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T cell-intrinsic role of Nod2 in promoting type 1 immunity to Toxoplasma gondii.

Shaw MH, Reimer T, Sánchez-Valdepeñas C, Warner N, Kim YG, Fresno M, Nuñez G - Nat. Immunol. (2009)

Bottom Line: Nod2 deficiency results in an impaired immune response to bacterial pathogens.Nod2(-/-) CD4(+) T cells had poor helper T cell differentiation, which was associated with impaired production of interleukin 2 (IL-2) and nuclear accumulation of the transcription factor subunit c-Rel.Our data demonstrate a T cell-intrinsic role for Nod2 signaling that is critical for host defense against T. gondii.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan, USA.

ABSTRACT
Nod2 belongs to the nucleotide-binding oligomerization domain receptor (NLR) family of proteins, which function as intracellular pathogen sensors in innate immune cells. Nod2 deficiency results in an impaired immune response to bacterial pathogens. However, how this protein promotes host defense against intracellular parasites is unknown. Here we found that Nod2(-/-) mice had less clearance of Toxoplasma gondii and lower interferon-gamma (IFN-gamma) production. Reconstitution of T cell-deficient mice with Nod2(-/-) T cells followed by T. gondii infection demonstrated a T cell-intrinsic defect. Nod2(-/-) CD4(+) T cells had poor helper T cell differentiation, which was associated with impaired production of interleukin 2 (IL-2) and nuclear accumulation of the transcription factor subunit c-Rel. Our data demonstrate a T cell-intrinsic role for Nod2 signaling that is critical for host defense against T. gondii.

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Nod2 interacts with c-Rel and enhances Il2 transcription(a) HEK293T cells were transiently transfected with 1μg of the indicated expression plasmids. Cells were harvested after 48 h and whole cell lysates were immunoprecipitated with antibodies against NIK or HA (Nod2) (left) or Flag (Nod2 or CARMA1) (right). Bcl10 was used as a specificity control. The immunoprecipitates were subjected to SDS-PAGE and immunoblotting with the indicated antibodies. (b) Jurkat cells were transiently transfected with a luciferase reporter driven by a CD28 response element, along with equivalent amounts of Nod2, NIK, and c-Rel expression constructs as indicated. The cells were stimulated with anti-CD3 and anti-CD28 and the average relative luciferase activity is shown (±s.d.). A renilla luciferase reporter construct was used to normalize for transfection efficiency and the results are representative of three independent experiments. (c) Nuclear and cytosolic fractions from purified T cells stimulated with plate-bound α-CD3 and α-CD28 for 1–9 h were subjected to SDS-PAGE, followed by immunoblotting with the indicated antibodies. The results are representative of three similar experiments.
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Figure 8: Nod2 interacts with c-Rel and enhances Il2 transcription(a) HEK293T cells were transiently transfected with 1μg of the indicated expression plasmids. Cells were harvested after 48 h and whole cell lysates were immunoprecipitated with antibodies against NIK or HA (Nod2) (left) or Flag (Nod2 or CARMA1) (right). Bcl10 was used as a specificity control. The immunoprecipitates were subjected to SDS-PAGE and immunoblotting with the indicated antibodies. (b) Jurkat cells were transiently transfected with a luciferase reporter driven by a CD28 response element, along with equivalent amounts of Nod2, NIK, and c-Rel expression constructs as indicated. The cells were stimulated with anti-CD3 and anti-CD28 and the average relative luciferase activity is shown (±s.d.). A renilla luciferase reporter construct was used to normalize for transfection efficiency and the results are representative of three independent experiments. (c) Nuclear and cytosolic fractions from purified T cells stimulated with plate-bound α-CD3 and α-CD28 for 1–9 h were subjected to SDS-PAGE, followed by immunoblotting with the indicated antibodies. The results are representative of three similar experiments.

