The DEL-1/β3 integrin axis promotes regulatory T cell responses during inflammation resolution

Abstract

FOXP3+CD4+ regulatory T cells (Tregs) are critical for immune homeostasis and respond to local tissue cues, which control their stability and function. We explored here whether developmental endothelial locus-1 (DEL-1), which, like Tregs, increases during resolution of inflammation, promotes Treg responses. DEL-1 enhanced Treg numbers and function at barrier sites (oral and lung mucosa). The underlying mechanism was dissected using mice lacking DEL-1 or expressing a point mutant thereof, or mice with T cell-specific deletion of the transcription factor RUNX1, identified by RNA sequencing analysis of the DEL-1-induced Treg transcriptome. Specifically, through interaction with αvβ3 integrin, DEL-1 promoted induction of RUNX1-dependent FOXP3 expression and conferred stability of FOXP3 expression upon Treg restimulation in the absence of exogenous TGF-β1. Consistently, DEL-1 enhanced the demethylation of the Treg-specific demethylated region (TSDR) in the mouse Foxp3 gene and the suppressive function of sorted induced Tregs. Similarly, DEL-1 increased RUNX1 and FOXP3 expression in human conventional T cells, promoting their conversion into induced Tregs with increased TSDR demethylation, enhanced stability, and suppressive activity. We thus uncovered a DEL-1/αvβ3/RUNX1 axis that promotes Treg responses at barrier sites and offers therapeutic options for modulating inflammatory/autoimmune disorders.

Keywords: Adaptive immunity; Autoimmunity; Inflammation; T cells.

Figure 1. DEL-1 deficiency decreases Treg numbers while it increases Th17 cells during resolution of inflammation.

Groups of littermate WT and Del1^KO mice were subjected to ligature-induced periodontitis (LIP) for 10 days and ligatures were removed on day 10 (to facilitate inflammation resolution) for 5 days. (A–D) FACS plots of Tregs in gingival tissues (A) and cLNs (C) on day 15 and bar graphs showing percentage of Tregs in CD4⁺ T cells (left), absolute numbers (middle), and FOXP3 MFI (right) of Tregs from gingival tissues (B) and cLNs (D) of littermate WT and Del1^KO mice on day 15 (n = 6–7 mice/group). (E) FACS plots of Th17 cells in gingival tissue on day 15 and (F) bar graphs showing percentage of Th17 cells in CD4⁺ T cells (left), absolute numbers (middle), and IL-17A MFI of Th17 cells (right) from gingival tissues of littermate WT and Del1^KO mice on day 15 (n = 7 mice/group). (G) FACS plot of Th17 cells in cLNs and (H) bar graphs showing percentage of Th17 cells in CD4⁺ T cells (left), absolute numbers (middle), and IL-17A MFI of Th17 cells (right) from cLNs of littermate WT and Del1^KO mice on day 15 (n = 6 mice for WT group and n = 9 for Del1^KO group). Data are means ± SD and are pooled from 2 independent experiments. ***P < 0.001, ****P < 0.0001 vs. WT mice by 2-tailed Student’s t test.

Figure 2. The DEL-1 EGF-like repeats (DEL-1[E1–E3]) are sufficient to upregulate the Treg/Th17 cell ratio.

(A–D) Groups of Del1^KO mice were subjected to ligature-induced periodontitis (LIP) for 10 days and ligatures were removed on day 10 to facilitate inflammation resolution. The mice were locally microinjected daily with DEL-1–Fc, DEL-1-[E1–E3]–Fc, or Fc control from day 10 to day 14 for a total of 5 doses. FACS plots of Tregs (top) and Th17 cells (bottom) in gingival tissues (A) and cLNs (C) of microinjected Del1^KO mice on day 15 and bar graphs showing the percentages and absolute numbers of Tregs (top left and middle) and Th17 cells (bottom left and middle) in CD4⁺ T cells, Treg/Th17 cell ratio (top right) from gingival tissues (B) and cLNs (D) (n = 6–7 mice/group). Data are means ± SD and are pooled from 2 independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Fc treatment group by 1-way ANOVA with Dunnett’s multiple-comparisons test for comparison with Fc control treatment.

