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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 production of Tregs in an inflammatory milieu is dependent on TLR-2 expression in Tregs and APC. (a,b) WT or Myd88−/− Tregs were stimulated with WT APC or Myd88−/− APC as indicated, without or with Pam3CSK4 or fixed Candida albicans (FCA) germ tube for four days. Foxp3 and IL-17A expression (a), and statistical representation of IL-17A expression in Foxp3+ Tregs (b). α-IL-6 antibodies were added under Th17 conditions without exogenous IL-6 (a, bottom panel). (c,d) WT or Tlr-2−/− Tregs were stimulated with Tlr-2−/− APC, without or with Pam3CSK4 or FCA for 4 days. Foxp3 and IL-17A expression in CD4 gated cells (c), and statistical representation of IL-17A expression (d), are shown. Data represent at least 3 independent experiments.
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pathogens-04-00090-f006: IL-17A production of Tregs in an inflammatory milieu is dependent on TLR-2 expression in Tregs and APC. (a,b) WT or Myd88−/− Tregs were stimulated with WT APC or Myd88−/− APC as indicated, without or with Pam3CSK4 or fixed Candida albicans (FCA) germ tube for four days. Foxp3 and IL-17A expression (a), and statistical representation of IL-17A expression in Foxp3+ Tregs (b). α-IL-6 antibodies were added under Th17 conditions without exogenous IL-6 (a, bottom panel). (c,d) WT or Tlr-2−/− Tregs were stimulated with Tlr-2−/− APC, without or with Pam3CSK4 or FCA for 4 days. Foxp3 and IL-17A expression in CD4 gated cells (c), and statistical representation of IL-17A expression (d), are shown. Data represent at least 3 independent experiments.

Mentions: We next investigated the requirement of TLR-2 receptor in induction of IL-17A in Foxp3+Tregs. Myd88 is the critical adaptor molecule downstream of TLR-2 ligand stimulation [35], and therefore we examined whether Myd88 is required for IL-17A up-regulation. We isolated WT or Myd88−/− Tregs and stimulated them under Th17 inducing conditions in the presence of WT APC with or without Pam3CSK4 and formaldehyde fixed Candida albicans hyphae (FCA). We found that the absence of Myd88 signaling in Tregs slightly reduced TLR-2 dependent IL-17A production in the presence of WT APC (Figure 6a, top two panels, 6b). We then stimulated Myd88−/− Tregs with Myd88−/− APC in the presence or absence of TLR-2 ligands. Absence of Myd88 in APC further reduced the IL-17A induction almost to the basal levels in Tregs (Figure 6a, third panel, 6b). Previous studies have shown TLR activation in APC can promote IL-6, a crucial cytokine for IL-17A induction in CD4 T cells [16,19]. Accordingly, when we stimulated Myd88−/− Tregs with WT APC in the presence of α-IL-6 neutralizing antibody, IL-17A was completely abolished in Tregs (Figure 6a, bottom panel, 6b). To further validate the direct TLR-2 signaling requirement in induction of IL-17A in Tregs, independent of TLR-2 expression in APC, we stimulated WT and Tlr-2−/− Tregs with Tlr-2−/− APC under Th17 inducing conditions. Only a fraction of both WT and Tlr-2−/− Tregs produced IL-17A (Figure 6c,d). However, Pam3CSK4 further promoted IL-17A expression only in WT Tregs but not in Tlr-2−/− Tregs. In the presence of FCA, IL-17A induction was only partially abrogated in Tlr-2−/− Tregs, showing that FCA mediated effects were only partially dependent on TLR-2 expression (Figure 6c,d). Taken together, these data show that under Th17 inducing conditions, maximal induction of IL-17A requires both direct and indirect TLR-2/Myd88 signaling on Tregs, as well as APC. IL-6 was a critical factor in the milieu for Tregs to produce IL-17A both in the presence and the absence of TLR-2 ligands.


