FLT3 ligand regulates expansion of regulatory T-cells induced by regulatory dendritic cells isolated from gut-associated lymphoid tissues through the Notch pathway

Abstract

Background: Regulatory dendritic cell (DCreg) subset exhibits a unique capacity for inducing immune tolerance among the variety subsets of dendritic cells (DCs) within gut-associated lymphoid tissues (GALTs). Fms-like tyrosine kinase 3 ligand (FLT3L) is involved in the differentiation of DCregs and the subsequent expansion of regulatory T-cells (Tregs) mediated by DCregs, though the precise mechanism remains poorly understood. This study aimed to explore the expansion mechanism of Treg induced by DCreg and the role of FLT3L in this process.

Methods: DCregs were distinguished from other DC subsets isolated from GALTs of BALB/c mice through a mixed lymphocyte reaction assay. The functions and mechanisms by which FLT3L promoted Treg expansion via DCregs were investigated in vitro through co-culture experiments involving DCregs and either CD4 + CD25 - T-cells or CD4 + CD25 + T-cells. Additionally, an in vivo experiment was conducted using a dextran sulfate sodium (DSS)-induced colitis model in mice.

Results: CD103 + CD11b + DC exhibited DCreg-like functionality and was identified as DCreg for subsequent investigation. Analysis of Foxp3 + Treg percentages within a co-culture system of CD4 + CD25 - T-cells and DCregs, with or without FLT3L, demonstrated the involvement of the FLT3/FLT3L axis in driving the differentiation of precursor T-cells into Foxp3 + Tregs induced by DCregs. Cell migration and co-culture assays revealed that the FLT3/FLT3L axis enhanced DCreg migration toward Tregs via the Rho pathway. Additionally, it was observed that DCregs could promote Treg proliferation through the Notch pathway, as inhibition of Notch signaling by DAPT (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester) suppressed Treg expansion within the co-culture system of DCregs and CD4 + T-cells or CD4 + CD25 + T-cells. Furthermore, the FLT3/FLT3L axis influenced JAG1 expression in DCregs, indirectly modulating Treg expansion. In vivo experiments further established that FLT3L promoted DCreg expansion and restored Treg balance in DSS-induced colitis models, thereby ameliorating colitis symptoms in mice.

Conclusion: The FLT3/FLT3L axis is integral to the maintenance of DCreg function in Treg expansion.

Keywords: Dendritic cells; Fms-like tyrosine kinase 3 ligand; Jagged1; Notch1; Regulatory T-cells.

Figure 1

MLR results of the four DC subsets isolated from GALTs of BALB/c mice. Among these four DC subsets, CD103⁺CD11b⁺ DCs and CD8α⁻CD11b⁺ DCs presented the immune suppression ability compared with the control group (CD4⁺ T-cell cultured alone). ^*P <0.05, ^†P <0.01. DCs: Dendritic cells; GALTs: Gut-associated lymphoid tissues; MLR: Mixed lymphocyte reaction; ns: Not siginificant.

Figure 2

Percentage of CD25⁺Foxp3⁺ T-cells analyzed by FC and results of cell migration. (A) Representative images of DCregs migration in the four groups (Crystal violet staining, Bars: 200 μm). (B) Comparison of the average migrated cell numbers of the four groups. Bars show the mean of migrated cell number selected randomly from five fields of view from each group, while the points show the migrated cell number of each field. Relative migration rate = Average number of migrated cells in experimental group/control group ×100%. ^*P <0.01, ^†P <0.001, by one-way ANOVA multiple comparisons. (C) The FACS plots showing the percentage of CD25⁺Foxp3⁺ T-cells in each group. ANOVA: Analysis of variance; DCregs: Regulatory dendritic cell; FACS: Fluorescence-activated cell sorting; FC: Flow cytometry.

Figure 3

Results of percentage of CD25⁺Foxp3⁺ T-cells analyzed by FC. Data shown are representative of two independent experiments gated on CD4⁺ T-cells. (A, B) The FACS plots showing the percentage of CD25⁺Foxp3⁺ T-cells in each group. FACS: Fluorescence-activated cell sorting; FC: Flow cytometry.

Figure 4

Percentage of CD25⁺Foxp3⁺ T-cells and expression level of Notch1 and JAG1. (A) Representative FACS plots showing the percentage of CD25⁺Foxp3⁺ T-cells in each group. (B) The Western blotting results of Notch1 and JAG1 in each group. (C) The RT-qPCR results of Notch1 and JAG1 in each group. DCreg: Regulatory dendritic cell; FACS: Fluorescence-activated cell sorting; FLT3L: Fms-like tyrosine kinase 3 ligand; RT-qPCR: Quantitative real-time polymerase chain reaction; Treg: Regulatory T-cells.

Figure 5

Results of DAI, CMDI and histopathological score, and FLT3 expression. (A) Representative histopatholotical images of colon tissue slices from each group (Hematoxylin & eosin staining, Original magnification ×100). (B) FLT3 indirect immunofluorescence stain images of colon tissue slices from each group (Original magnification ×400). (C) Results of DAI scores of each group in the modeling period. Compared with DSS group, ^*P <0.05, ^†P <0.01; compared with DSS+DCreg group, ^‡P <0.01. (D) Comparison of CMDI scores of each group. (E) Comparison of histopathological scores of each group. ^*P <0.05, ^†P <0.01. (C–E): Data were represented as mean ± SEM (n = 5), the values were compared by one-way ANOVA multiple tests. (F) Comparison of FLT3 expression in colons of each group. Data were represented as mean ± SEM (n = 3), the values were compared by one-way ANOVA multiple tests. ^*P <0.05, ^†P <0.01. ANOVA: Analysis of variance; CMDI: Colon macroscopic damage index; DAI: Disease activity index; DCreg: Regulatory dendritic cell; DSS: Dextran sulfate sodium; FLT3L: Fms-like tyrosine kinase 3 ligand; ns: Not significant; SEM: Standard error of the mean.

Figure 6

Percentage of DCreg, CD4⁺ T cell and Foxp3⁺ Treg of colon LP and MLN. (A) Representative FACS plots showing the percentage of CD4⁺ T cell and Foxp3⁺ Treg of colon LP and MLN of each group. (B) Representative FACS plots showing the percentage of DCreg (CD103⁺ DC11b⁺ DC) of colon LP and MLN of each group. (C) Comparison of the percentage of DCreg, CD4⁺ T cell and Foxp3⁺ Treg of colon LP and MLN. Data were represented as mean ± SEM (n = 5). ^*P <0.05, ^†P <0.01 by one-way ANOVA multiple comparisons. ANOVA: Analysis of variance; DC: Dendritic cell; DCreg: Regulatory dendritic cell; DSS: Dextran sulfate sodium; FACS: Fluorescence-activated cell sorting; FLT3L: Fms-like tyrosine kinase 3 ligand; LP: Lamina propria; MLN: Mesenteric lymph node; ns: Not significant; PBS: Phosphate buffered saline.