Figure 3. DEL-1 directly promotes de novo Treg differentiation via its RGD motif.

(A and B) Naive splenic CD4⁺ cells were differentiated, or not, into Th17 cells under pathogenic conditions (see Methods) in the presence of DEL-1–Fc or Fc control. (A) FACS plots and (B) percentage of IL-17A⁺ cells in CD4⁺ T cells (n = 6 replicates). (C–E) Naive splenic CD4⁺ cells were differentiated, or not, into Tregs in the presence of DEL-1–Fc, DEL-1[RGE]–Fc, or Fc control (10 μg/mL). (C) FACS plots and (D) percentage of FOXP3⁺ cells in CD4⁺ T cells (n = 7 replicates). (E) Relative mRNA expression of Foxp3 in Tregs (n = 6 replicates). (F) Suppression of CFSE-labeled CD4⁺CD25^– T cell (Tconv) division by purified DEL-1–Fc−iTregs or Fc-iTregs. Numbers on x axis indicate CD4⁺CFSE⁺cell/iTreg ratio (n = 6 replicates). (G) Naive splenic CD4⁺ T cells were differentiated into Tregs in the presence of DEL-1–Fc or Fc control (10 μg/mL). CD4⁺CD25⁺ cells were sorted and restimulated for 4 days in medium containing IL-2 (40 ng/mL) and FOXP3 expression was assessed. (H and I) Naive splenic CD4⁺ cells were differentiated into Tregs. CD4⁺CD25⁺ cells were sorted and restimulated for 4 days with DEL-1–Fc or Fc control in medium containing IL-2 (40 ng/mL) without (H) or with TGF-β1 (5 ng/mL) (I), and FOXP3 expression was assessed. (J) Naive splenic CD4⁺ T cells were differentiated for 4 days to Tregs in the presence of DEL-1–Fc or Fc control (10 μg/mL). iTregs (CD4⁺CD25⁺) were sorted and evaluated for their methylation status of the Foxp3 CNS2 (n = 9 mice). All CD4⁺ T cells were isolated from WT mice. Data are means ± SD and are pooled from 2 (B and D–I) or 5 (J) independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Fc control (B and D–J) by 1-way ANOVA with Dunnett’s post-test for comparison with Fc control (B, D, and E), 2-way ANOVA with Holm-Šidák post hoc test for comparison with Fc control (F), or 2-tailed Student’s t test for comparison with Fc control (G–J). NS, not significant.

Figure 4. The DEL-1 RGD motif is critical for Treg responses in vivo.

(A–D) Groups of Del1^KO mice were subjected to ligature-induced periodontitis (LIP) for 10 days, at which time the ligatures were removed. The mice were then locally microinjected daily with DEL-1–Fc, DEL-1[RGE]–Fc, or Fc control from day 10 to day 14 for a total of 5 doses. Percentages and absolute numbers of Tregs (A and C) and Th17 cells and Treg/Th17 cell ratio (B and D) from gingival tissues (A and B) and cLNs (C and D) on day 15 (n = 6 mice/group). (E–H) Groups of WT, Del1^KO, and Del1^RGE/RGE mice were subjected to LIP for 10 days and ligatures were removed on day 10 for 5 days. Percentages and absolute numbers of Tregs (E and G) and Th17 cells and Treg/Th17 cell ratio (F and H) from gingival tissues (E and F) and cLNs (G and H) of WT, Del1^KO, and Del1^RGE/RGE mice on day 15 (n = 6 mice/group). Data are means ± SD and are pooled from 2 independent experiments. **P < 0.01; ***P < 0.001; ****P < 0.0001 by 1-way ANOVA with Dunnett’s post hoc test vs. Fc control group (A–D) or vs. WT mice (E–H).

Figure 5. Del1^KO and Del1^RGE/RGE mice have impaired Treg induction and resolution of inflammation in a model of acute lung injury.

