Response gene to complement 32 is required for C5b-9 induced cell cycle activation in endothelial cells

Proliferation of vascular endothelial cells (EC) and smooth muscle cells (SMC) is a critical event in angiogenesis and atherosclerosis. We previously showed that the C5b-9 assembly during complement activation induces cell cycle in human aortic EC (AEC) and SMC. C5b-9 can induce the expression of Response Gene to Complement (RGC)-32 and over expression of this gene leads to cell cycle activation. Therefore, the present study was carried out to test the requirement of endogenous RGC-32 for the cell cycle activation induced by C5b-9 by knocking-down its expression using siRNA. We identified two RGC-32 siRNAs that can markedly reduce the expression of RGC-32 mRNA in AEC. RGC-32 silencing in these cells abolished DNA synthesis induced by C5b-9 and serum growth factors, indicating the requirement of RGC-32 activity for S-phase entry. RGC-32 siRNA knockdown also significantly reduced the C5b-9 induced CDC2 activation and Akt phosphorylation. CDC2 does not play a role in G1/S transition in HeLa cells stably overexpressing RGC-32. RGC-32 was found to physically associate with Akt and was phosphorylated by Akt in vitro. Mutation of RGC-32 protein at Ser 45 and Ser 47 prevented Akt mediated phosphorylation. In addition, RGC-32 was found to regulate the release of growth factors from AEC. All these data together suggest that cell cycle induction by C5b-9 in AEC is RGC-32 dependent and this is in part through regulation of Akt and growth factor release.     

Epigenetic modifications induced by RGC-32 in colon cancer

First described as a cell cycle activator, RGC-32 is both an activator and a substrate for CDC2. Deregulation of RGC-32 expression has been detected in a wide variety of human cancers. We have now shown that RGC-32 is expressed in precancerous states, and its expression is significantly higher in adenomas than in normal colon tissue. The expression of RGC-32 was higher in advanced stages of colon cancer than in precancerous states or the initial stages of colon cancer. In order to identify the genes that are regulated by RGC-32, we used gene array analysis to investigate the effect of RGC-32 knockdown on gene expression in the SW480 colon cancer cell line. Of the 230 genes that were differentially regulated after RGC-32 knockdown, a group of genes involved in chromatin assembly were the most significantly regulated in these cells: RGC-32 knockdown induced an increase in acetylation of histones H2B lysine 5 (H2BK5), H2BK15, H3K9, H3K18, and H4K8. RGC-32 silencing was also associated with decreased expression of SIRT1 and decreased trimethylation of histone H3K27 (H3K27me3). In addition, RGC-32 knockdown caused a significantly higher percentage of SW480 cells to enter S phase and subsequently G2/M. These data suggest that RGC-32 may contribute to the development of colon cancer by regulating chromatin assembly.     

RGC-32 and diseases: the first 20 years

The response gene to complement (RGC)-32 acts as a cell cycle regulator and mediator of TGF-β effects. However, recent studies have revealed other functions for RGC-32 in diverse processes such as cellular migration, differentiation, and fibrosis. In addition to its induction by complement activation and the C5b-9 terminal complement complex, RGC-32 expression is also stimulated by growth factors, hormones, and cytokines. RGC-32 is induced by TGF-β through Smad3 and RhoA signaling and plays an important role in cell differentiation. In particular, RGC-32 is essential for the differentiation of Th17 cells. RGC-32^−/− mice display an attenuated experimental autoimmune encephalomyelitis phenotype that is accompanied by decreased central nervous system inflammation and reductions in IL-17- and GM-CSF-producing CD4⁺ T cells. Accumulating evidence has drawn attention to the deregulated expression of RGC-32 in human cancers, atherogenesis, metabolic disorders, and autoimmune disease. Furthermore, RGC-32 is a potential therapeutic target in multiple sclerosis and other Th17-mediated autoimmune diseases. A better understanding of the mechanism(s) by which RGC-32 contributes to the pathogenesis of all these diseases will provide new insights into its therapeutic potential.

