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TLR-4 cooperates with Dectin-1 and mannose receptor to expand Th17 and Tc17 cells induced by Paracoccidioides brasiliensis stimulated dendritic cells.

Loures FV, Araújo EF, Feriotti C, Bazan SB, Calich VL - Front Microbiol (2015)

Bottom Line: The concomitant use of diverse pattern recognition receptors (PRRs) by innate immune cells can result in synergistic or inhibitory activities that profoundly influence anti-microbial immunity.In addition, treatment of WT, TLR-2(-/-), and TLR-4(-/-) DCs by anti-Dectin-1 antibodies or antigen presentation by Dectin-1(-/-) DCs led to decreased lymphoproliferation and impaired Th17 and Tc17 expansion.These responses were also inhibited by anti-MR treatment of DCs, but a synergistic action on Th17/Tc17 differentiation was mediated by TLR-4 and MR.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo , São Paulo, Brazil.

ABSTRACT
The concomitant use of diverse pattern recognition receptors (PRRs) by innate immune cells can result in synergistic or inhibitory activities that profoundly influence anti-microbial immunity. Dectin-1 and the mannose receptor (MR) are C-type lectin receptors (CLRs) previously reported to cooperate with toll-like receptors (TLRs) signaling in the initial inflammatory response and in the induction of adaptive Th17 and Tc17 immunity mediated by CD4(+) and CD8(+) T cells, respectively. The protective immunity against paracoccidioidomycosis, the most prevalent fungal infection of Latin America, was previously shown to be influenced by these T cell subsets motivating us to study the contribution of TLRs, Dectin-1, and MR to the development of Th17/Tc17 immunity. First, curdlan a specific Dectin-1 agonist was used to characterize the influence of this receptor in the proliferative response and Th17/Tc17 differentiation of naïve lymphocytes induced by Paracoccidioides brasiliensis activated dendritic cells (DCs) from C57BL/6 mice. Then, wild type (WT), Dectin-1(-/-), TLR-2(-/-), and TLR-4(-/-) DCs treated or untreated with anti-Dectin-1 and anti-MR antibodies were used to investigate the contribution of these receptors in lymphocyte activation and differentiation. We verified that curdlan induces an enhanced lymphocyte proliferation and development of IL-17 producing CD4(+) and CD8(+) T cells. In addition, treatment of WT, TLR-2(-/-), and TLR-4(-/-) DCs by anti-Dectin-1 antibodies or antigen presentation by Dectin-1(-/-) DCs led to decreased lymphoproliferation and impaired Th17 and Tc17 expansion. These responses were also inhibited by anti-MR treatment of DCs, but a synergistic action on Th17/Tc17 differentiation was mediated by TLR-4 and MR. Taken together, our results indicate that diverse TLRs and CLRs are involved in the induction of lymphocyte proliferation and Th17/Tc17 differentiation mediated by P. brasiliensis activated DCs, but a synergist action was restricted to Dectin-1, TLR-4, and MR.

No MeSH data available.


Related in: MedlinePlus

Influence of Dectin-1 receptor in the lymphocyte proliferation, activation and development of IL-17 producing T cells. BMDCs from WT and Dectin-1–/– C57BL/6 mice were cultured as described and then infected with viable P. brasiliensis yeasts for 2 h. Splenic lymphocytes from uninfected WT and Dectin-1–/– mice were previously labeled with CFSE (5 mM) and co-cultivated with DCs. After 5 days, the cells were adjusted to 1 × 106, labeled with specific anti-CD4, CD8, CD25, and CD69 antibodies and analyzed by flow cytometry. (A) Proliferative response of CD4+ and CD8+ T cells. (B,C) The leukocyte population was gated by FSC/SSC analysis and gated cells were analyzed for CD4+CD25+(B) and CD8+CD69+(C) expression. Some cultures were re-stimulated with PMA/ionomycin for 6 h and subjected to intracellular staining for IL-17. The results are expressed as frequency of IL-17-positive cells (D). Data are means ± SD of five wells per group from three independent experiments (*P < 0.05).
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Figure 4: Influence of Dectin-1 receptor in the lymphocyte proliferation, activation and development of IL-17 producing T cells. BMDCs from WT and Dectin-1–/– C57BL/6 mice were cultured as described and then infected with viable P. brasiliensis yeasts for 2 h. Splenic lymphocytes from uninfected WT and Dectin-1–/– mice were previously labeled with CFSE (5 mM) and co-cultivated with DCs. After 5 days, the cells were adjusted to 1 × 106, labeled with specific anti-CD4, CD8, CD25, and CD69 antibodies and analyzed by flow cytometry. (A) Proliferative response of CD4+ and CD8+ T cells. (B,C) The leukocyte population was gated by FSC/SSC analysis and gated cells were analyzed for CD4+CD25+(B) and CD8+CD69+(C) expression. Some cultures were re-stimulated with PMA/ionomycin for 6 h and subjected to intracellular staining for IL-17. The results are expressed as frequency of IL-17-positive cells (D). Data are means ± SD of five wells per group from three independent experiments (*P < 0.05).

