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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

TLR-2 ligands induce proliferation but not plasticity in Tregs stimulated under Th17 conditions. CPD-670 dilution (proliferation) (a), or Foxp3 expression (b), of Treg cells co-cultured with conventional CD4+ T cells under Th17 conditions for four days, with or without Pam3CSK4, HKCA, or by themselves without (d4-no IL-2) or with IL-2 (d4-IL-2). CD45.2 Tregs are gated in the analyses. (c) Tregs were stimulated as in “a”. Foxp3 and IL-17A expression of cells gated on CPD670+CD45.2 Tregs (upper panel), or CPD670-Foxp3-CD45.1 Teff (lower panel) in co-cultures. (d) Foxp3 and CD4 expression (upper panel), of naïve cells that were stimulated under Th0 (Th0) or Th17 (Th17) conditions, or Tregs that were stimulated under Th17 (Th17 Treg) conditions. Foxp3 and IL-17A expression (lower panel), of the Foxp3+ cells in the upper panel. (e) Proliferation suppression of the CD4 responder cells (as in “2c”) co-cultured with GFP+ Treg cells isolated from cultures stimulated as in “a”. Data represent three independent experiments.
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pathogens-04-00090-f004: TLR-2 ligands induce proliferation but not plasticity in Tregs stimulated under Th17 conditions. CPD-670 dilution (proliferation) (a), or Foxp3 expression (b), of Treg cells co-cultured with conventional CD4+ T cells under Th17 conditions for four days, with or without Pam3CSK4, HKCA, or by themselves without (d4-no IL-2) or with IL-2 (d4-IL-2). CD45.2 Tregs are gated in the analyses. (c) Tregs were stimulated as in “a”. Foxp3 and IL-17A expression of cells gated on CPD670+CD45.2 Tregs (upper panel), or CPD670-Foxp3-CD45.1 Teff (lower panel) in co-cultures. (d) Foxp3 and CD4 expression (upper panel), of naïve cells that were stimulated under Th0 (Th0) or Th17 (Th17) conditions, or Tregs that were stimulated under Th17 (Th17 Treg) conditions. Foxp3 and IL-17A expression (lower panel), of the Foxp3+ cells in the upper panel. (e) Proliferation suppression of the CD4 responder cells (as in “2c”) co-cultured with GFP+ Treg cells isolated from cultures stimulated as in “a”. Data represent three independent experiments.

Mentions: We then sought to investigate whether TLR-2 ligands may promote IL-17A in Tregs stimulated under Th17 inducing conditions in vitro. Although TLR-2 ligands have been shown to reduce Foxp3 expression and suppressive properties in Tregs [15], whether IL-17A is induced in Tregs is unknown. However, Strober and colleagues have shown that Tregs produce IL-17A in Th17 inducing conditions in vitro [32]. Therefore, we stimulated Tregs in co-cultures along with Tcons at a ratio of 1:10 under Th17 inducing conditions. Tcon cells proliferated in to Th17 effectors (Teff). As controls, we stimulated Tregs in Th0 conditions. To distinguish Tregs and Teff in co-cultures, we labeled Tregs using CPD670 in addition to CD45.2 congenic marker on Tregs in co-cultures (Figure S3a). We gated on CPD670+CD45.2+Tregs (Figure S3a), and examined their proliferation, Foxp3 expression and IL-17A expression. Four days after stimulation, Pam3CSK4 and HKCA increased the proliferation of Tregs (Figure 4a). Although ~20% of the cells lost Foxp3 expression completely, many of these cells were non-proliferating Tregs, which did not dilute CPD670 (Figure S3b). These cells were found in the presence and the absence of TLR-2 ligands (Figure S3b), likely due to the fact that IL-6 that can transiently reprogram Tregs to lose Foxp3 expression [22,33]. However, when proliferating Tregs were examined, TLR-2 ligands did not lead to reduction in Foxp3 expression (Figure 4b). As expected, in the absence of IL-2, Tregs showed poor survivability and reduced Foxp3 expression ([30], Figure 4b). We also observed that a small fraction of the Foxp3+ cells (~3%) consistently expressed IL-17A (Figure 4c). However, TLR-2 ligands increased the frequency and the levels of IL-17A substantially (Figure 4c, upper panel). We did not observe IL-17A expression in control Th0 stimulated Tregs (Figure 4c, upper panel). To verify the specificity of the staining, we used Tregs from Il17a-/-cre mice, which barely showed IL-17A expression (Figure S4). Under Th0 and Th17 polarizing conditions, a small fraction of Teffs also induced Foxp3 expression (iTregs) (Figure 4d, upper panel). iTregs that were induced in Th0 conditions did not express IL-17A, while iTregs induced under Th17 polarizing conditions up-regulated IL-17A, similar to natural Tregs (Figure 4d). These data show that both in vitro induced iTregs and natural Tregs isolated from mice are capable of producing IL-17A under Th17 inducing conditions.