Mentions: The blunted LIP, T cell-driven colitis and IL-2 secretion by Nod2−/− T cells suggests that Nod2 may function downstream of either TCR or co-stimulatory signals. As TCR-driven phosphorylation of Jnk, p38 and Erk MAP kinases was similar in wild-type and Nod2−/− T cells (Supplementary Figure 5), we investigated whether Nod2 is involved in the CD28 costimulatory signaling pathway. The production of IL-2 in response to CD28 co-stimulation depends on the activation of the NF-κB subunit, c-Rel, which interacts with the NF-κB binding sites as well as CD28-responsive elements (CD28RE) in the proximal Il2 promoter region15, 16. The relevance of c-Rel for IL-2 production is underscored by the observation that T cells from c-Rel-deficient mice exhibit a profound deficiency in IL-2 production17. The ability of c-Rel to interact with the CD28RE to induce IL-2 production is regulated by NF-κB inducing kinase, NIK18 (also called MAP3KI4). T cells from alymphoplasia (aly/aly) mice that bear a NIK mutation also exhibit defects in IL-2 secretion in response to CD3 and CD28 stimulation19. In light of these findings, we hypothesized that Nod2 may interact with c-Rel and/or NIK in T lymphocytes to promote IL-2 production. To determine if Nod2 can interact with c-Rel or NIK, HEK293T cells were transiently transfected with expression vectors encoding c-Rel, NIK and/or Nod2. As previously reported, NIK coimmunoprecipitated with Nod2 (Figure 8a)20. Interestingly, c-Rel was also present in Nod2 immunoprecipitates (Figure 8a). When Nod2, NIK and c-Rel were simultaneously coexpressed, both NIK and c-Rel coimmunoprecipitated with Nod2 (Figure 8a), suggesting that these three proteins can form a tertiary complex. As a specificity control, Bcl10, which is known to play a role in T cell receptor signaling, associated with CARMA1 as previously reported21, 22; however, no interaction was observed between Bcl10 and Nod2 (Figure 8a)


T cell-intrinsic role of Nod2 in promoting type 1 immunity to Toxoplasma gondii.

Shaw MH, Reimer T, Sánchez-Valdepeñas C, Warner N, Kim YG, Fresno M, Nuñez G - Nat. Immunol. (2009)

Nod2 interacts with c-Rel and enhances Il2 transcription(a) HEK293T cells were transiently transfected with 1μg of the indicated expression plasmids. Cells were harvested after 48 h and whole cell lysates were immunoprecipitated with antibodies against NIK or HA (Nod2) (left) or Flag (Nod2 or CARMA1) (right). Bcl10 was used as a specificity control. The immunoprecipitates were subjected to SDS-PAGE and immunoblotting with the indicated antibodies. (b) Jurkat cells were transiently transfected with a luciferase reporter driven by a CD28 response element, along with equivalent amounts of Nod2, NIK, and c-Rel expression constructs as indicated. The cells were stimulated with anti-CD3 and anti-CD28 and the average relative luciferase activity is shown (±s.d.). A renilla luciferase reporter construct was used to normalize for transfection efficiency and the results are representative of three independent experiments. (c) Nuclear and cytosolic fractions from purified T cells stimulated with plate-bound α-CD3 and α-CD28 for 1–9 h were subjected to SDS-PAGE, followed by immunoblotting with the indicated antibodies. The results are representative of three similar experiments.
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Related In: Results  -  Collection

