Depletion of B2 cells did not affect the number of CD4+ T cells and CD8+ T cells in the spleen
Depletion of B2 cells did not affect the number of CD4+ T cells and CD8+ T cells in the spleen. light-chain and heavy-chain variable genes suggesting a decreased repertoire of B cells after BAFF neutralization. Further, the B cell activation and the phagocytosis pathways were impaired in the WAT of BAFF-neutralized mice. multiple mechanisms and are well-studied (20). Interestingly, IgG autoAbs generated in response to nerve injury clear myelin debris and promote axonal growth supporting a beneficial role of autoAbs (21). Winer et?al. demonstrated the presence of IgG autoAbs against intracellular proteins normally expressed in multiple tissues and the serum of obese and IR individuals (2). Frasca et?al., later reported that the plasma of obese individuals is rich in IgG autoAbs targeting intracellular proteins of adipocytes (22). Interestingly, IgG Rabbit polyclonal to Src.This gene is highly similar to the v-src gene of Rous sarcoma virus.This proto-oncogene may play a role in the regulation of embryonic development and cell growth.The protein encoded by this gene is a tyrosine-protein kinase whose activity can be inhibited by phosphorylation by c-SRC kinase.Mutations in this gene could be involved in the malignant progression of colon cancer.Two transcript variants encoding the same protein have been found for this gene. deposition around dead adipocytes is found in the CLSs of obese mice (2). While macrophages can clear dead adipocytes by exophagy and efferocytosis, it is unknown if adipocyte-specific autoAbs are generated in C57BL/6J mice, the widely studied murine models of diet-induced obesity and insulin resistance. It is also unknown if the autoAbs are deposited on the surface of dead adipocytes and promote their clearance by macrophages, thereby affecting WAT remodeling and systemic IR. Here, we show that HFD-induced obesity resulted in the generation of IgG autoAbs that bind to apoptotic 3T3-L1 adipocytes. model. (A) Representative fluorescent images of apoptotic 3T3-L1 adipocytes incubated with plasma from NCD or HFD-fed mice stained for nucleus (Hoechst-blue), apoptosis (YoPro-green), and IgG (pink, anti-IgG antibody). White arrows indicate cells positive, for Hoechst, YoPro, and IgG. (B) Schematic of phagocytosis experiment used for confocal imaging. (C) 3D confocal images of macrophages (DiI-red) co-cultured with apoptotic 3T3-L1 adipocytes (BODIPY-green) for 0 hours (0hr) and 6 hours (6hr). Parts of the images ALK2-IN-2 were digitally enlarged to show the localization of BODIPY stains inside the macrophages with yellow arrows. (D) Schematic of phagocytosis experiment used for flow cytometry. In this experiment, macrophages were stained with CellTrace Violet, and adipocytes ALK2-IN-2 were stained with three dyes: BODIPY, CypHer5E, and DiI. (E, F) Flow cytometry of ALK2-IN-2 ALK2-IN-2 macrophages after co-cultured with apoptotic adipocytes in the presence of IgG-rich plasma fraction from NCD or HFD-fed mice. (E) The amount of apoptotic cell uptake was assessed by median fluorescent intensity (MFI) of BODIPY, CypHer5e, and DiI of macrophages. (F) The percentage of phagocytic macrophages was determined by the percent of BODIPY, CypHer5e, and DiI-positive macrophages. Values are expressed as means + SEM. *, p<0.05; **, p<0.01; ***, p<0.001 ****, p<0.0001 by parametric unpaired t-test. n=3 wells per treatment, 3 images each (A), n=1 well per treatment, 10 images each (C), n=6 (E, F). Scale bars: 100 m (A); 40 m (C). AutoAbs are necessary for the recognition and removal of dead cells through antibody-dependent cellular phagocytosis (ADCP) (27). We examined if the increase in IgG autoAbs found in obese mice affected the rate of phagocytic removal of dead adipocytes. To do so we developed an phagocytosis assay utilizing apoptotic 3T3-L1 adipocytes. To confirm the efficacy of the method, apoptotic 3T3-L1 adipocytes were stained with BODIPY, a fluorescent neutral lipid dye, and incubated with murine bone-marrow-derived macrophages stained with DiI, a fluorescent membrane dye ( Figure?1B ). Phagocytosis was confirmed after 6 hours with confocal microscopy based on the uptake of BODIPY into the macrophages ( Figure?1C ). For quantitative analysis of phagocytosis, we developed a flow cytometry-based assay. Since IgM antibodies can also bind to dead cells and promote their clearance by macrophages (28), we fractionated the mouse plasma samples using size exclusion chromatography and pooled 155 kDa molecular weight.