     

Heme Oxygenase-1 expression in M-CSF-polarized M2 macrophages contributes to LPS-induced IL-10 release

The shift between pro-inflammatory (M1) and anti-inflammatory (M2) states of macrophage polarization allows the resolution of inflammatory processes as well as the maintenance of a basal anti-inflammatory environment in tissues continuously exposed to harmless antigens (e.g., lung and gut). To identify markers for the anti-inflammatory state of macrophages, expression profiling was performed on human macrophages polarized by either GM-CSF or M-CSF, which lead to the generation of TNF-α and IL-12p40-producing pro-inflammatory macrophages [M1 (GM-CSF)] or IL-10-producing anti-inflammatory macrophages [M2 (M-CSF)] upon exposure to LPS, respectively. A different iron metabolism gene signature was detected in both macrophage types, with the heme regulatory molecules CD163 and Heme Oxygenase-1 (HO-1) being preferentially expressed by M2 (M-CSF) macrophages. M1-polarizing cytokines (GM-CSF, IFNγ) inhibited, while IL-4 enhanced, the M-CSF-driven HO-1 expression. In agreement with this in vitro data, HO-1 expression in metastatic melanoma was primarily detected in CD163⁺ tumor-associated macrophages, which are known to exhibit an M2-skewed polarization phenotype. In contrast to the HO-1 inhibitor tin protoporphyrin (SnPP), the administration of cobalt protoporphyrin (CoPP), a potent inducer of HO-1 resulted in increased LPS-triggered IL-10 release from M2 (M-CSF) macrophages. The data suggests that HO-1 is important for the anti-inflammatory activities of M-CSF-polarized M2 macrophages. Moreover, since M2 (M-CSF) macrophages also express higher levels of the CD163 scavenger receptor, the CD163/HO-1/IL-10 axis appears to contribute to the generation of an immunosuppressive environment within the tumor stroma.     

Haemophilus ducreyi-Induced Interleukin-10 Promotes a Mixed M1 and M2 Activation Program in Human Macrophages

During microbial infection, macrophages are polarized to classically activated (M1) or alternatively activated (M2) cells in response to microbial components and host immune mediators. Proper polarization of macrophages is critical for bacterial clearance. To study the role of macrophage polarization during Haemophilus ducreyi infection, we analyzed a panel of macrophage surface markers in skin biopsy specimens of pustules obtained from experimentally infected volunteers. Lesional macrophages expressed markers characteristic of both M1 and M2 polarization. Monocyte-derived macrophages (MDM) also expressed a mixed M1 and M2 profile of surface markers and cytokines/chemokines upon infection with H. ducreyi in vitro. Endogenous interleukin 10 (IL-10) produced by infected MDM downregulated and enhanced expression of several M1 and M2 markers, respectively. Bacterial uptake, mediated mainly by class A scavenger receptors, and activation of mitogen-activated protein kinase and phosphoinositide 3-kinase signaling pathways were required for H. ducreyi-induced IL-10 production in MDM. Compared to M1 cells, IL-10-polarized M2 cells displayed enhanced phagocytic activity against H. ducreyi and similar bacterial killing. Thus, IL-10-modulated macrophage polarization may contribute to H. ducreyi clearance during human infection.     

Role of response gene to complement 32 in diseases

The role of response gene to complement (RGC)-32 as a cell cycle regulator has been attributed to its ability to activate cdc2 kinases and to induce S-phase entry and mitosis. However, recent studies revealed novel functions for RGC-32 in diverse processes such as cellular differentiation, inflammation, and fibrosis. Besides responding to C5b-9 stimulation, RGC-32 expression is also induced by growth factors, hormones, and cytokines. Transforming growth factor β activates RGC-32 through Smad and RhoA signaling, thus initiating smooth muscle cell differentiation. Accumulating evidence has drawn attention to the deregulated expression of RGC-32 in human malignancies, hyper-immunoglobulin E syndrome, and fibrosis. RCG-32 expression is up-regulated in cutaneous T cell lymphoma and colon, ovarian, and breast cancer, but down-regulated in invasive prostate cancer, multiple myeloma, and drug-resistant glioblastoma. A better understanding of the mechanism by which RGC-32 contributes to the pathogenesis of these diseases will provide new insights into its therapeutic potential. In this review we provide an overview of this field and discuss the most recent research on RGC-32.     

