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, 6 (4), 234-42

A Commensal Symbiotic Factor Derived From Bacteroides Fragilis Promotes Human CD39(+)Foxp3(+) T Cells and Treg Function

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A Commensal Symbiotic Factor Derived From Bacteroides Fragilis Promotes Human CD39(+)Foxp3(+) T Cells and Treg Function

Kiel M Telesford et al. Gut Microbes.

Abstract

Polysaccharide A (PSA) derived from the human commensal Bacteroides fragilis is a symbiosis factor that stimulates immunologic development within mammalian hosts. PSA rebalances skewed systemic T helper responses and promotes T regulatory cells (Tregs). However, PSA-mediated induction of Foxp3 in humans has not been reported. In mice, PSA-generated Foxp3(+) Tregs dampen Th17 activity thereby facilitating bacterial intestinal colonization while the increased presence and function of these regulatory cells may guard against pathological organ-specific inflammation in hosts. We herein demonstrate that PSA induces expression of Foxp3 along with CD39 among naïve CD4 T cells in vitro while promoting IL-10 secretion. PSA-activated dendritic cells are essential for the mediation of this regulatory response. When cultured with isolated Foxp3(+) Tregs, PSA enriched Foxp3 expression, enhanced the frequency of CD39(+)HLA-DR(+) cells, and increased suppressive function as measured by decreased TNFα expression by LPS-stimulated monocytes. Our findings are the first to demonstrate in vitro induction of human CD4(+)Foxp3(+) T cells and enhanced suppressive function of circulating Foxp3(+) Tregs by a human commensal bacterial symbiotic factor. Use of PSA for the treatment of human autoimmune diseases, in particular multiple sclerosis and inflammatory bowel disease, may represent a new paradigm in the approach to treating autoimmune disease.

Keywords: B. fragilis, Bacteroides fragilis; Bacteroides fragilis; DC, Dendritic cell; Foxp3; GF, Germ Free; MS, Multiple sclerosis; NCD4, Naïve CD4; PBMCs, Peripheral blood mononuclear cells; PSA, Polysaccharide A; SPF, Specific pathogen free; Sp1, Streptococcus pneumoniae polysaccharide type 1; T regulatory cells; Treg, T regulatory cell; ZPS, Zwitterionic polysaccharide.; autoimmunity; commensal microbiota; dendritic cell; ectonuclease; multiple sclerosis; pDC, Plasmacytoid dendritic cell; zwitterionic polysaccharide.

Figures

Figure 1.
Figure 1.
Dendritic Cells mediate PSA induction of Human CD39+Foxp3+ CD4+ T cells. PSA-mediated induction of CD39+Foxp3+ T cells was observed in the presence of DCs but not other APCs. 3 × 104 NCD4s were cultured in the presence or absence of 25 µg/ml PSA or Sp1 and 100 U/ml IL-2, alongside 5 × 103 of one of 3 primary autologous APC populations: CD19+ B cells, CD14+ monocytes, or total blood dendritic cells. (A) Fold induction of Foxp3+ frequency alone or in the presence of autologous DCs, n = 4. (B) Representative histograms showing frequency of Foxp3+ cells (red line) as an overlay against isotype control (shaded blue), panels were pre-gated on CD4+ cells. (C) Replicates of CD4+Foxp3+ frequency, n = 3. (D) Representative FACS plots showing frequency of CD25 and CD39 cells, pre-gated on Foxp3+ cells. Positive cells (red line) are overlaid against respective isotype controls (shaded blue). (E) PSA dose response of CD4+CD39+Foxp3+ T cells, n = 1 (Bars represent means of 4 culture wells per condition, error bars represent standard error of the mean). (F) Individual repeats of CD39+Foxp3+ T cells, n = 4–14 per condition. P values were calculated using 2-tailed Student's t-test (A, C) or one-way ANOVA using Dunnett's multiple comparison test (E, F).
Figure 2.
Figure 2.
PSA induction of CD39+Foxp3+ T cells requires engagement of HLA-DR and costimulatory molecules. Blocking HLA-DR or costimulatory molecules limited PSA-mediated induction of CD39+Foxp3+ T cells and cytokine production. (A) 5 × 104 DCs were cultured with 25 µg/ml PSA for approximately 16 hours before being stained for surface expression of the above markers, n = 3–6. DCs were incubated with 10 µg/ml of blocking antibody specific to CD86, PD-L1, HLA-DR or CD40L for 30 min prior to co-culture with 3 × 104 autologous NCD4s in the presence or absence of 25 µg/ml PSA and 100 U/ml IL-2. Supernatants were assessed by ELISA for IL-10 or IFNγ. (B) CD4+CD39+Foxp3+ T cell frequency, n = 6–10 per condition (C) IL-10 and IFNγ production, n = 4–10 per condition. Error bars reflect standard error of the mean representing independent experiments, each using cells from different individuals. P values were calculated using 2-tailed Student's t-test (A) or one-way ANOVA using Dunnett's multiple comparison test (B, C).
Figure 3.
Figure 3.
PSA increases the frequency of CD39-expressing Tregs and Treg function. PSA exposure significantly enhanced the frequency of CD39 and HLA-DR-expressing circulating Tregs and resulted in greater capacity to suppress monocyte secretion of TNFα. 1 × 104 Tregs were cultured in the presence or absence of PSA and 100 U/ml IL-2, alongside 5 × 103 autologous DCs. (A) Representative histograms of different Treg-associated markers. Positive cells (red line) are overlaid against respective isotype controls (shaded blue) (B) CD39 MFI, n = 11 (left), representative CD39+Foxp3+FACS panel pre-gated on CD4+ cells (center), replicates of CD39+Foxp3+Treg frequency, n = 12 (right). (C) HLA-DR MFI, n = 7 (left), Representative CD39+HLA-DR+ Treg FACS panel pre-gated on CD4+Foxp3+ cells (center), replicates of CD39+ HLA-DR+ Treg frequency, n = 7 (right). (D) Foxp3 MFI of CD4+ Tregs, n = 12 (E) Suppression of LPS-induced TNFα production, n = 3. Error bars reflect standard error of the mean representing independent experiments, each using cells from different individuals. P values were calculated using 2-tailed Student's t-test (B–D) or one-way ANOVA using Dunnett's multiple comparison test (E).

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