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TLR-2 Signaling Promotes IL-17A Production in CD4+CD25+Foxp3+ Regulatory Cells during Oropharyngeal Candidiasis.

Bhaskaran N, Cohen S, Zhang Y, Weinberg A, Pandiyan P - Pathogens (2015)

Bottom Line: Here we show that CD4+Foxp3+Tregs produce the effector cytokine IL-17A during oropharyngeal candidiasis (OPC) and inflammatory bowel disease in a TLR-2/Myd88 signaling dependent manner.The minimal and transient loss of Foxp3 expression and suppressive properties are due to the presence of IL-6 in the milieu, but not the direct effect of TLR-2 signaling in Tregs.These IL-17A producing Tregs may be relevant in mucosal infections and inflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, School of Dental Medicine, Case Western Reserve University, Cleveland, OH 44106, USA. nxb160@case.edu.

ABSTRACT
Recent studies show that CD4+CD25+Foxp3+ regulatory cells (Tregs) produce effector cytokines under inflammatory conditions. However, the direct role of microbial agents that serve as toll-like receptor (TLR) ligands in the induction of effector cytokines in Tregs is less clear. Here we show that CD4+Foxp3+Tregs produce the effector cytokine IL-17A during oropharyngeal candidiasis (OPC) and inflammatory bowel disease in a TLR-2/Myd88 signaling dependent manner. TLR-2 ligands promote proliferation in Tregs in the presence and absence of TCR signals and inflammatory cytokines in vitro. The proliferation is directly dependent on TLR-2 expression in Tregs. Consistent with this, Tlr2-/- mice harbor fewer thymically derived Tregs and peripheral Tregs under homeostatic conditions in vivo. However, under Th17 inducing conditions, IL-6 and TLR-2 signaling both in Tregs as well as antigen presenting cells (APC) are critical for maximal ROR-γt and IL-17A up-regulation in Foxp3+ Tregs. The minimal and transient loss of Foxp3 expression and suppressive properties are due to the presence of IL-6 in the milieu, but not the direct effect of TLR-2 signaling in Tregs. Taken together, our data reveal that TLR-2 signaling promotes not only proliferation, but also IL-17A in Tregs, depending on the cytokine milieu. These IL-17A producing Tregs may be relevant in mucosal infections and inflammation.

No MeSH data available.


Related in: MedlinePlus

IL-17A expression in CD4+ Foxp3+ Tregs requires TLR-2 signaling in Tregs during C. albicans infection in vivo. WT C57BL/6 or Tlr-2−/− mice infected with C. albicans, and tissues were isolated as in “1a”. Flow cytometric plots gated on CD4+ cells showing IL-17A expression (a), and statistical analyses of IL-17A expression in CD4+Foxp3+ cells (b) are shown. Data represent triplicate experiments. (c,d) Foxp3YFPcre or Myd88 fl°x Foxp3YFPcre mice were infected, and cells were isolated as in “1a”. Flow cytometric plots gated on or CD4+ cells showing IL-17A expression (c), and statistical analyses of IL-17A expression in CD4+Foxp3-cells (d, upper panel) and CD4+Foxp3+ cells (d, lower panel) are shown.
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pathogens-04-00090-f007: IL-17A expression in CD4+ Foxp3+ Tregs requires TLR-2 signaling in Tregs during C. albicans infection in vivo. WT C57BL/6 or Tlr-2−/− mice infected with C. albicans, and tissues were isolated as in “1a”. Flow cytometric plots gated on CD4+ cells showing IL-17A expression (a), and statistical analyses of IL-17A expression in CD4+Foxp3+ cells (b) are shown. Data represent triplicate experiments. (c,d) Foxp3YFPcre or Myd88 fl°x Foxp3YFPcre mice were infected, and cells were isolated as in “1a”. Flow cytometric plots gated on or CD4+ cells showing IL-17A expression (c), and statistical analyses of IL-17A expression in CD4+Foxp3-cells (d, upper panel) and CD4+Foxp3+ cells (d, lower panel) are shown.