Dual role of Response gene to complement-32 in multiple sclerosis

Response gene to complement (RGC)-32 is a novel molecule that plays an important role in cell proliferation. We investigated the expression of RGC-32 in multiple sclerosis (MS) brain and in peripheral blood mononuclear cells (PBMCs) obtained from patients with relapsing–remitting multiple sclerosis. We found that CD3⁺, CD68⁺, and glial fibrillar acidic protein (GFAP)⁺ cells in MS plaques co-localized with RGC-32. Our results show a statistically significant decrease in RGC-32 mRNA expression in PBMCs during relapses when compared to the levels in stable MS patients. This decrease might be useful in predicting disease activity in patients with relapsing–remitting MS. RGC-32 expression was also correlated with that of FasL mRNA during relapses. FasL mRNA expression was significantly reduced after RGC-32 silencing, indicating a role for RGC-32 in the regulation of FasL expression. In addition, the expression of Akt1, cyclin D1, and IL-21 mRNA was significantly increased during MS relapses when compared to levels in healthy controls. Furthermore, we investigated the role of RGC-32 in TGF-β-induced extracellular matrix expression in astrocytes. Blockage of RGC-32 using small interfering RNA significantly inhibits TGF-β induction of procollagen I, fibronectin and of the reactive astrocyte marker α-smooth muscle actin (α-SMA). Our data suggest that RGC-32 plays a dual role in MS, both as a regulator of T-cells mediated apoptosis and as a promoter of TGF-β-mediated profibrotic effects in astrocytes.     

Overexpression of RGC-32 in colon cancer and other tumors

Tumors often exhibit deregulation of the cell cycle and overexpression of cyclins and cyclin-dependent kinases (CDKs). Response gene to complement (RGC)-32 is a substrate and regulator of CDC2 and its overexpression induces cell cycle activation. We investigated RGC-32 mRNA and protein expression in tumors with special emphasis in colon carcinoma. By using an expression array technique we found that 19% of tumor tissues showed increased RGC-32 mRNA expression over the levels of corresponding normal tissues. On the other hand, an increased RGC-32 protein was found in 70% of colon adenocarcinoma samples tested. In colon carcinomas, two major patterns of RGC-32 immunoreactivity were seen: staining of malignant epithelial cells only in some tumors and RGC-32 reactivity of both malignant epithelia as well as cells in the interstitium in others. Colonic epithelium obtained from normal individuals was consistently negative for RGC-32 protein. Overexpression of RGC-32 protein was found in other tumors including prostate, bladder, breast, lung, and other digestive tract tumors. RGC-32 expression was present in the same malignant epithelial cells that also expressed the proliferation marker Ki-67. Our data suggest that RGC-32 overexpression might be part of the deregulation of the cell cycle that is required for the growth of tumor cells.     

