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Expression of leukosialin (CD43) defines a major intrahepatic T cell subset associated with protective responses in visceral leishmaniasis.

Nico D, Maran N, Santos L, Ramos-Junior ES, Mantuano NR, Coutinho JL, Vale AM, Freire-de-Lima CG, Todeschini A, Rodrigues JC, Palatnik-de-Sousa CB, Morrot A - Parasit Vectors (2015)

Bottom Line: IFN-γ-producing T cells are involved in protection against the disease.We further showed that expression of CD43 defines a major intrahepatic CD4⁺ and CD8⁺ T cell subsets with pro-inflammatory phenotypes and leads to increased levels of IFN-γ secretion by activated splenocytes.Our findings point to a role of CD43 in the development of host resistance to visceral leishmaniasis.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Imunologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro (UFRJ), CCS - Sala D1-035, Av. Carlos Chagas Filho, 373 - Cidade Universitária, CEP 21.941-902, Ilha do Fundão, Rio de Janeiro, RJ, Brazil. morrot@micro.ufrj.br.

ABSTRACT

Background: Leishmaniasis is a neglected vector-borne tropical disease caused by Leishmania protozoa that are transmitted to mammalian hosts by infected sand flies. Infection is associated with distinct clinical manifestations that include cutaneous, mucocutaneous and visceral lesions. Visceral leishmaniasis (VL) is the most severe form of the disease and is considered second in terms of mortality and fourth in terms of morbidity among tropical diseases. IFN-γ-producing T cells are involved in protection against the disease.

Methods: CD43⁺/⁺ and CD43⁻/⁻ mice on a C57BL/6 background were intravenously injected with 5 × 10 ⁷ amastigotes of Leishmania (L.) infantum chagasi, and 30 days after infection the clinical signs of disease were examined; the splenocytes were isolated and assayed for cytokine production; and the livers were removed for phenotypic analysis of T cell subsets by flow cytometry.

Results: We report that mice lacking CD43 display increased susceptibility to infection by Leishmania (L.) infantum chagasi, with higher parasite burdens than wild-type mice. The increased susceptibility of CD43⁻/⁻ mice were associated with a weakened delayed hypersensitivity response and reduced levels of IgG2a antibodies to leishmania antigens. We further showed that expression of CD43 defines a major intrahepatic CD4⁺ and CD8⁺ T cell subsets with pro-inflammatory phenotypes and leads to increased levels of IFN-γ secretion by activated splenocytes.

Conclusions: Our findings point to a role of CD43 in the development of host resistance to visceral leishmaniasis.

No MeSH data available.


Related in: MedlinePlus

Intrahepatic CD43+CD4+and CD43+CD4+T cells exibit a pro-inflammatory phenotype in visceral leishmaniasis. Livers were removed from CD43+/+ wild-type mice infected with 5 × 107 amastigotes of Leishmania (L.) infantum chagasi at 30 DPI and CD43+CD4+ and CD43+CD8+ T cell subsets were purified by flow cytometry. To measure cytokine transcripts, total mRNA from highly purified (>98%) T cell subsets (as indicated) were processed for RT-PCR. The results represent (A,F) IFN-γ transcripts, (B,G) TNF-α transcripts, (C,H) IL-17 transcripts, (D,I) IL-10 transcripts, and (E,J) TGF-β transcripts, standardized with the housekeeping gene GAPDH, calculated as 2-ΔCt and presented as mean values ± SE. All experiments were performed in triplicate and the data shown are representatives of three independent experiments using five mice per group. Indicated differences between groups are significant *(p<0.05), **(p<0.01), ***(p<0.001).
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Fig5: Intrahepatic CD43+CD4+and CD43+CD4+T cells exibit a pro-inflammatory phenotype in visceral leishmaniasis. Livers were removed from CD43+/+ wild-type mice infected with 5 × 107 amastigotes of Leishmania (L.) infantum chagasi at 30 DPI and CD43+CD4+ and CD43+CD8+ T cell subsets were purified by flow cytometry. To measure cytokine transcripts, total mRNA from highly purified (>98%) T cell subsets (as indicated) were processed for RT-PCR. The results represent (A,F) IFN-γ transcripts, (B,G) TNF-α transcripts, (C,H) IL-17 transcripts, (D,I) IL-10 transcripts, and (E,J) TGF-β transcripts, standardized with the housekeeping gene GAPDH, calculated as 2-ΔCt and presented as mean values ± SE. All experiments were performed in triplicate and the data shown are representatives of three independent experiments using five mice per group. Indicated differences between groups are significant *(p<0.05), **(p<0.01), ***(p<0.001).