Mentions: To better evaluate the role of Dectin-1 in the activation of lymphocytes and differentiation of Th17/Tc17 cells, Dectin-1–/– and WT mice were used in co-culture experiments as described above. Thus, BMDCs from WT and Dectin-1–/– mice were infected with viable P. brasiliensis yeast cells for 2 h. In parallel, splenic lymphocytes from uninfected WT and Dectin-1–/– mice were previously labeled or not with CFSE and co-cultured with P. brasiliensis-infected DCs. After 5 days, lymphocytes were analyzed by flow cytometry. Lower proliferative responses of CD8+ T cells were observed in Dectin-1–/– cultures in comparison with Dectin-1-sufficient WT cultures. No differences in the proliferation of CD4+ T cells were observed between the two analyzed groups (Figure 4A). Using Dectin-1–/– cells, a lower frequency of CD4+, CD4+CD25+, and CD8+CD69+ T lymphocytes (Figures 4B,C) was observed when compared with WT cells. Furthermore, a lower frequency of IL-17-producing CD4+ and CD8+ T cells was expanded in Dectin-1–/– cultures (Figure 4D). Combined, our data indicate that Dectin-1 is important for the proliferation of T cells and the induction of Th17/Tc17 cells by P. brasiliensis-infected DCs. Furthermore, even in the absence of Dectin-1 expression, some Th17/Tc17 cells were expanded, indicating that other PRRs may be involved in this response. This led us to investigate the cooperation among TLR-2, TLR-4, Dectin-1, and MR in the activation of T cells and differentiation of IL-17-producing cells.


TLR-4 cooperates with Dectin-1 and mannose receptor to expand Th17 and Tc17 cells induced by Paracoccidioides brasiliensis stimulated dendritic cells.

Loures FV, Araújo EF, Feriotti C, Bazan SB, Calich VL - Front Microbiol (2015)

Influence of Dectin-1 receptor in the lymphocyte proliferation, activation and development of IL-17 producing T cells. BMDCs from WT and Dectin-1–/– C57BL/6 mice were cultured as described and then infected with viable P. brasiliensis yeasts for 2 h. Splenic lymphocytes from uninfected WT and Dectin-1–/– mice were previously labeled with CFSE (5 mM) and co-cultivated with DCs. After 5 days, the cells were adjusted to 1 × 106, labeled with specific anti-CD4, CD8, CD25, and CD69 antibodies and analyzed by flow cytometry. (A) Proliferative response of CD4+ and CD8+ T cells. (B,C) The leukocyte population was gated by FSC/SSC analysis and gated cells were analyzed for CD4+CD25+(B) and CD8+CD69+(C) expression. Some cultures were re-stimulated with PMA/ionomycin for 6 h and subjected to intracellular staining for IL-17. The results are expressed as frequency of IL-17-positive cells (D). Data are means ± SD of five wells per group from three independent experiments (*P < 0.05).
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Figure 4: Influence of Dectin-1 receptor in the lymphocyte proliferation, activation and development of IL-17 producing T cells. BMDCs from WT and Dectin-1–/– C57BL/6 mice were cultured as described and then infected with viable P. brasiliensis yeasts for 2 h. Splenic lymphocytes from uninfected WT and Dectin-1–/– mice were previously labeled with CFSE (5 mM) and co-cultivated with DCs. After 5 days, the cells were adjusted to 1 × 106, labeled with specific anti-CD4, CD8, CD25, and CD69 antibodies and analyzed by flow cytometry. (A) Proliferative response of CD4+ and CD8+ T cells. (B,C) The leukocyte population was gated by FSC/SSC analysis and gated cells were analyzed for CD4+CD25+(B) and CD8+CD69+(C) expression. Some cultures were re-stimulated with PMA/ionomycin for 6 h and subjected to intracellular staining for IL-17. The results are expressed as frequency of IL-17-positive cells (D). Data are means ± SD of five wells per group from three independent experiments (*P < 0.05).
Mentions: To better evaluate the role of Dectin-1 in the activation of lymphocytes and differentiation of Th17/Tc17 cells, Dectin-1–/– and WT mice were used in co-culture experiments as described above. Thus, BMDCs from WT and Dectin-1–/– mice were infected with viable P. brasiliensis yeast cells for 2 h. In parallel, splenic lymphocytes from uninfected WT and Dectin-1–/– mice were previously labeled or not with CFSE and co-cultured with P. brasiliensis-infected DCs. After 5 days, lymphocytes were analyzed by flow cytometry. Lower proliferative responses of CD8+ T cells were observed in Dectin-1–/– cultures in comparison with Dectin-1-sufficient WT cultures. No differences in the proliferation of CD4+ T cells were observed between the two analyzed groups (Figure 4A). Using Dectin-1–/– cells, a lower frequency of CD4+, CD4+CD25+, and CD8+CD69+ T lymphocytes (Figures 4B,C) was observed when compared with WT cells. Furthermore, a lower frequency of IL-17-producing CD4+ and CD8+ T cells was expanded in Dectin-1–/– cultures (Figure 4D). Combined, our data indicate that Dectin-1 is important for the proliferation of T cells and the induction of Th17/Tc17 cells by P. brasiliensis-infected DCs. Furthermore, even in the absence of Dectin-1 expression, some Th17/Tc17 cells were expanded, indicating that other PRRs may be involved in this response. This led us to investigate the cooperation among TLR-2, TLR-4, Dectin-1, and MR in the activation of T cells and differentiation of IL-17-producing cells.