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 production of Tregs in an inflammatory milieu is dependent on TLR-2 expression in Tregs and APC. (a,b) WT or Myd88−/− Tregs were stimulated with WT APC or Myd88−/− APC as indicated, without or with Pam3CSK4 or fixed Candida albicans (FCA) germ tube for four days. Foxp3 and IL-17A expression (a), and statistical representation of IL-17A expression in Foxp3+ Tregs (b). α-IL-6 antibodies were added under Th17 conditions without exogenous IL-6 (a, bottom panel). (c,d) WT or Tlr-2−/− Tregs were stimulated with Tlr-2−/− APC, without or with Pam3CSK4 or FCA for 4 days. Foxp3 and IL-17A expression in CD4 gated cells (c), and statistical representation of IL-17A expression (d), are shown. Data represent at least 3 independent experiments.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4384074&req=5

pathogens-04-00090-f006: IL-17A production of Tregs in an inflammatory milieu is dependent on TLR-2 expression in Tregs and APC. (a,b) WT or Myd88−/− Tregs were stimulated with WT APC or Myd88−/− APC as indicated, without or with Pam3CSK4 or fixed Candida albicans (FCA) germ tube for four days. Foxp3 and IL-17A expression (a), and statistical representation of IL-17A expression in Foxp3+ Tregs (b). α-IL-6 antibodies were added under Th17 conditions without exogenous IL-6 (a, bottom panel). (c,d) WT or Tlr-2−/− Tregs were stimulated with Tlr-2−/− APC, without or with Pam3CSK4 or FCA for 4 days. Foxp3 and IL-17A expression in CD4 gated cells (c), and statistical representation of IL-17A expression (d), are shown. Data represent at least 3 independent experiments.
Mentions: We next investigated the requirement of TLR-2 receptor in induction of IL-17A in Foxp3+Tregs. Myd88 is the critical adaptor molecule downstream of TLR-2 ligand stimulation [35], and therefore we examined whether Myd88 is required for IL-17A up-regulation. We isolated WT or Myd88−/− Tregs and stimulated them under Th17 inducing conditions in the presence of WT APC with or without Pam3CSK4 and formaldehyde fixed Candida albicans hyphae (FCA). We found that the absence of Myd88 signaling in Tregs slightly reduced TLR-2 dependent IL-17A production in the presence of WT APC (Figure 6a, top two panels, 6b). We then stimulated Myd88−/− Tregs with Myd88−/− APC in the presence or absence of TLR-2 ligands. Absence of Myd88 in APC further reduced the IL-17A induction almost to the basal levels in Tregs (Figure 6a, third panel, 6b). Previous studies have shown TLR activation in APC can promote IL-6, a crucial cytokine for IL-17A induction in CD4 T cells [16,19]. Accordingly, when we stimulated Myd88−/− Tregs with WT APC in the presence of α-IL-6 neutralizing antibody, IL-17A was completely abolished in Tregs (Figure 6a, bottom panel, 6b). To further validate the direct TLR-2 signaling requirement in induction of IL-17A in Tregs, independent of TLR-2 expression in APC, we stimulated WT and Tlr-2−/− Tregs with Tlr-2−/− APC under Th17 inducing conditions. Only a fraction of both WT and Tlr-2−/− Tregs produced IL-17A (Figure 6c,d). However, Pam3CSK4 further promoted IL-17A expression only in WT Tregs but not in Tlr-2−/− Tregs. In the presence of FCA, IL-17A induction was only partially abrogated in Tlr-2−/− Tregs, showing that FCA mediated effects were only partially dependent on TLR-2 expression (Figure 6c,d). Taken together, these data show that under Th17 inducing conditions, maximal induction of IL-17A requires both direct and indirect TLR-2/Myd88 signaling on Tregs, as well as APC. IL-6 was a critical factor in the milieu for Tregs to produce IL-17A both in the presence and the absence of TLR-2 ligands.

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