Mentions: Since the CD43 signaling pathway is involved in the T-bet-dependent expression of IFN-γ by type-1 T cells, we investigated the cytokine profiles of the T cell subsets based on the expression of the CD43 marker. Immunosuppression of antigen-specific immune responses due to impairment of DC activation, and CD4+ and CD8+ T cell exhaustion has been demonstrated in visceral leishmaniasis [24-26]. Indeed we found in our model that splenic T cells obtained from wild-type infected mice at 30 DPI secreted neither IFN-γ nor TNF-α upon polyclonal stimulation with an anti-CD3 stimulus (Additional file 1: Figure S1). Therefore, in order to assess the polarization profiles of the intrahepatic T cell subsets during infection we measured changes in the levels of cytokine transcripts. Total RNA was isolated from CD4+ and CD8+ T cells sorted by FACS based on the expression of the CD43 marker and purified from the livers of wild-type mice at 30 days post-infection. Real-time RT-PCR analysis revealed a significantly higher level of induction of the pro-inflammatory cytokines IFN-γ and TNF-α in the CD43+ subset than in the CD43− population (Figure 5). At the same time we detected a greater reduction of IL-17 and the regulatory TGF-β and IL-10 cytokines in the CD43+ T cell subsets, suggesting a role of CD43+ T cells in inflammatory responses during infection (Figure 5).Figure 5


Expression of leukosialin (CD43) defines a major intrahepatic T cell subset associated with protective responses in visceral leishmaniasis.

Nico D, Maran N, Santos L, Ramos-Junior ES, Mantuano NR, Coutinho JL, Vale AM, Freire-de-Lima CG, Todeschini A, Rodrigues JC, Palatnik-de-Sousa CB, Morrot A - Parasit Vectors (2015)