(A–G) Groups of WT, Del1^KO, or Del1^RGE/RGE mice were intratracheally instilled with Escherichia coli LPS at 3.75 μg/g bodyweight or PBS control. (A) Relative mRNA expression of Del1, Il17a, and Il6 in WT mice at the indicated time points (n = 6 mice/group). (B) BAL from WT, Del1^KO, or Del1^RGE/RGE mice was analyzed for total protein concentration (left) and total cell numbers (right) on day 10 after LPS instillation (n = 6 mice/group). (C) H&E staining of lung sections (left) and lung injury scoring (right) from WT, Del1^KO, or Del1^RGE/RGE mice on day 10 after LPS instillation (n = 6 mice/group). Scale bars: 200 μm (top panels) and 100 μm (bottom panels). (D and E) BAL from WT, Del1^KO, or Del1^RGE/RGE mice was analyzed for (D) the percentage and absolute numbers of Tregs and (E) Th17 cells in CD4⁺ T cells and Treg/Th17 cell ratio (n = 6 mice/group). (F) The percentage and absolute numbers of Tregs and (G) Th17 cells in CD4⁺ T cells and Treg/Th17 cell ratio in mediastinal LNs from WT, Del1^KO, or Del1^RGE/RGE mice on day 10 (n = 6–9 mice/group as indicated). Data are means ± SD and are pooled from 2 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by 1-way ANOVA with Dunnett’s post hoc test for comparison between indicated groups.

Figure 6. DEL-1 enhances FOXP3 expression in Tregs in a β3 integrin–dependent manner.

(A) FACS plots of CD51 (αv) and CD61 (β3) expression on mouse naive splenic CD4⁺ T cells. (B and C) Naive splenic CD4⁺ cells isolated from WT mice were differentiated, or not, into Tregs in medium containing anti-CD3/anti-CD28, TGF-β1 (5 ng/mL), IL-2 (40 ng/mL), and Fc control or DEL-1–Fc (10 μg/mL) in the presence or not of IgG control or anti-αvβ3 antibody (10 μg/mL; added 15 minutes before DEL-1–Fc treatment). Shown are (B) FACS plots and (C) data analysis of the percentage of FOXP3⁺ cells in CD4⁺ T cells from the in vitro culture system on day 4 (n = 6 replicates from 2 separate cell isolations). (D and E) Naive splenic CD4⁺ cells isolated from WT and Itgal^KO mice were differentiated, or not, into Tregs in medium containing anti-CD3/anti-CD28, TGF-β1 (5 ng/mL), IL-2 (40 ng/mL), and Fc control or DEL-1–Fc (10 μg/mL). Shown are (D) FACS plots and (E) data analysis of the percentage of FOXP3⁺ cells in CD4⁺ T cells from the in vitro culture system on day 4 (n = 6 replicates from 2 separate cell isolations). Data are means ± SD and are pooled from 2 independent experiments. ****P < 0.0001 between indicated groups by 1-way ANOVA with Dunnett’s post hoc test for comparisons with DEL-1–Fc treatment (C) or by 2-tailed Student’s t test (E). NS, not significant.

Figure 7. RUNX1 is required for the ability of DEL-1 to induce Treg differentiation.