SUMO特异性蛋白酶3通过调控巨噬细胞极化促进磷酸钙诱导的小鼠腹主动脉瘤形成

目的:探讨SUMO特异性蛋白酶3(SENP3)调控巨噬细胞极化在磷酸钙诱导的小鼠腹主动脉瘤(AAA)形成中的作用。方法:(1)分离C57BL/6背景的Senp3flox/flox(野生型,WT)小鼠和Senp3flox/flox;Lyz2-Cre(SENP3单核细胞特异性敲除,即条件性敲除,c KO)小鼠骨髓来源的单核细胞(BMDMs),分别诱导其M1/M2型分化,采用RT-q PCR、Western blot和细胞免疫荧光比较SENP3表达以及M1/M2型巨噬细胞分布差异。(2)8~12周龄雄性WT小鼠和c KO小鼠用改良型磷酸钙诱导14 d构建小鼠AAA模型,比较两组小鼠的成瘤率和生存率;RT-q PCR和Western blot检测SENP3、白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNFα)、IL-6及基质金属蛋白酶9(MMP-9)在两组小鼠AAA组织中的表达差异;二氢乙啶染色检测组织中活性氧簇(ROS)生成差异。(3)为探讨其中机制,通过Western blot和免疫共沉淀验证SENP3与丝裂原活化蛋白激酶激酶7(MKK7)的相互作用;在BMDMs中转染FlagMKK7野生型(Flag-MKK7 WT)和SUMO修饰位点K18突变体(Flag-MKK7 K18R mutant)后诱导BMDMs进行M1极化,用Western blot检测p-JNK和MMP-9表达的差异。结果:(1)SENP3在M1型巨噬细胞中表达显著升高(P<0.01),在M2型巨噬细胞中显著下调(P<0.01);SENP3在M1向M2型巨噬细胞转化过程中表达显著下降(P<0.01),而在M2向M1型巨噬细胞转化时表达显著上调(P<0.01);c KO组BMDMs经M1/M2型诱导后,巨噬细胞M1型少于WT组,而M2型多于WT组;(2)SENP3在AAA组织中表达上调(P<0.05);c KO小鼠磷酸钙诱导后成瘤率显著降低(P<0.01),生存率显著提高(P<0.05),最大腹主动脉外直径显著减小(P<0.01);c KO小鼠AAA组织中IL-1β、IL-6和TNFα表达显著下调(P<0.01),ROS生成减少,MMP-9表达也显著下调(P<0.05)。(3)M1巨噬细胞中,MKK7的SUMO2/3修饰减少,与SENP3的相互作用增强;突变回补实验说明,转染Flag-MKK7 K18R mutant的M1型巨噬细胞中p-JNK和MMP-9表达显著上调(P<0.05)。结论:SENP3通过去SUMO化修饰MKK7,激活MAPK/JNK通路,调控巨噬细胞的极化,上调MMP-9的表达,从而促进AAA形成。     

Transient depletion of CD4+ CD25+ regulatory T cells results in multiple autoimmune diseases in wild‐type and B‐cell‐deficient NOD mice

Plasticity is a hallmark of macrophages, and in response to environmental signals these cells undergo different forms of polarized activation, the extremes of which are called classic (M1) and alternative (M2). Rapamycin (RAPA) is crucial for survival and functions of myeloid phagocytes, but its effects on macrophage polarization are not yet studied. To address this issue, human macrophages obtained from six normal blood donors were polarized to M1 or M2 in vitro by lipopolysaccharide plus interferon-γ or interleukin-4 (IL-4), respectively. The presence of RAPA (10 ng/ml) induced macrophage apoptosis in M2 but not in M1. Beyond the impact on survival in M2, RAPA reduced CXCR4, CD206 and CD209 expression and stem cell growth factor-β, CCL18 and CCL13 release. In contrast, in M1 RAPA increased CD86 and CCR7 expression and IL-6, tumour necrosis factor-α and IL-1β release but reduced CD206 and CD209 expression and IL-10, vascular endothelial growth factor and CCL18 release. In view of the in vitro data, we examined the in vivo effect of RAPA monotherapy (0·1 mg/kg/day) in 12 patients who were treated for at least 1 month before islet transplant. Cytokine release by Toll-like receptor 4-stimulated peripheral blood mononuclear cells showed a clear shift to an M1-like profile. Moreover, macrophage polarization 21 days after treatment showed a significant quantitative shift to M1. These results suggest a role of mammalian target of rapamycin (mTOR) into the molecular mechanisms of macrophage polarization and propose new therapeutic strategies for human M2-related diseases through mTOR inhibitor treatment.     

