Commensal DNA limits regulatory T cell conversion and is a natural adjuvant of intestinal immune responses - PubMed (original) (raw)

Commensal DNA limits regulatory T cell conversion and is a natural adjuvant of intestinal immune responses

Jason A Hall et al. Immunity. 2008.

Abstract

The intestinal tract is in intimate contact with the commensal microflora. Nevertheless, how commensals communicate with cells to ensure immune homeostasis is still unclear. In this study, we found that gut flora DNA (gfDNA) plays a major role in intestinal homeostasis through Toll-like receptor 9 (TLR9) engagement. Tlr9(-/-) mice displayed increased frequencies of CD4(+)Foxp3(+) regulatory T (Treg) cells within intestinal effector sites and reduced constitutive IL-17- and IFN-gamma-producing effector T (Teff) cells. Complementing this, gfDNA limited lamina propria dendritic cell-induced Treg cell conversion in vitro. Further, Treg/Teff cell disequilibrium in Tlr9(-/-) mice led to impaired immune responses to oral infection and to oral vaccination. Impaired intestinal immune responses were recapitulated in mice treated with antibiotics and were reversible after reconstitution with gfDNA. Together, these data point to gfDNA as a natural adjuvant for priming intestinal responses via modulation of Treg/Teff cell equilibrium.

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Figures

Figure 1

Figure 1. TLR9 signaling regulates Treg frequency and steady-state Teff cytokine production in gut associated lymphoid tissue of uninfected mice

(A)Comparative assessment of CD4+Foxp3+ Treg in spleen, mesenteric lymph node (MLN), intestinal epithelium lymphocyte (IEL), intestinal lamina propria (LP) and Peyer’s patch (PP) compartments in age-matched naïve WT and TLR9−/− mice. CD4+TCR-β+-gated cells were analyzed for expression of Foxp3 and CD25 by flow cytometry. Numbers in quadrants refer to the percentage of each subset. (B) Treg percentages in naïve WT (●) and TLR9−/− (○) mice. Each dot represents the results from one experiment (three mice pooled per group) and crossbars depict the mean of three independent experiments (*, p<0.05 compared with WT mice). (C) Loss of TLR9 reduces the basal frequency of IL-17 and IFN-γ producing CD4+ T cells in the LP and IFN-γ producing CD8α+ T cells in the IEL compartments. Numbers in quadrants refer to the percentage of each subset. (D) Absolute numbers of CD4+ and/or CD8α+ T lymphocytes producing IFN-γ and IL-17 in naïve WT (●) and TLR9−/− (○) mice. Each dot represents one mouse and each bar the mean of three mice analyzed (*, p<0.05; **, p<0.01; ***, p<0.001 compared with WT mice). For C and D, data shown are representative of two independent experiments with similar results.

Figure 2

Figure 2. TLR9−/− mice mount impaired protective responses to E. cuniculi following oral infection and respond poorly to oral vaccination

WT and TLR9−/− mice were infected with E. cuniculi spores i.p. (A–B) or through oral route (C–D). (A and C) ELISA of IFN-γ from i.p. infected and IFN-γ and IL-17 from orally infected mice in supernatants of bulk leukocyte preparations from 11 d post-infected WT and TLR9−/− mice restimulated with uninfected (DC) or _E. cuniculi_-infected BMDC (inf. DC). (B and D) Parasite loads are increased in orally infected TLR9−/− mice. Parasite loads were measured in the duodenum and liver of i.p. (B) or orally (D) infected mice 11 d post-infection by quantitative real-time PCR. Data shown are representative of three independent experiments with five mice per group (**, p<0.01). (E) WT and TLR9−/− mice were orally immunized with a mixture of OVA and the mutant E. coli LT(R129G) on d 0 and d 7, and treated with anti-CD25 mAb or isotype control antibody. On d 14, IFN-γ and IL-17 secretion by LP cells were evaluated by ELISA after in vitro restimulation with BMDC infected with recombinant vaccinia virus expressing OVA. For A, C and E, histograms represent the mean cytokine concentration of triplicate wells ± SD and are representative of at least two independent experiments with similar results (*, p<0.05; **, p<0.01; ***, p<0.001;.n.s., non significant; ND, not detected).

Figure 3

Figure 3. Expression of TLR9 by hematopoietic cells is sufficient to negatively modulate levels of Treg and positively favors immune responses in the GALT

Levels of Foxp3+ Treg at homeostasis (A and B) and immune responses against E. cuniculi at 11 d after oral infection (C) were analyzed in BM chimeric mice, in which the hematopoietic or the nonhematopoietic compartment lacks TLR9 expression. (A) Comparative assessment of CD4+Foxp3+ Treg in the intestinal lamina propria (LP) of naïve irradiated WT and TLR9−/− mice reconstituted with BM cells from WT or TLR9−/− congenic mice. Percentages of CD4+TCR-β+Foxp3+ were evaluated in the donor (CD45.1+) compartment. (B) Foxp3+ Treg percentages in naive irradiated WT or TLR9−/− mice reconstituted with BM cells from WT (●) or TLR9−/− (○) congenic mice. Percentages of CD4+TCR-β+Foxp3+ were evaluated in the donor (CD45.1+) compartment. Each dot represents one mouse and each bar the mean of three mice analyzed. (C) ELISA measurement of IFN-γ production by spleen and IEL compartments after in vitro restimulation with uninfected (DC) or _E. cuniculi_-infected (inf. DC) BMDC. Histograms represent the mean cytokine concentration from triplicate wells ± SD. Data shown are representative of two independent experiments with similar results (*, p<0.05; **, p<0.01; ***, p<0.001; ND, not detected).