(A and B) Naive splenic CD4⁺ cells from WT mice were differentiated into Tregs in the presence of DEL-1–Fc or Fc control for 3 days. RNA-seq analysis of the Treg transcriptome is presented as expression in the DEL-1–Fc group relative to the Fc control (log₂ values) (n = 3 biological repeats/group). (A) MA (left) and volcano (right) plots show the distribution of gene expression. (B) Heatmaps show the significantly regulated genes (FDR < 0.05) annotated with ontology terms (Gene Ontology [GO] and PANTHER protein class). (C) Relative mRNA expression of Runx1, Runx3, and Cbfb in Tregs in in vitro culture on day 3 by qPCR (n = 6 replicates; 2 separate cell isolations). (D) MFI of RUNX1 in RUNX1⁺FOXP3⁺ iTregs (n = 6 replicates; 2 separate cell isolations). (E and F) Naive splenic CD4⁺ cells from CD4-Cre⁺ Runx1^fl/fl mice or CD4-Cre^– Runx1^fl/fl littermate controls were differentiated, or not, into Tregs in the presence of Fc control or DEL-1–Fc for 4 days. (E) FACS plots and (F) percentage of FOXP3⁺ cells in CD4⁺ T cells from the in vitro culture system on day 4 (n = 6 replicates; 2 separate cell isolations). (G–L) Groups of littermate Del1^WT CD4-Cre⁺ Runx1^fl/fl and Del1^KO CD4-Cre⁺ Runx1^fl/fl mice were subjected to ligature-induced periodontitis for 10 days and ligatures were removed on day 10 for 5 days. FACS plots of Tregs (top) and Th17 cells (bottom) in gingival tissues (G) and cLNs (J) and percentages and absolute numbers of Tregs (H and K) and Th17 cells as well as Treg/Th17 cell ratio (I and L) in gingival tissues (H and I) and cLNs (K and L) of littermate Del1^WT CD4-Cre⁺ Runx1^fl/fl and Del1^KO CD4-Cre⁺ Runx1^fl/fl mice on day 15 (n = 6 mice/group). Data are means ± SD and are pooled from 2 independent experiments. ****P < 0.0001 by 1-way ANOVA with Dunnett’s post hoc test for comparison with Fc control (C) or 2-tailed Student’s t test (D, F, H, I, K, and L). NS, not significant.

Figure 8. DEL-1 increases FOXP3E2 expression and the suppressive capacity of human iTregs.

(A–E) Human Tconv cells were stimulated with anti-CD3/anti-CD28 (0.1 beads/cell) in the presence of DEL-1–Fc or Fc control for 36 hours. (A and C) Representative FACS plots of FOXP3E2⁺ cells (A) or FOXP3⁺ cells (C) in CD4⁺CD25⁺ cells. (B and D) Percentage (left) and MFI (right) of FOXP3E2⁺ cells (B) or FOXP3⁺ cells (D) in CD4⁺CD25⁺ cells (n = 17 from 13 [A and B] or 11 [C and D] independent experiments). (E) Relative mRNA expression of RUNX1 (up) (n = 8 from 8 independent experiments) and CBFB (bottom) (n = 7 from 7 independent experiments) measured at 24 hours of Tconv cell stimulation during iTreg generation. (F–I) CFSE-labeled CD4⁺ T cells were cultured for 72 hours with anti-CD3/anti-CD28 (0.2 beads/cell) alone or in the presence of FACS-isolated DEL-1–Fc–iTregs or Fc control–iTregs (F and G), or long-term-cultured (10 days) DEL-1–Fc–iTregs or Fc control–iTregs (H and I). Representative FACS plots of proliferation of CFSE-labeled CD4⁺ T cells. Numbers in plots indicate percentage of CFSE dilution in CD4⁺ T cells cultured alone (top left); numbers above bracketed lines indicate percentage of CFSE dilution in CD4⁺ T cells cultured with either FACS-isolated DEL-1–Fc–iTregs or Fc control–treated iTregs (F), or long-term-cultured (10 days) DEL-1–Fc–iTregs or Fc control–iTregs (H). Percentage of iTreg suppression in the above conditions (G, n = 29 from 5 independent experiments; I, n = 12 from 4 independent experiments). (J) Methylation of CpG island of FOXP3 CNS2 evaluated in DEL-1–Fc–iTregs or Fc control–iTregs cultured for 10 days in the presence anti-CD3/anti-CD28 (0.1 beads/cell) and IL-2 (50 IU/mL) (n = 6 from 4 independent experiments). (B, D, E, and J) Lines connect paired data for each individual. (G and I) Data are means ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001 vs. Fc control (B, D, E, G, I, and J) by 2-tailed, paired Wilcoxon’s test (B, D, E, G, and I) or 2-tailed, paired Student’s t test (J).