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)

TLR-2 ligands induce proliferation but not plasticity in Tregs stimulated under Th17 conditions. CPD-670 dilution (proliferation) (a), or Foxp3 expression (b), of Treg cells co-cultured with conventional CD4+ T cells under Th17 conditions for four days, with or without Pam3CSK4, HKCA, or by themselves without (d4-no IL-2) or with IL-2 (d4-IL-2). CD45.2 Tregs are gated in the analyses. (c) Tregs were stimulated as in “a”. Foxp3 and IL-17A expression of cells gated on CPD670+CD45.2 Tregs (upper panel), or CPD670-Foxp3-CD45.1 Teff (lower panel) in co-cultures. (d) Foxp3 and CD4 expression (upper panel), of naïve cells that were stimulated under Th0 (Th0) or Th17 (Th17) conditions, or Tregs that were stimulated under Th17 (Th17 Treg) conditions. Foxp3 and IL-17A expression (lower panel), of the Foxp3+ cells in the upper panel. (e) Proliferation suppression of the CD4 responder cells (as in “2c”) co-cultured with GFP+ Treg cells isolated from cultures stimulated as in “a”. Data represent three 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-f004: TLR-2 ligands induce proliferation but not plasticity in Tregs stimulated under Th17 conditions. CPD-670 dilution (proliferation) (a), or Foxp3 expression (b), of Treg cells co-cultured with conventional CD4+ T cells under Th17 conditions for four days, with or without Pam3CSK4, HKCA, or by themselves without (d4-no IL-2) or with IL-2 (d4-IL-2). CD45.2 Tregs are gated in the analyses. (c) Tregs were stimulated as in “a”. Foxp3 and IL-17A expression of cells gated on CPD670+CD45.2 Tregs (upper panel), or CPD670-Foxp3-CD45.1 Teff (lower panel) in co-cultures. (d) Foxp3 and CD4 expression (upper panel), of naïve cells that were stimulated under Th0 (Th0) or Th17 (Th17) conditions, or Tregs that were stimulated under Th17 (Th17 Treg) conditions. Foxp3 and IL-17A expression (lower panel), of the Foxp3+ cells in the upper panel. (e) Proliferation suppression of the CD4 responder cells (as in “2c”) co-cultured with GFP+ Treg cells isolated from cultures stimulated as in “a”. Data represent three independent experiments.
Mentions: We then sought to investigate whether TLR-2 ligands may promote IL-17A in Tregs stimulated under Th17 inducing conditions in vitro. Although TLR-2 ligands have been shown to reduce Foxp3 expression and suppressive properties in Tregs [15], whether IL-17A is induced in Tregs is unknown. However, Strober and colleagues have shown that Tregs produce IL-17A in Th17 inducing conditions in vitro [32]. Therefore, we stimulated Tregs in co-cultures along with Tcons at a ratio of 1:10 under Th17 inducing conditions. Tcon cells proliferated in to Th17 effectors (Teff). As controls, we stimulated Tregs in Th0 conditions. To distinguish Tregs and Teff in co-cultures, we labeled Tregs using CPD670 in addition to CD45.2 congenic marker on Tregs in co-cultures (Figure S3a). We gated on CPD670+CD45.2+Tregs (Figure S3a), and examined their proliferation, Foxp3 expression and IL-17A expression. Four days after stimulation, Pam3CSK4 and HKCA increased the proliferation of Tregs (Figure 4a). Although ~20% of the cells lost Foxp3 expression completely, many of these cells were non-proliferating Tregs, which did not dilute CPD670 (Figure S3b). These cells were found in the presence and the absence of TLR-2 ligands (Figure S3b), likely due to the fact that IL-6 that can transiently reprogram Tregs to lose Foxp3 expression [22,33]. However, when proliferating Tregs were examined, TLR-2 ligands did not lead to reduction in Foxp3 expression (Figure 4b). As expected, in the absence of IL-2, Tregs showed poor survivability and reduced Foxp3 expression ([30], Figure 4b). We also observed that a small fraction of the Foxp3+ cells (~3%) consistently expressed IL-17A (Figure 4c). However, TLR-2 ligands increased the frequency and the levels of IL-17A substantially (Figure 4c, upper panel). We did not observe IL-17A expression in control Th0 stimulated Tregs (Figure 4c, upper panel). To verify the specificity of the staining, we used Tregs from Il17a-/-cre mice, which barely showed IL-17A expression (Figure S4). Under Th0 and Th17 polarizing conditions, a small fraction of Teffs also induced Foxp3 expression (iTregs) (Figure 4d, upper panel). iTregs that were induced in Th0 conditions did not express IL-17A, while iTregs induced under Th17 polarizing conditions up-regulated IL-17A, similar to natural Tregs (Figure 4d). These data show that both in vitro induced iTregs and natural Tregs isolated from mice are capable of producing IL-17A under Th17 inducing conditions.

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