Effect Of Autoimmune Regulator On Macroautophagy And Cellular Phagocytosis

Part 1 Autoimmune Regulator and MacroautophayPurposeAutoimmune regulator (AIRE) has the potential to induce central immune tolerance. However its function in peripheral immune tolerance has so far remained unknown. Assuming that macroautophagy may have a role in the induction of immune tolerance, we intended to explore the expression of AIRE in periphery and its possible effect upon the macroautophagy of THP-1 human monocytes.MethodspEGFP-AIRE, the eukaryotic expression vector, was identified by double restriction enzyme digestion, then amplified by PCR and verified identified by DNA sequencing analysis. Using density gradient centrifugation and adhesive culture, peripheral blood mononuclear cells (PBMC) and monocytes were separated from peripheral blood. The phorbol ester (PMA) was adopted to induce the differentiation of THP-1 cells, which then was morphologically indentified by Wright-Giemsa staining and transfected by the Llipofectamine 2000. AIRE expression was inhibited by siRNA. Under the fluorescence microscope, the expression of GFP-AIRE fusion protein was observed and the subcellular location of proteins was indicated. The transfection efficiency was confirmed by RT-PCR and Western Blot. The intracellular formation and localization of autophagosome was indicated by indirect immunofluorescence staining. Macroautophagy was inhibited by PI3K inhibitor (wortmannin); the protein expression of LC3B-â…¡and p62/SQSTM1 were detected by Western Blot.Results Specific bands appeared after plasmid DNA was treated by restriction enzyme digestion, PCR amplification and agarose gel electrophoresis. According to the result of DNA sequencing, the compliance rate is as high as 99% with that of AIRE (NM₀00383.2) (BLAST scores 1698). AIRE mRNA expression existed in PBMC, monocytes, U937 and THP-1 cells; AIRE protein expression is low in monocytes and THP-1 cells. Followed by induction, THP-1 cells present a series of typical manifestations of mature differentiation: an increased adherence to tissue culture plastic, expanded cell body, morphological diversity, irregular nucleus, phagocytic vacuoles and cellular debris in cytoplasm.48 hours after plasmid transfection, the dot-like GFP-AIRE fusion protein was spotted in the nucleus under fluorescence microscopy; in mRNA and protein level, AIRE showed significantly high expression while AIRE siRNA markedly inhibited the GFP-AIRE fusion protein expression as well as AIRE mRNA and protein expression. The over-expression of AIRE upregulated the LC3B-â…¡expression and promoted the formation of autophagosome. But this effect could be counteracted by macroautophagy inhibitors and AIRE siRNA. Neither over-expression of AIRE nor macroautophagy inhibitors and AIRE siRNA had any effect on the expression of p62/SQSTM1.ConclusionThe correct construction of pEGFP-AIRE, the eukaryotic expression vector, can be applied to the in vitro research of transfection. AIRE can be expressed in peripheral monocytes and by upregulating both the expression of LC3B-â…¡and the formation of autophagosome, it is able to promote monocytic macroautophagy. The signal transduction pathway through which AIRE mediated monocytic macroautophagy may bear no relation to p62/SQSTM1 protein. It is probably by promoting monocytic macroautophagy that AIRE functions in peripheral immune tolerance. Part 2 Autoimmune Regulator and Phagocytosis of Apoptotic CellsPurposeAn opportune clearance of apoptotic cells, which are a major source of self-antigenic components, is of great significance for maintaining immunological homeostasis. As two different forms of programmed cell death, autophagic cell death and apoptosis share many common regulators. Based on the findings of the first part in this study, we continue to explore the function of autoimmune regulator (AIRE) in peripheral immune tolerance by focusing on the effect AIRE has upon phagocytosis of apoptotic cells by non-professional phagocytes-epithelial 16HBE cells.Methods16HBE cells were transfected by pEGFP-AIRE, the same plasmid in Part 1, using FuGENE HD transfection reagent. The transfection efficiency was verified by fluorescence microscopy, RT-PCR and Western Blot. The apoptosis of HL-60 cells was first induced by the CPT (Camptothecin), then double stained by Annexin V-FITC/propidium iodide (PI) and finally detected by flow cytometry. Phagocytosis of apoptotic cells was displayed by myeloperoxidase (MPO) staining. Both RT-PCR and Western Blot were adopted to detect the expression of the phagocytosis-related gene (Rac 1).Results48 hours after the pEGFP-AIRE transfection, we observed the predominant location of the GFP-AIRE fusion proteins in the nuclear dots by using fluorescence microscope; through RT-PCR and Western Blot assays, AIRE mRNA and protein expression were both significantly high. Compared to the negative control group with no apoptosis induction, the data from flow cytometry indicated that the percentage of apoptosis was approximately increased threefold [(4.08±0.26)% Vs (13.46±1.71)%] in HL-60 cells induced by camptothecin. Being incubated with apoptotic cells for 1 hour, the AIRE-transfected 16HBE cells enjoyed a phagocytosis rate of (25.5±3.67)%, while those transfected by empty vector had a phagocytosis rate of (6.25±1.58)%, indicating a significant differences (P<0.05). However, over-expression of AIRE had little effect on 16HBE phagocytosis of non-apoptotic cells [(1.0±0.67)%] but upregulated the Rac 1 mRNA and protein expression.Conclusion16HBE cells possess a basic phagocytic rate in removing apoptotic cells and thus can be used as an alternative model in the study of phagocytosis of apoptotic cells in vitro. MPO staining is an optional testing method since it provides a sharp contrast in detecting engulfment of MPO-positive target cells in vitro. AIRE can promote the phagocytosis of apoptotic cells by epithelial cells. The upregulation of the expression of Rac 1 by AIRE can strengthen the phagocytic clearance of apoptotic cells and thus prevent the exposure of self-antigens, suggesting that AIRE might play a role in peripheral immune tolerance.