Controlled release of cytokines using silk-biomaterials for macrophage polarization

Polarization of macrophages into an inflammatory (M1) or anti-inflammatory (M2) phenotype is important for clearing pathogens and wound repair, however chronic activation of either type of macrophage has been implicated in several diseases. Methods to locally control the polarization of macrophages is of great interest for biomedical implants and tissue engineering. To that end, silk protein was used to form biopolymer films that release either IFN-γ or IL-4 to control the polarization of macrophages. Modulation of the solubility of the silk films through regulation of β-sheet (crystalline) content enabled a short-term release (4–8 h) of either cytokine, with smaller amounts released out to 24 h. Altering the solubility of the films was accomplished by varying the time that the films were exposed to water vapor. The released IFN-γ or IL-4 induced polarization of THP-1 derived macrophages into the M1 or M2 phenotypes, respectively. The silk biomaterials were able to release enough IFN-γ or IL-4 to repolarize the macrophage from M1 to M2 and vice versa, demonstrating the well-established plasticity of macrophages. High β-sheet content films that are not soluble and do not release the trapped cytokines were also able to polarize macrophages that adhered to the surface through degradation of the silk protein. Chemically conjugating IFN-γ to silk films through disulfide bonds allowed for longer-term release to 10 days. The release of covalently attached IFN-γ from the films was also able to polarize M1 macrophages in vitro. Thus, the strategy described here offers new approaches to utilizing biomaterials for directing the polarization of macrophages.     

Spleen-derived macrophages are readily polarized into classically activated (M1) or alternatively activated (M2) states

Bone marrow derived macrophages (BM-MΦ) that differentiate from precursor cells can be polarized into classically activated pro-inflammatory (M1) or alternatively activated (M2) states depending upon the cytokine microenvironment. We questioned whether tissue MΦ, such as spleen-derived MΦ (Sp-MΦ), have the ability to differentiate into M1 or M2 cells. We show in response to activation with IFN-gamma (IFN-γ) and lipopolysaccharide (LPS), that the Sp-MΦ readily acquired an M1 status indicated by up-regulation of iNOS mRNA, nitric oxide (NO) production, and the co-stimulatory molecule CD86. Conversely, Sp-MΦ exposed to IL-4 exhibited increased levels of mannose receptor (CD 206), arginase-1 (Arg)-1 mRNA expression, and significant urea production typical of M2 cells. At this stage of differentiation, the M2 Sp-MΦ were more efficient at phagocytosis of cell-associated antigens than their M1 counterparts. This polarization was not indefinite as the cells could revert back to their original state upon the removal of stimuli and exhibited flexibility to convert from M2 to M1. Remarkably, both M1 and M2 Sp-MΦ induced more CD4 expression on their cells surface after stimulation. We also demonstrate that adherent macrophages cultured for a short term in IL-4 enhances ARG-1 and YM-1 mRNA along with increasing urea producing capacity: traits indicative of an M2 phenotype. Moreover, in response to in vivo virus infection, the adherent macrophages obtained from spleens rapidly express iNOS. These results provide new evidence for the polarization capabilities of Sp-MΦ when exposed to pro-inflammatory or anti-inflammatory cytokines.     

A method to differentiate chicken monocytes into macrophages with proinflammatory properties

