Tollip Orchestrates Macrophage Polarization To Alleviate Intestinal Mucosal Inflammation

Background and ObjectionDysfunction of the innate immune system and impairment of mucosal repair are two important pathophysiological features of inflammatory bowel disease(IBD).Although many treatments have been frequently used in clinical therapeutic strategies,IBD therapy remains a complex problem because of the limited treatment options available.Previous studies have found that there are a large number of macrophages in the intestinal tissue of patients with IBD,and there is a polarization imbalance of these macrophages.Macrophage polarization regulation is a promising strategy for the treatment of IBD.Tollip is an important negative regulator of immune response mediated through Toll-like receptors(TLRs).A previous study by our group found that the expression of Tollip was downregulated in the colonic tissue of patients with IBD.The purpose of this study was to construct a molecular targeted immunotherapy method by delivering the Tollip gene to intestinal macrophages to regulate the polarization of macrophages and to provide a candidate method for clinical treatment of IBDMethods and results1.To construct nanoparticles that can target intestinal macrophages as gene carriers.By linking mannose-modified trimethyl chitosan(MTC)with Tollip-expressing plasmids via ionic cross-linking,MTC-Tollip nanoparticles with a targeting function were formed.Observed by electron microscopy,the results show that MTC-Ns is spherical and its surface is smooth.Through the particle size and zeta potential measuring instrument,we found that the particle size distribution of MTC-NPs is uniform and the potential is positive.Gel electrophoresis showed that MTC-NPs had high entrapment efficiency and strong stability.The cytotoxicity test showed that MTC-NPs had no obvious toxicity.Immunofluorescence and PCR detection showed that MTC-Tollip could target mouse intestinal macrophages and upregulate the expression of Tollip mRNA in intestinal tissue.2.MTC-Tollip alleviates DSS-induced colitis in miceThe mouse of acute colitis was stimulated by 3%DSS and then treated with MTC-Tollip.The results represented that compared with the DSS model group,MTC-Tollip nanoparticle treatment reduced body weight loss,hematochezia,colon shortening and histopathological scores.Immunohistochemical staining and PCR detection of tight junction protein in mouse intestinal tissue,which showed that MTC-Tollip treatment protected the intestinal barrier.The results of immunohistochemical staining and immunofluorescence of intestinal tissue showed that after MTC-Tollip treatment,M1 macrophages in intestinal tissue were decreased and M2 macrophages were increased.3.Tollip orchestrates macrophage polarizationMouse peritoneal macrophages were overexpressed by Tollip-expressing lentivirus in vitro.Macrophages were stimulated with LPS,the proportion of M1 macrophages was detected by flow cytometry,and TLR-related pathways and NF-κB transcription were detected by Western bloting.It was found that overexpression of Tollip could inhibit M1 polarization of macrophages by reducing the transcription of NF-κB.Using IL-4-treated macrophages,the proportion of M2-type macrophages was detected by flow cytometry,and M2 polarization-related signaling pathways were detected by Western bloting.We found that overexpressed Tollip promoted IL-4-induced M2 polarization by cooperating with AKT activation.Further detection of peritoneal macrophages in Tollip knockout mice showed that Tollip deletion aggravated the M1-type polarization of macrophages through LPS-induced and decreased the M2-type polarization of macrophages through IL-4-induced.Conclusions1.Mannose-modified trimethyl chitosan as carriers can deliver the Tollip gene to mouse intestinal macrophages and upregulate the expression of Tollip.2.MTC-Tollip nanoparticles can effectively alleviate DSS-induced experimental colitis in mice by reducing M1 and increasing M2 macrophages in intestinal tissue.3.In vitro,Tollip decreased the activation of NF-κB and inhibited the M1 polarization of macrophages stimulated by LPS through negatively regulating the TLR signaling pathway.Tollip promotes M2 polarization of macrophages stimulated by IL-4 by up-regulating the phosphorylation of AKT.