Mentions: Lastly, we examined the requirement of TLR-2 expression for inducing IL-17A in Tregs during OPC in vivo. Although TLR-2 signaling promotes Treg proliferation during systemic candidiasis, IL-17A induction was not assessed in Tregs during the infection [21]. To determine the role of TLR-2 signaling in induction of IL-17A in Foxp3+Tregs in vivo, we orally infected WT and Tlr-2−/− mice and measured IL-17A production in CD4+ T cells. We found reduced frequency of Tregs in Tlr-2−/− mice compared to WT mice infiltrating the draining lymphnodes and MOIL during infection (Figure 7a). Importantly, among those Foxp3+CD4+ Tregs the frequency of IL-17A producers was at least three to five times decreased in Tlr-2−/− mice than in WT mice (Figure 7a,b). These data show that TLR-2 signaling enhances proliferation and maximal IL-17A induction in Tregs. Consistent to the role of TLR-2 signaling in promoting IL-17A in effector CD4 cells [14], we also found reduced IL-17A production among Foxp3-effector CD4+ cells in Tlr-2−/− mice, when compared to the WT mice (Figure 7a). To further confirm the direct role of TLR signaling in Tregs, we used Myd88fl°x Foxp3YFPcre mice, in which Myd88 gene was conditionally deleted in Foxp3+ cells. Compared to the control Foxp3YFPcre infected mice, the induction of IL-17A was significantly reduced in Myd88fl°x Foxp3YFPcre mice in Foxp3+ cells (Figure 7c,d lower panel). Although there was a slight decrease in IL-17A production even in the effector cells in MOIL, the frequency of IL-17A producing effector cells was unchanged between the groups in CLN and ALN (Figure 7c,d upper panel). Taken together, these results reveal that direct TLR-2 signaling in Tregs plays a crucial role in increasing IL-17A+Foxp3+ Tregs during OPC in vivo.


TLR-2 Signaling Promotes IL-17A Production in CD4+CD25+Foxp3+ Regulatory Cells during Oropharyngeal Candidiasis.

Bhaskaran N, Cohen S, Zhang Y, Weinberg A, Pandiyan P - Pathogens (2015)

IL-17A expression in CD4+ Foxp3+ Tregs requires TLR-2 signaling in Tregs during C. albicans infection in vivo. WT C57BL/6 or Tlr-2−/− mice infected with C. albicans, and tissues were isolated as in “1a”. Flow cytometric plots gated on CD4+ cells showing IL-17A expression (a), and statistical analyses of IL-17A expression in CD4+Foxp3+ cells (b) are shown. Data represent triplicate experiments. (c,d) Foxp3YFPcre or Myd88 fl°x Foxp3YFPcre mice were infected, and cells were isolated as in “1a”. Flow cytometric plots gated on or CD4+ cells showing IL-17A expression (c), and statistical analyses of IL-17A expression in CD4+Foxp3-cells (d, upper panel) and CD4+Foxp3+ cells (d, lower panel) are shown.
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getmorefigures.php?uid=PMC4384074&req=5