Macrophages are part of the first line of defense against invading pathogens. In mammals, the in vitro culture of macrophages from blood monocytes or bone marrow cells is well established, including culturing conditions to differentiate them towards M1 or M2-like macrophages. In chicken, monocyte-derived macrophages have been used in several studies, but there is no uniform protocol or actual characterization of these cells. Therefore, to generate proinflammatory M1-like macrophages, in this study blood monocytes were differentiated using GM-CSF for 4 days and characterized based on cell morphology, surface marker expression and cytokine expression response to TLRs stimulation at each (daily) time point. Cell morphology showed that one-day-cultured cells contained a mixture of cell populations, while the homogenous population of cells on day 3 and day 4 were flat and had a ‘fried-egg’ like shape, similar to human M1 macrophages. In addition, cell surface marker staining showed that 3 and 4- days-cultured cells expressed a high level of MRC1L-B (KUL01) and MHC-II. Furthermore, LPS stimulation of the cultured cells induced gene expression of the proinflammatory cytokines IL-1β, IL-6 and IL-8 after 3 days of culture. Finally, it was shown that day 3 macrophages were able to phagocytose avian pathogenic E. coli (APEC) and respond by nitric oxide production. Overall, our systematic characterization of the monocyte derived cells from blood showed that a 3-days culture was optimal to obtain pro-inflammatory M1 like macrophages, increasing our knowledge about chicken macrophage polarization and providing useful information for studies on chicken macrophage phenotypes.     

MiR-146a modulates macrophage polarization in systemic juvenile idiopathic arthritis by targeting INHBA

Monocytes from patients with systemic juvenile idiopathic arthritis (SJIA) have both features of classical activated M1 and alternatively activated M2 macrophages. An increasing number of studies have indicated that microRNAs (miRNAs) are critical regulators of monocyte polarization. Here, we focused on miR-146a expression in SJIA and investigated the function of miR-146a in monocyte polarization. We found that miR-146a expression was highly up-regulated in SJIA monocytes and correlated with the systemic features. miR-146a was expressed at a higher level in monocytes polarized with M2 conditions than those polarized with M1 conditions. miR-146a overexpression significantly decreased the production of M1 phenotype markers such as IL-6, IL-12, TNF-α, CD86 and iNOS in M1 macrophages, but increased the production of M2 marker genes such as Arg1, CCL17, CCL22 and CD206 in M2 macrophages. Conversely, knockdown of miR-146a promoted M1 macrophage polarization but diminished M2 macrophage polarization. We subsequently demonstrated that miR-146a targeted the 3′-untranslated region (UTR) of INHBA to inhibit its expression. Additionally, INHBA overexpression rescued the reduced IL-6, IL-12, and TNF-α levels induced by miR-146a overexpression in M1 macrophages, and rescued the increased Arg1, CCL17, and CCL22 levels induced by miR-146a overexpression in M2 macrophages. Similarly, the effects of miR-146a inhibition in monocyte polarization were all partly reversed by INHBA inhibition. Taken together, the data suggest that miR-146a serves as a molecular regulator in monocyte polarization and might play an important role in monocytes from patients with SJIA.     

Schistosoma japonicum infection induces macrophage polarization

The role of macrophages （MФ） as the first line of host defense is well accepted. These cells play a central role in orchestrating crucial functions during schistosomal infection. Thus, understanding the functional diversity of these cells in the process of infection as well as the mechanisms underlying these events is crucial for developing disease control strategies. In this study, we adopted a Mqb polarization recognition system. M1 macrophage was characterized by expressing CD16/32, IL-12 and iNOS. M2 macrophage was characterized by expressing CD206, IL-10 and arg-1. In vivo （mouse peritoneal macrophages of different infection stages were obtained） and in vitro （different S. japonicum antigens were used to stimulate RAW264.7） were characterized by using the above mentioned system. NCA and ACA stimulated RAW264.7 express significantly higher levels of IL-12 while significantly higher levels of IL-10 were detected after soluble egg antigen （SEA） stimulation. The results showed that dramatic changes of antigen in the microenvironment before and after egg production led to macrophage polarization. Furthermore, through TLR blocking experiments, the TLR4 signaling pathway was found to play a role in the process of macrophage polarization toward M1. Our data suggest that macrophage polarization during S. japonicum infection had significant effects on host immune responses to S. japonicum.     

