis of bacteria and particulate matter was significantly higher in EPO-over expression transgenic TG6 mice(26). In TG6 mice, studies showed that macrophage phagocytosis of red blood cell was significantly higher than that in wild type control(27). The mechanism by which macrophages uptake red blood cells was similar to phagocytosis of apoptotic cell, both of them are PS externalization and Mer-mediated phagocytosis processes, and therefore these results suggest that EPO could promote macrophage phagocytosis of apoptotic cells(28, 29).Clinical study suggests that the dysfunction of EPO/EPOR signaling pathway in SLE patients is closely related to the severity of SLE disease. Study found that in patients with SLE, serum EPO concentration was significantly lower than that of healthy people(30). Anti-EPO antibodies were detected in 46% of SLE patients. ADA in anti-EPO antibody positive SLE patients was significantly higher than the anti-EPO antibody negative SLE patients, suggesting the potential relationship between insufficiencient EPO signaling pathway and SLE autoimmune reaction(30). In addition to the anti-EPO antibodies, studies found that patients with SLE also have anti-EPOR antibodies(31). In SLE patients with anti-EPOR antibodies, the ADA concentration and disease activity showed higher(32). Another study confirmed that the anti-EPOR antibodies in serum of SLE patients were able to neutralize the activity of EPO in vivo. All of these results demonstrate the dysfunction of EPO/EPOR signaling in SLE patients may related to the autoimmune severity.Our current study showed that the "find me" molecule S1 P from apoptotic cell was able to induce the expression and activation of EPO/EPOR pathway in macrophages. EPO/EPOR promotes the phagocytosis of apoptotic cells by inducing the expression of PPARγ in macrophages. The phagocytosis of apoptotic cell by EPOR knocking out macrophage was significantly reduced. Correspondingly EPOR-c KO mice occurred age dependent apoptotic cell accumulation and systematic auto-immune symptoms.In the first part of the study, the in vitro experiments found that apoptotic cell conditioned medium(ACCM) and S1 P increased expression of mice peritoneal macrophages HIF-1α, EPO, EPOR and p Jak2. ACCM lack of S1 P did not induce macrophage EPO/EPOR signaling pathway. Blocking macrophage S1PR1 can inhibit the the effect of ACCM and S1 P. In vivo experiments, the expression and activation of EPO/EPOR signaling pathway in macrophages was also induced by S1 P through S1PR1. Using dexamethasone to induce endogenous apoptosis can induce the expression and activation of EPO/EPOR signaling pathway in macrophages. Under physiological conditions, the expression of EPO in mice spleen and the expression of EPOR were significantly decreased after blocking S1PR1 or blocking cell apoptosis. The above results indicate that the expression and activation of EPO/EPOR pathway in macrophages can be induced by apoptotic cells derived S1 P.In the second part, we studied the role of EPO/EPOR pathway in regulation of macrophage phagocytosis of apoptotic cells. The in vitro study showed that the phagocytosis of apoptotic cells was significantly decreased in EPOR knockout macrophages, whereas exogenous rh EPO promoted macrophage phagocytosis of apoptotic cells. Injection of apoptotic cells in vivo found significant decrease of macrophage phagocytosis of apoptosis cells in EPOR-c KO mice while exogenous rh EPO promote WT mouse peritoneal and spleen macrophage in vivo phagocytosis. These results demonstrate that EPO/EPOR pathway regulates macrophage phagocytosis of apoptotic cells. Detection of inflammatory factors found rh EPO inhibited macrophages TNF-a, IL-6, MCP-1 secretion but promoted TGF-β secretion both in vivo and in vitro. In 55-week-old EPOR-c KO mice, accumulation of apoptotic cells was observed in the spleen, peripheral blood, thymus, kidney tissues.The third part of the study shows that rh EPO significantly induce the expression of PPARγ in WT macrophages. In the peritoneal macrophages of EPOR-c KO mice, PPARγ was significantly decreased. PPARγ agonists can promote EPOR-c KO mice macrophage phagocytosis of apoptotic cells, but rhEPO can not promote PPARγ-cKO mice macrophages phagocytosis, indicating that rhEPO promotes macrophage phagocytosis of apoptotic cells by PPARγ pathway. In addition, the study found that rh EPO can induce WT macrophages but not PPARγ deficient macrophages expression of Mfge8, Mertk, CD36 and Gas6, further suggesting that EPO upregulates macrophages phagocytosis of apoptotic cell through PPARγ.In the fourth part, results show that ACCM and S1 P could induce macrophage PPARγ expression and induce WT macrophage phagocytosis of apoptotic cells but not EPOR-cKO or PPARγ-c KO macrophages. S1 P induce WT macrophage expression of Gas6, Mertk, CD36 and Mfge8, but not in EPOR-c KO macrophages, further suggesting that S1 P and ACCM upregulates macrophages phagocytosis of apoptotic cell through PPARγ.In the fifth part, we studied the age dependant SLE like autoimmunity in EPOR-cKO mice. We found that in 40- and 55-week-old EPOR-cKO mice, serum ANA, ADA and anti-Sm antibody was significantly higher than control. A large number of IgG, Ig A and C3 were deposited in the kidney of 55 weeks old EPOR-cKO mice with impaired renal functions. In addition, in 55-week-old EPOR-cKO mice, skin appear obvious IgG deposition, lung occurres significant inflammatory cell infiltration. These results showed that EPOR-c KO mice occurrs age dependent SLE like autoimmune disease.In the sixth part, rhEPO was used to treat mice SLE model induced by pristan(33). The phagocytosis of apoptotic cells was significantly increased after rh EPO treatment, and the accumulation of apoptotic cells were significantly decreased. rh EPO treatment reduces serum ADA and ANA titers, kidney IgG deposition, serum IL-6, MCP-1 and TNF-α level. Further study showed that PPARγ agonist rosiglitazone significantly reduced the autoimmune severity of EPOR-c KO mice, while rh EPO was ineffective in the treatment of autoimmune symptoms of PPAR-cKO mice, further suggesting that EPO/EPOR acts through PPARγ.In summary, our study showed that apoptotic cells derived S1 P induces the expression of macrophage HIF-1α through S1PR1, HIF-1 alpha further induces the expression and activation of EPO/EPOR pathway of macrophages. EPO/EPOR signaling can promote macrophage phagocytosis of apoptotic cells by inducing PPARγ. In EPOR-c KO mice, EPOR deficiency impaired macrophage phagocytosis of apoptotic cells. EPOR-c KO mice appeared age dependent apoptotic cell accumulation and SLE like systemic autoimmune symptoms.