Figure 4

Figure 4. Engagement of TLR9 signaling potently limits Treg conversion

FACS-sorted naïve CD4+CD25−CD44loFoxp3− T cells isolated from Foxp3eGFP mice were cultured in Treg polarizing conditions with WT LpDCs in the presence of the indicated TLR ligands. (A) Dot plots gated on viable CD4+7-AAD− T cells illustrate α4β7 versus Foxp3 expression after culture in presence or absence of CpG (10 μg/ml). (B) PGN (TLR2), LPS (TLR4), Flgn (TLR5) or CpG (TLR9) were added in the culture at starting concentrations of 2 μg/ml, 10 μg/ml, 1 μg/ml and 10 μg/ml, respectively. Two subsequent five-fold dilutions of ligand were tested (gray wedge). Results were normalized to conditions plated in the absence of TLR ligands with 100% equaling conversion in Treg polarizing conditions. Cross bars indicate the high and lows of duplicate cultures. (C) CFSE dilution analysis of naïve CD4+CD25−CD44lo T cells isolated from TLR9−/− mice, cultured as in (A) and analyzed for CD4 and Foxp3 expression at the indicated time points. Overlay of the histograms for the CFSE dilution profiles of the CD4+Foxp3− T cells, indicated in the boxed in regions (solid line: control; dotted line: in presence of CpG). Data shown are representative of three independent experiments with similar results.

Figure 5

Figure 5. Engagement of TLR9 limits Treg conversion and enhances Teff activity

(A)5×104 purified LpDCs or SpDCs were stimulated for 18 hrs in the presence or absence of CpG (10 μg/ml) in complete media containing 40 ng/ml of GM-CSF (CM). Recovered supernatant was assessed for IL-6 by ELISA. (B) CD4+CD25−CD44loFoxp3− T cells from TLR9−/− Foxp3eGFP mice were cultured in Treg polarizing conditions in the presence or absence of CpG for 6 days and then restimulated for assessment of intracellular cytokine production. (C–E) Naïve CD4+CD25−CD44lo T cells from Myd88−/− mice were cultured in Treg polarizing conditions for 6 days in the presence or absence of CpG. In wells containing CpG, antibodies to IL-6 and IL-6 receptor α (αIL-6), IL-4 (α-IL-4) and IFN-γ (α-IFN-γ) were added at the start of culture as indicated. (C) Measurement of IL-17 by ELISA for previously described conditions. (D) Dot plots gated on viable CD4+ T cells show the percentages of Foxp3+ cells. (E) Summary of results of D normalized to baseline Treg conversion. Cross bars indicate the high and lows of duplicate cultures.. Data shown are representative of at least three independent experiments with similar results. (F) Blockade of LpDC induced Treg conversion by IL-6, IL-4 and IFN-γ. Naïve CD4+CD25−CD44lo T cells were cocultured with LpDCs in Treg polarizing conditions and various doses of IL-6, IL-4 or IFN-γ or a combination of all 3 cytokines. Cytokine concentration is provided on the x axis. In wells containing the combined cytokines, the same concentration of each cytokine was used. Data are representative of 2 independent experiments.

Figure 6

Figure 6. DNA enriched from the gut flora suppresses Treg conversion in a TLR9 dependent manner

(A) CD4+CD25−CD44lo T cells isolated from WT or TLR9−/− mice were cultured in Treg polarizing conditions with LpDCs from WT or TLR9−/− mice, respectively. In some culture wells, 10μg/ml of CpG, DNA enriched from murine gut flora (gfDNA) or gfDNA formulated with the cationic liposome, DOTAP, was added. CD4, Foxp3 and α4β7 expression was analyzed on day 6. (B) Summary of results from (A) normalized to baseline Treg conversion. Error bars represent the SD of triplicate cultures (**, p<0.01; ***, p<0.001).

Figure 7

Figure 7. Gut floral DNA restores immune responses in antibiotic treated mice orally infected with E. cuniculi

(A) 3 weeks old mice received a cocktail of antibiotics (ATB) in the drinking water for 6 weeks in conjunction with oral weekly treatments of PBS alone or containing 100 μg of CpG, 500 μg gfDNA, or 25 mg/kg of lipopolysaccharide from Escherichia coli (LPS). Control mice received no ATB treatment. At 6 wks post-treatment, mice were infected orally with E. cuniculi. (B) ELISA of IFN-γ and IL-17 in supernatants of bulk leukocyte preparations from IEL and LP of 11 d post-infected WT and TLR9−/− mice restimulated with _E. cuniculi_-infected BMDC for 72 hrs. Histograms represent the mean cytokine concentration ± SD. This experiment is representative of two independent experiments with similar results (*, p<0.05; **, p<0.01; ***, p<0.001). (C) 8 wk-old mice were treated and infected as described in (A) and parasite burden was evaluated in duodenum and liver 11 d post-infection by quantitative real-time PCR. Each dot represents one mouse and each bar the mean of three or four mice analyzed (*, p<0.05; **, p<0.01; ***, p<0.001; n.s., non significant).

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