Coxiella burnetii, the agent of Q fever, stimulates an atypical M2 activation program in human macrophages

Coxiella burnetii is an obligate intracellular bacterium, responsible for Q fever, which survives in macrophages by interfering with their microbicidal competence. As functional polarization of macrophages is critical for their microbicidal activity, we studied the activation program of monocyte-derived macrophages (MDM) stimulated with C. burnetii. This program was markedly distinct from that induced by lipopolysaccharides (LPS), a canonical inducer of M1 polarization. Indeed, C. burnetii up-regulated the expression of genes associated with M2 polarization, including TGF-beta1, IL-1 receptor antagonist (IL-1ra), CCL18, the mannose receptor and arginase-1, and only up-regulated the expression of two genes associated with M1 polarization, namely IL-6 and CXCL8. In contrast, C. burnetii down-regulated the expression of genes associated with M1 polarization such as TNF, CD80, CCR7 and TLR-2. Functional analyses showed that C. burnetii-stimulated MDM produced high levels of TGF-beta1 and CCL18, and expressed the mannose receptor and arginase-1, the latter being associated with the prevention of nitric oxide production by MDM. Finally, C. burnetii induced the release of IL-6 and CXCL8 at a lower level than LPS-stimulated MDM. Our results suggest that C. burnetii stimulated an atypical M2 activation program that may account for the persistence of C. burnetii in macrophages.     

RGC-32 regulates reactive astrocytosis and extracellular matrix deposition in experimental autoimmune encephalomyelitis

Extracellular matrix (ECM) deposition in active demyelinating multiple sclerosis (MS) lesions may impede axonal regeneration and can modify immune reactions. Response gene to complement (RGC)-32 plays an important role in the mediation of TGF-β downstream effects, but its role in gliosis has not been investigated. To gain more insight into the role played by RGC-32 in gliosis, we investigated its involvement in TGF-β-induced ECM expression and the upregulation of the reactive astrocyte markers α-smooth muscle actin (α-SMA) and nestin. In cultured neonatal rat astrocytes, collagens I, IV, and V, fibronectin, α-SMA, and nestin were significantly induced by TGF-β stimulation, and RGC-32 silencing resulted in a significant reduction in their expression. Using astrocytes isolated from RGC-32 knock-out (KO) mice, we found that the expression of TGF-β-induced collagens I, IV, and V, fibronectin, and α-SMA was significantly reduced in RGC-32 KO mice when compared with wild-type (WT) mice. SIS3 inhibition of Smad3 phosphorylation was also associated with a significant reduction in RGC-32 nuclear translocation and TGF-β-induced collagen I expression. In addition, during experimental autoimmune encephalomyelitis (EAE), RGC-32 KO mouse astrocytes displayed an elongated, bipolar phenotype, resembling immature astrocytes and glial progenitors whereas those from WT mice had a reactive, hypertrophied phenotype. Taken together, our data demonstrate that RGC-32 plays an important role in mediating TGF-β-induced reactive astrogliosis in EAE. Therefore, RGC-32 may represent a new target for therapeutic intervention in MS.     

IL-10+IL-4 能提高M2 巨噬细胞表型和嗜酸性粒细胞的迁移

目的:研究论证IL-10 是否能通过诱导IL-4 来提高M2。方法:用C57BL/6J 鼠中的骨髓细胞诱导M-CSF 诱导的骨髓源巨噬细胞,利用小鼠全基因组版本进行转录,进而嗜酸性粒细胞的迁移实验,体内巨噬细胞的转移实验。结果:研究发现在M-CSF 诱导的BMDMs 中通过IL-4、IL-10 及IL-4+IL-10 诱导来分析M2 巨噬细胞,IL-10 通过IL-4 来提高M2a 标志物的表达,此外,IL-4 和IL-10 诱导产生CCL24 时起协同作用。在GM-CSF 诱导的BMDMs 中CCL24 的表达提高。CCL24 是CCR3 的激动剂和嗜酸性粒细胞的趋化因子。在体外,IL-4+IL-10 激活的巨噬细胞产生大量的CCL24,并且能提高嗜酸性粒细胞的迁移。这个过程能被抗CCL24 抗体抑制。IL-4+IL-10 激活的巨噬细胞转移到C57BL/6J 小鼠的腹膜,这能增加嗜酸性粒细胞浸润腹膜腔。结论:IL-4+IL-10 激活的巨噬细胞能增强M2a 巨噬细胞相关基因的表达,提高CCL24 的产生和嗜酸性粒细胞的浸润,导致嗜酸性粒细胞相关疾病的发生。     

