The Role And Mechanism Of Flt3L In Murine Experimental Colitis

Background: Fms-like tyrosine kinase 3 ligand(Flt3L) is a key DC growth factor. Flt3 L and its receptor(Flt3) instruct haematopoetic progenitors along a DC developmental pathway to give rise to multiple DC subsets, especially p DCs. As a member of dendritic cells which are specialized in capture, processing and presentation of antigens to T cells, p DCs are important in the regulation of innate and adaptive immune responses. Dysfunction of the intestinal immune system plays an important role in inflammatory bowel disease(IBD) pathogenesis. p DCs comprise a subset of intestinal dendritic cells, such as the mesenteric lymph node(MLN)and the intestine lamina propria(LP). However, its role in inflammatory bowel diseases is poorly characterized.Objective: To explore the role of Flt3 L in TNBS-induced murine experimental colitis and clarify its mechanism.Methods: 1. Production of recombinant GST-Flt3 L protectine : The plasmid PGEX-4T3-m Flt3 L was previously constructed in our lab. E.coli cells were grown in LB medium containing an appropriate antibiotic and induced for GST-Flt3 L protein expression by addition of IPTG. GST-Flt3 L fusion protein was purified by glutathione-affinity chromatography. Then GST-Flt3 L fusion protein was dectected by SDS-PAGE and Western Blot.2. Induction of experimental colitis mouse model:Mice were administered 0.1ml of TNBS in 50% ethanol intrarectally to induce inflammatory injury of colon. Then recorded body weight of mice daily. On the fourth day, the mice were sacrificed and tissue samples were collected.3. Intraperitoneal injection of Flt3 L before induction of colitis:Mice received daily intraperitoneal recombinant Flt3-Ligand injections(10 mg/injection) for ten consecutive days. The control group were performed by the same technique. Colitis mouse model were induced on the eleventh day.4. Collected tissue samples of the experimental group and the control groups, then dectected some parameters.5. In vitro mouse bone marrow–derived plasmacytoid dendritic cells(BM-p DCs) were generated, then p DCs were puried by negative selection using microbeads. Puried p DCs were stimulated with Cp G-A or TNBS. CD4+ T cells isolated from mouse spleen were then added to the culture for 96 h. Treg cells proliferation was assessed by flow cytometry and IL-10 was measured in the culture supernatant by ELISA.6. Detection methods and indices(1) Use Western Blot to dectect GST-Flt3 L fusion protein.(2) Assess the severity of body weight, colon length, macroscopic damage score in colitis mice.(3) The microscopic damage and score were dectected by H&E staining.(4) Single-cell suspensions of mouse spleen and mesenteric lymph nodes were obtained to analyze p DCs frequency by flow cytometry.(5) Treg cells frequency in cell culture was dectected by flow cytometry.(6) IL-10 was measured in the culture supernatants using a Mouse IL-10 ELISA kit.Results:1. The GST-Flt3 L fusion protein was successfully purified.2. The TNBS-induced experimental colitis mouse model was successfully established.3. In contrast to control groups, Flt3 L could expand the frequency of p DCs in mouse spleen and mesenteric lymph nodes, and attenuated the severity of colitis.4. p DCs preferentially induced regulatory T cells in DC-CD4+ T cell coculture and enhanced IL-10 secretion.Conclusion:Flt3L could improve the frequency of p DCs in vivo. p DCs promote CD4+ T cells to differentiate into Treg cells and specifically stimulate IL-10 secretion. Our findings provide that Flt3 L could attenuate the severity of TNBS-induced mice colitis.