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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-Ξ³t 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

Pam3CSK4 and HKCA induce proliferation of Tregs in a TLR-2 dependent manner. (a) Live cell counts (PIneg and FSChigh) of Treg cells from WT (grey) or Tlr-2βˆ’/βˆ’ (black) mice stimulated with various TLR-2 ligands for three days as in β€œ2a”. CD4+CD25+Foxp3+ (b), or CD4+ Foxp3+Nrp1+ (d), frequencies in WT (left) and Tlr-2βˆ’/βˆ’ (right) cells isolated ex vivo. (c) Statistical analyses showing Treg frequencies in WT and Tlr-2βˆ’/βˆ’ mice, as determined in β€œb”.
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pathogens-04-00090-f003: Pam3CSK4 and HKCA induce proliferation of Tregs in a TLR-2 dependent manner. (a) Live cell counts (PIneg and FSChigh) of Treg cells from WT (grey) or Tlr-2βˆ’/βˆ’ (black) mice stimulated with various TLR-2 ligands for three days as in β€œ2a”. CD4+CD25+Foxp3+ (b), or CD4+ Foxp3+Nrp1+ (d), frequencies in WT (left) and Tlr-2βˆ’/βˆ’ (right) cells isolated ex vivo. (c) Statistical analyses showing Treg frequencies in WT and Tlr-2βˆ’/βˆ’ mice, as determined in β€œb”.

Mentions: We then sought to confirm that proliferation induced by TLR-2 ligands was dependent on TLR-2 expression in Tregs. We isolated Tregs from the spleens of WT or Tlr-2βˆ’/βˆ’ mice and stimulated with IL-2 and TLR-2 ligands. On day 3 after stimulation, HKCA and Pam3CSK4 increased the Treg cell numbers in WT Tregs but not in Tlr-2βˆ’/βˆ’ Tregs, showing that they induced proliferation in TLR-2 dependent manner in Tregs (Figure 3a). If TLR-2 signaling induced proliferation of Tregs in the absence of TCR ligation or inflammatory cytokines, TLR-2 agonists in gut commensal microbes and other endogenous ligands may also promote Treg proliferation under homeostatic conditions in vivo. To test this idea, we examined the frequency of CD4+CD25+Foxp3+ Tregs in various organs, including SPLN, MLN, payer’s patches (PP), gut lamina propria (LP), and the gut intra epithelial lymphocytes (IEL) in WT and Tlr-2βˆ’/βˆ’ mice, using flow cytometry. We found that the frequency of CD4+CD25+Foxp3+ cells was significantly reduced in Tlr-2βˆ’/βˆ’ mice when compared to the WT mice (Figure 3b,c). A previous study showed that the frequency of CD4+CD25+ cells was lower in the peripheral blood of Tlr-2βˆ’/βˆ’ mice than in WT mice [21]. However, neither Foxp3 expression nor the frequency of thymically derived Tregs (tTregs) and peripheral Tregs (pTregs) was examined. Therefore we evaluated the expression of neuropilin-1 (Nrp-1), a marker on tTregs in peripheral organs and mucosal tissues. The frequency of both the Treg populations were decreased slightly in SPLN, but dramatically in CLN, MLN, PP and LP of Tlr-2βˆ’/βˆ’ mice, when compared to the WT mice (Figure 3d). Interestingly, in payer’s patches and the intestinal lamina propria, the reduction of Tregs in Tlr-2βˆ’/βˆ’ mice was more pronounced in the Nrp-1βˆ’ pTreg compartment (Figure 3d). These results highlight the importance of TLR-2 ligands in promoting tTreg and pTreg homeostatic proliferation under steady-state conditions 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)

Pam3CSK4 and HKCA induce proliferation of Tregs in a TLR-2 dependent manner. (a) Live cell counts (PIneg and FSChigh) of Treg cells from WT (grey) or Tlr-2βˆ’/βˆ’ (black) mice stimulated with various TLR-2 ligands for three days as in β€œ2a”. CD4+CD25+Foxp3+ (b), or CD4+ Foxp3+Nrp1+ (d), frequencies in WT (left) and Tlr-2βˆ’/βˆ’ (right) cells isolated ex vivo. (c) Statistical analyses showing Treg frequencies in WT and Tlr-2βˆ’/βˆ’ mice, as determined in β€œb”.
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Related In: Results  -  Collection

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

pathogens-04-00090-f003: Pam3CSK4 and HKCA induce proliferation of Tregs in a TLR-2 dependent manner. (a) Live cell counts (PIneg and FSChigh) of Treg cells from WT (grey) or Tlr-2βˆ’/βˆ’ (black) mice stimulated with various TLR-2 ligands for three days as in β€œ2a”. CD4+CD25+Foxp3+ (b), or CD4+ Foxp3+Nrp1+ (d), frequencies in WT (left) and Tlr-2βˆ’/βˆ’ (right) cells isolated ex vivo. (c) Statistical analyses showing Treg frequencies in WT and Tlr-2βˆ’/βˆ’ mice, as determined in β€œb”.
Mentions: We then sought to confirm that proliferation induced by TLR-2 ligands was dependent on TLR-2 expression in Tregs. We isolated Tregs from the spleens of WT or Tlr-2βˆ’/βˆ’ mice and stimulated with IL-2 and TLR-2 ligands. On day 3 after stimulation, HKCA and Pam3CSK4 increased the Treg cell numbers in WT Tregs but not in Tlr-2βˆ’/βˆ’ Tregs, showing that they induced proliferation in TLR-2 dependent manner in Tregs (Figure 3a). If TLR-2 signaling induced proliferation of Tregs in the absence of TCR ligation or inflammatory cytokines, TLR-2 agonists in gut commensal microbes and other endogenous ligands may also promote Treg proliferation under homeostatic conditions in vivo. To test this idea, we examined the frequency of CD4+CD25+Foxp3+ Tregs in various organs, including SPLN, MLN, payer’s patches (PP), gut lamina propria (LP), and the gut intra epithelial lymphocytes (IEL) in WT and Tlr-2βˆ’/βˆ’ mice, using flow cytometry. We found that the frequency of CD4+CD25+Foxp3+ cells was significantly reduced in Tlr-2βˆ’/βˆ’ mice when compared to the WT mice (Figure 3b,c). A previous study showed that the frequency of CD4+CD25+ cells was lower in the peripheral blood of Tlr-2βˆ’/βˆ’ mice than in WT mice [21]. However, neither Foxp3 expression nor the frequency of thymically derived Tregs (tTregs) and peripheral Tregs (pTregs) was examined. Therefore we evaluated the expression of neuropilin-1 (Nrp-1), a marker on tTregs in peripheral organs and mucosal tissues. The frequency of both the Treg populations were decreased slightly in SPLN, but dramatically in CLN, MLN, PP and LP of Tlr-2βˆ’/βˆ’ mice, when compared to the WT mice (Figure 3d). Interestingly, in payer’s patches and the intestinal lamina propria, the reduction of Tregs in Tlr-2βˆ’/βˆ’ mice was more pronounced in the Nrp-1βˆ’ pTreg compartment (Figure 3d). These results highlight the importance of TLR-2 ligands in promoting tTreg and pTreg homeostatic proliferation under steady-state conditions 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-Ξ³t 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