Macrophage polarization in response to wear particles in vitro

Total joint replacement is a highly successful surgical procedure for treatment of patients with disabling arthritis and joint dysfunction. However, over time, with high levels of activity and usage of the joint, implant wear particles are generated from the articulating surfaces. These wear particles can lead to activation of an inflammatory reaction, and subsequent bone resorption around the implant （periprosthetic osteolysis）. Cells of the monocyte/macrophage lineage orchestrate this chronic inflammatory response, which is dominated by a pro-inflammatory （M 1） macrophage phenotype rather than an anti-inflammatory pro-tissue healing （M2） macrophage phenotype. While it has been shown that interleukin-4 （IL-4） selectively polarizes macrophages towards an M2 anti-inflammatory phenotype which promotes bone healing, rather than inflammation, little is known about the time course in which this occurs or conditions in which repolarization through I L-4 is most effective. The goal of this work was to study the time course of murine macrophage polarization and cytokine release in response to challenge with combinations of polymethyl methacrylate （PMMA） particles, lipopolysaccharide （LPS） and IL-4 in vitro. Treatment of particle-challenged monocyte/macrophages with IL-4 led to an initial suppression of pro-inflammatory cytokines and inducible nitric oxide synthase （iNOS） production and subsequent polarization into an M2 anti-inflammatory phenotype. This result was optimized when IL-4 was delivered before PMMA particle challenge, to an M 1 phenotype rather than to uncommitted （MO） macrophages. The effects of this polarization were sustained over a 5-day time course. Polarization of M1 macrophages into an M2 phenotype may be a strategy to mitigate wear particle associated periprosthetic osteolysis.     

Vascular endothelial growth factor-induced skin carcinogenesis depends on recruitment and alternative activation of macrophages

Inflammation contributes to tumour growth, invasion and angiogenesis. We investigated the contribution of macrophages and their polarization to tumour progression in a model of VEGF-A-induced skin carcinogenesis. Transfection of the human non-tumourigenic keratinocyte cell line HaCaT with murine VEGF-A leads to malignant tumour growth in vivo. The resulting tumours are characterized by extensive vascularization, invasive growth and high numbers of M2-polarized macrophages that crucially contribute to the establishment of the malignant phenotype. Accordingly, macrophage depletion from tumour-bearing animals resulted in reduced tumour growth, inhibition of invasion, decreased proliferation and reduced angiogenesis. In vitro, VEGF-A exerted a chemo-attracting effect on macrophages, but did not induce M2 polarization. We identified IL-4 and IL-10 as the factors involved in M2 polarization. These factors were produced by tumour cells (IL-10) and macrophages (IL-4) in vivo. Addition of recombinant IL-4 and IL-10 in vitro induced a pro-invasive M2 macrophage phenotype and inhibition of the IL-4 receptor in vivo blocked M2 polarization of macrophages, resulting in a less aggressive tumour phenotype. Thus, we provide evidence that M2 macrophages are crucial for the development of VEGF-A-induced skin tumours and that VEGF-A contributes to malignant tumour growth, not only by enhancing angiogenesis but also by establishing an anti-inflammatory microenvironment. However, VEGF-A alone is not sufficient to create a tumour-promoting microenvironment and requires the presence of IL-4 and IL-10 to induce M2 polarization of macrophages.