Bottom Line: The concomitant use of diverse pattern recognition receptors (PRRs) by innate immune cells can result in synergistic or inhibitory activities that profoundly influence anti-microbial immunity.In addition, treatment of WT, TLR-2(-/-), and TLR-4(-/-) DCs by anti-Dectin-1 antibodies or antigen presentation by Dectin-1(-/-) DCs led to decreased lymphoproliferation and impaired Th17 and Tc17 expansion.These responses were also inhibited by anti-MR treatment of DCs, but a synergistic action on Th17/Tc17 differentiation was mediated by TLR-4 and MR.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo , São Paulo, Brazil.

ABSTRACT
The concomitant use of diverse pattern recognition receptors (PRRs) by innate immune cells can result in synergistic or inhibitory activities that profoundly influence anti-microbial immunity. Dectin-1 and the mannose receptor (MR) are C-type lectin receptors (CLRs) previously reported to cooperate with toll-like receptors (TLRs) signaling in the initial inflammatory response and in the induction of adaptive Th17 and Tc17 immunity mediated by CD4(+) and CD8(+) T cells, respectively. The protective immunity against paracoccidioidomycosis, the most prevalent fungal infection of Latin America, was previously shown to be influenced by these T cell subsets motivating us to study the contribution of TLRs, Dectin-1, and MR to the development of Th17/Tc17 immunity. First, curdlan a specific Dectin-1 agonist was used to characterize the influence of this receptor in the proliferative response and Th17/Tc17 differentiation of naïve lymphocytes induced by Paracoccidioides brasiliensis activated dendritic cells (DCs) from C57BL/6 mice. Then, wild type (WT), Dectin-1(-/-), TLR-2(-/-), and TLR-4(-/-) DCs treated or untreated with anti-Dectin-1 and anti-MR antibodies were used to investigate the contribution of these receptors in lymphocyte activation and differentiation. We verified that curdlan induces an enhanced lymphocyte proliferation and development of IL-17 producing CD4(+) and CD8(+) T cells. In addition, treatment of WT, TLR-2(-/-), and TLR-4(-/-) DCs by anti-Dectin-1 antibodies or antigen presentation by Dectin-1(-/-) DCs led to decreased lymphoproliferation and impaired Th17 and Tc17 expansion. These responses were also inhibited by anti-MR treatment of DCs, but a synergistic action on Th17/Tc17 differentiation was mediated by TLR-4 and MR. Taken together, our results indicate that diverse TLRs and CLRs are involved in the induction of lymphocyte proliferation and Th17/Tc17 differentiation mediated by P. brasiliensis activated DCs, but a synergist action was restricted to Dectin-1, TLR-4, and MR.

No MeSH data available.


Related in: MedlinePlus