Intrahepatic CD43+CD4+and CD43+CD4+T cells exibit a pro-inflammatory phenotype in visceral leishmaniasis. Livers were removed from CD43+/+ wild-type mice infected with 5 × 107 amastigotes of Leishmania (L.) infantum chagasi at 30 DPI and CD43+CD4+ and CD43+CD8+ T cell subsets were purified by flow cytometry. To measure cytokine transcripts, total mRNA from highly purified (>98%) T cell subsets (as indicated) were processed for RT-PCR. The results represent (A,F) IFN-γ transcripts, (B,G) TNF-α transcripts, (C,H) IL-17 transcripts, (D,I) IL-10 transcripts, and (E,J) TGF-β transcripts, standardized with the housekeeping gene GAPDH, calculated as 2-ΔCt and presented as mean values ± SE. All experiments were performed in triplicate and the data shown are representatives of three independent experiments using five mice per group. Indicated differences between groups are significant *(p<0.05), **(p<0.01), ***(p<0.001).
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Fig5: Intrahepatic CD43+CD4+and CD43+CD4+T cells exibit a pro-inflammatory phenotype in visceral leishmaniasis. Livers were removed from CD43+/+ wild-type mice infected with 5 × 107 amastigotes of Leishmania (L.) infantum chagasi at 30 DPI and CD43+CD4+ and CD43+CD8+ T cell subsets were purified by flow cytometry. To measure cytokine transcripts, total mRNA from highly purified (>98%) T cell subsets (as indicated) were processed for RT-PCR. The results represent (A,F) IFN-γ transcripts, (B,G) TNF-α transcripts, (C,H) IL-17 transcripts, (D,I) IL-10 transcripts, and (E,J) TGF-β transcripts, standardized with the housekeeping gene GAPDH, calculated as 2-ΔCt and presented as mean values ± SE. All experiments were performed in triplicate and the data shown are representatives of three independent experiments using five mice per group. Indicated differences between groups are significant *(p<0.05), **(p<0.01), ***(p<0.001).
Mentions: Since the CD43 signaling pathway is involved in the T-bet-dependent expression of IFN-γ by type-1 T cells, we investigated the cytokine profiles of the T cell subsets based on the expression of the CD43 marker. Immunosuppression of antigen-specific immune responses due to impairment of DC activation, and CD4+ and CD8+ T cell exhaustion has been demonstrated in visceral leishmaniasis [24-26]. Indeed we found in our model that splenic T cells obtained from wild-type infected mice at 30 DPI secreted neither IFN-γ nor TNF-α upon polyclonal stimulation with an anti-CD3 stimulus (Additional file 1: Figure S1). Therefore, in order to assess the polarization profiles of the intrahepatic T cell subsets during infection we measured changes in the levels of cytokine transcripts. Total RNA was isolated from CD4+ and CD8+ T cells sorted by FACS based on the expression of the CD43 marker and purified from the livers of wild-type mice at 30 days post-infection. Real-time RT-PCR analysis revealed a significantly higher level of induction of the pro-inflammatory cytokines IFN-γ and TNF-α in the CD43+ subset than in the CD43− population (Figure 5). At the same time we detected a greater reduction of IL-17 and the regulatory TGF-β and IL-10 cytokines in the CD43+ T cell subsets, suggesting a role of CD43+ T cells in inflammatory responses during infection (Figure 5).Figure 5

Bottom Line: IFN-γ-producing T cells are involved in protection against the disease.We further showed that expression of CD43 defines a major intrahepatic CD4⁺ and CD8⁺ T cell subsets with pro-inflammatory phenotypes and leads to increased levels of IFN-γ secretion by activated splenocytes.Our findings point to a role of CD43 in the development of host resistance to visceral leishmaniasis.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Imunologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro (UFRJ), CCS - Sala D1-035, Av. Carlos Chagas Filho, 373 - Cidade Universitária, CEP 21.941-902, Ilha do Fundão, Rio de Janeiro, RJ, Brazil. morrot@micro.ufrj.br.

ABSTRACT

Background: Leishmaniasis is a neglected vector-borne tropical disease caused by Leishmania protozoa that are transmitted to mammalian hosts by infected sand flies. Infection is associated with distinct clinical manifestations that include cutaneous, mucocutaneous and visceral lesions. Visceral leishmaniasis (VL) is the most severe form of the disease and is considered second in terms of mortality and fourth in terms of morbidity among tropical diseases. IFN-γ-producing T cells are involved in protection against the disease.

Methods: CD43⁺/⁺ and CD43⁻/⁻ mice on a C57BL/6 background were intravenously injected with 5 × 10 ⁷ amastigotes of Leishmania (L.) infantum chagasi, and 30 days after infection the clinical signs of disease were examined; the splenocytes were isolated and assayed for cytokine production; and the livers were removed for phenotypic analysis of T cell subsets by flow cytometry.

Results: We report that mice lacking CD43 display increased susceptibility to infection by Leishmania (L.) infantum chagasi, with higher parasite burdens than wild-type mice. The increased susceptibility of CD43⁻/⁻ mice were associated with a weakened delayed hypersensitivity response and reduced levels of IgG2a antibodies to leishmania antigens. We further showed that expression of CD43 defines a major intrahepatic CD4⁺ and CD8⁺ T cell subsets with pro-inflammatory phenotypes and leads to increased levels of IFN-γ secretion by activated splenocytes.

Conclusions: Our findings point to a role of CD43 in the development of host resistance to visceral leishmaniasis.

No MeSH data available.


Related in: MedlinePlus