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Figure 8: Nod2 interacts with c-Rel and enhances Il2 transcription(a) HEK293T cells were transiently transfected with 1μg of the indicated expression plasmids. Cells were harvested after 48 h and whole cell lysates were immunoprecipitated with antibodies against NIK or HA (Nod2) (left) or Flag (Nod2 or CARMA1) (right). Bcl10 was used as a specificity control. The immunoprecipitates were subjected to SDS-PAGE and immunoblotting with the indicated antibodies. (b) Jurkat cells were transiently transfected with a luciferase reporter driven by a CD28 response element, along with equivalent amounts of Nod2, NIK, and c-Rel expression constructs as indicated. The cells were stimulated with anti-CD3 and anti-CD28 and the average relative luciferase activity is shown (±s.d.). A renilla luciferase reporter construct was used to normalize for transfection efficiency and the results are representative of three independent experiments. (c) Nuclear and cytosolic fractions from purified T cells stimulated with plate-bound α-CD3 and α-CD28 for 1–9 h were subjected to SDS-PAGE, followed by immunoblotting with the indicated antibodies. The results are representative of three similar experiments.
Mentions: The blunted LIP, T cell-driven colitis and IL-2 secretion by Nod2−/− T cells suggests that Nod2 may function downstream of either TCR or co-stimulatory signals. As TCR-driven phosphorylation of Jnk, p38 and Erk MAP kinases was similar in wild-type and Nod2−/− T cells (Supplementary Figure 5), we investigated whether Nod2 is involved in the CD28 costimulatory signaling pathway. The production of IL-2 in response to CD28 co-stimulation depends on the activation of the NF-κB subunit, c-Rel, which interacts with the NF-κB binding sites as well as CD28-responsive elements (CD28RE) in the proximal Il2 promoter region15, 16. The relevance of c-Rel for IL-2 production is underscored by the observation that T cells from c-Rel-deficient mice exhibit a profound deficiency in IL-2 production17. The ability of c-Rel to interact with the CD28RE to induce IL-2 production is regulated by NF-κB inducing kinase, NIK18 (also called MAP3KI4). T cells from alymphoplasia (aly/aly) mice that bear a NIK mutation also exhibit defects in IL-2 secretion in response to CD3 and CD28 stimulation19. In light of these findings, we hypothesized that Nod2 may interact with c-Rel and/or NIK in T lymphocytes to promote IL-2 production. To determine if Nod2 can interact with c-Rel or NIK, HEK293T cells were transiently transfected with expression vectors encoding c-Rel, NIK and/or Nod2. As previously reported, NIK coimmunoprecipitated with Nod2 (Figure 8a)20. Interestingly, c-Rel was also present in Nod2 immunoprecipitates (Figure 8a). When Nod2, NIK and c-Rel were simultaneously coexpressed, both NIK and c-Rel coimmunoprecipitated with Nod2 (Figure 8a), suggesting that these three proteins can form a tertiary complex. As a specificity control, Bcl10, which is known to play a role in T cell receptor signaling, associated with CARMA1 as previously reported21, 22; however, no interaction was observed between Bcl10 and Nod2 (Figure 8a)

Bottom Line: Nod2 deficiency results in an impaired immune response to bacterial pathogens.Nod2(-/-) CD4(+) T cells had poor helper T cell differentiation, which was associated with impaired production of interleukin 2 (IL-2) and nuclear accumulation of the transcription factor subunit c-Rel.Our data demonstrate a T cell-intrinsic role for Nod2 signaling that is critical for host defense against T. gondii.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan, USA.

ABSTRACT
Nod2 belongs to the nucleotide-binding oligomerization domain receptor (NLR) family of proteins, which function as intracellular pathogen sensors in innate immune cells. Nod2 deficiency results in an impaired immune response to bacterial pathogens. However, how this protein promotes host defense against intracellular parasites is unknown. Here we found that Nod2(-/-) mice had less clearance of Toxoplasma gondii and lower interferon-gamma (IFN-gamma) production. Reconstitution of T cell-deficient mice with Nod2(-/-) T cells followed by T. gondii infection demonstrated a T cell-intrinsic defect. Nod2(-/-) CD4(+) T cells had poor helper T cell differentiation, which was associated with impaired production of interleukin 2 (IL-2) and nuclear accumulation of the transcription factor subunit c-Rel. Our data demonstrate a T cell-intrinsic role for Nod2 signaling that is critical for host defense against T. gondii.

Show MeSH
Related in: MedlinePlus