pathogens-04-00090-f007: IL-17A expression in CD4+ Foxp3+ Tregs requires TLR-2 signaling in Tregs during C. albicans infection in vivo. WT C57BL/6 or Tlr-2−/− mice infected with C. albicans, and tissues were isolated as in “1a”. Flow cytometric plots gated on CD4+ cells showing IL-17A expression (a), and statistical analyses of IL-17A expression in CD4+Foxp3+ cells (b) are shown. Data represent triplicate experiments. (c,d) Foxp3YFPcre or Myd88 fl°x Foxp3YFPcre mice were infected, and cells were isolated as in “1a”. Flow cytometric plots gated on or CD4+ cells showing IL-17A expression (c), and statistical analyses of IL-17A expression in CD4+Foxp3-cells (d, upper panel) and CD4+Foxp3+ cells (d, lower panel) are shown.
Mentions: Lastly, we examined the requirement of TLR-2 expression for inducing IL-17A in Tregs during OPC in vivo. Although TLR-2 signaling promotes Treg proliferation during systemic candidiasis, IL-17A induction was not assessed in Tregs during the infection [21]. To determine the role of TLR-2 signaling in induction of IL-17A in Foxp3+Tregs in vivo, we orally infected WT and Tlr-2−/− mice and measured IL-17A production in CD4+ T cells. We found reduced frequency of Tregs in Tlr-2−/− mice compared to WT mice infiltrating the draining lymphnodes and MOIL during infection (Figure 7a). Importantly, among those Foxp3+CD4+ Tregs the frequency of IL-17A producers was at least three to five times decreased in Tlr-2−/− mice than in WT mice (Figure 7a,b). These data show that TLR-2 signaling enhances proliferation and maximal IL-17A induction in Tregs. Consistent to the role of TLR-2 signaling in promoting IL-17A in effector CD4 cells [14], we also found reduced IL-17A production among Foxp3-effector CD4+ cells in Tlr-2−/− mice, when compared to the WT mice (Figure 7a). To further confirm the direct role of TLR signaling in Tregs, we used Myd88fl°x Foxp3YFPcre mice, in which Myd88 gene was conditionally deleted in Foxp3+ cells. Compared to the control Foxp3YFPcre infected mice, the induction of IL-17A was significantly reduced in Myd88fl°x Foxp3YFPcre mice in Foxp3+ cells (Figure 7c,d lower panel). Although there was a slight decrease in IL-17A production even in the effector cells in MOIL, the frequency of IL-17A producing effector cells was unchanged between the groups in CLN and ALN (Figure 7c,d upper panel). Taken together, these results reveal that direct TLR-2 signaling in Tregs plays a crucial role in increasing IL-17A+Foxp3+ Tregs during OPC in vivo.

Bottom Line: Here we show that CD4+Foxp3+Tregs produce the effector cytokine IL-17A during oropharyngeal candidiasis (OPC) and inflammatory bowel disease in a TLR-2/Myd88 signaling dependent manner.The minimal and transient loss of Foxp3 expression and suppressive properties are due to the presence of IL-6 in the milieu, but not the direct effect of TLR-2 signaling in Tregs.These IL-17A producing Tregs may be relevant in mucosal infections and inflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, School of Dental Medicine, Case Western Reserve University, Cleveland, OH 44106, USA. nxb160@case.edu.

ABSTRACT
Recent studies show that CD4+CD25+Foxp3+ regulatory cells (Tregs) produce effector cytokines under inflammatory conditions. However, the direct role of microbial agents that serve as toll-like receptor (TLR) ligands in the induction of effector cytokines in Tregs is less clear. Here we show that CD4+Foxp3+Tregs produce the effector cytokine IL-17A during oropharyngeal candidiasis (OPC) and inflammatory bowel disease in a TLR-2/Myd88 signaling dependent manner. TLR-2 ligands promote proliferation in Tregs in the presence and absence of TCR signals and inflammatory cytokines in vitro. The proliferation is directly dependent on TLR-2 expression in Tregs. Consistent with this, Tlr2-/- mice harbor fewer thymically derived Tregs and peripheral Tregs under homeostatic conditions in vivo. However, under Th17 inducing conditions, IL-6 and TLR-2 signaling both in Tregs as well as antigen presenting cells (APC) are critical for maximal ROR-γt and IL-17A up-regulation in Foxp3+ Tregs. The minimal and transient loss of Foxp3 expression and suppressive properties are due to the presence of IL-6 in the milieu, but not the direct effect of TLR-2 signaling in Tregs. Taken together, our data reveal that TLR-2 signaling promotes not only proliferation, but also IL-17A in Tregs, depending on the cytokine milieu. These IL-17A producing Tregs may be relevant in mucosal infections and inflammation.

No MeSH data available.


Related in: MedlinePlus