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Nod2 suppresses Borrelia burgdorferi mediated murine Lyme arthritis and carditis through the induction of tolerance.

Petnicki-Ocwieja T, DeFrancesco AS, Chung E, Darcy CT, Bronson RT, Kobayashi KS, Hu LT - PLoS ONE (2011)

Bottom Line: The internalization of Borrelia burgdorferi, the causative agent of Lyme disease, by phagocytes is essential for an effective activation of the immune response to this pathogen.In vitro stimulation of Nod2 deficient bone marrow derived macrophages (BMDM) resulted in decreased induction of multiple cytokines, interferons and interferon regulated genes compared with wild-type cells.However, B. burgdorferi infection of Nod2 deficient mice resulted in increased rather than decreased arthritis and carditis compared to control mice.

View Article: PubMed Central - PubMed

Affiliation: Division of Geographic Medicine and Infectious Diseases, Tufts Medical Center, Boston, Massachusetts, United States of America.

ABSTRACT
The internalization of Borrelia burgdorferi, the causative agent of Lyme disease, by phagocytes is essential for an effective activation of the immune response to this pathogen. The intracellular, cytosolic receptor Nod2 has been shown to play varying roles in either enhancing or attenuating inflammation in response to different infectious agents. We examined the role of Nod2 in responses to B. burgdorferi. In vitro stimulation of Nod2 deficient bone marrow derived macrophages (BMDM) resulted in decreased induction of multiple cytokines, interferons and interferon regulated genes compared with wild-type cells. However, B. burgdorferi infection of Nod2 deficient mice resulted in increased rather than decreased arthritis and carditis compared to control mice. We explored multiple potential mechanisms for the paradoxical response in in vivo versus in vitro systems and found that prolonged stimulation with a Nod2 ligand, muramyl dipeptide (MDP), resulted in tolerance to stimulation by B. burgdorferi. This tolerance was lost with stimulation of Nod2 deficient cells that cannot respond to MDP. Cytokine patterns in the tolerance model closely paralleled cytokine profiles in infected Nod2 deficient mice. We propose a model where Nod2 has an enhancing role in activating inflammation in early infection, but moderates inflammation after prolonged exposure to the organism through induction of tolerance.

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Nod2 mediates the activation of cytokines in response to B. burgdorferi.BMDM from wild type (B6) and Nod2 deficient mice were stimulated with B. burgdorferi at the designated MOIs for 6 hours. Cytokine levels in supernatants were measures by ELISA; bars represent mean percent change ± s.e.m. of three independent experiments. Values for wild type (B6) cells for each experiment were normalized to 100% and other values are shown relative to B6. Because of the normalization, the s.e.m. for B6 is zero and no error bars are visible, * p<0.05. Raw data for each of the conditions showed that wild type cells secreted a mean of 6.34, 3.51 and 1.8 ng/ml of TNF-α at MOIs of 100, 10 and 1 respectively, while Nod2 deficient cells secreted a mean of 3.52, 2.35 and 1.3 ng/ml at the same MOIs. For IL-6, wild type cells secreted 1.6 ng/ml of IL-6 for MOI 1, while Nod2 deficient cells secreted a mean of 0.58 ng/ml for IL-6 for MOI 1.
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pone-0017414-g005: Nod2 mediates the activation of cytokines in response to B. burgdorferi.BMDM from wild type (B6) and Nod2 deficient mice were stimulated with B. burgdorferi at the designated MOIs for 6 hours. Cytokine levels in supernatants were measures by ELISA; bars represent mean percent change ± s.e.m. of three independent experiments. Values for wild type (B6) cells for each experiment were normalized to 100% and other values are shown relative to B6. Because of the normalization, the s.e.m. for B6 is zero and no error bars are visible, * p<0.05. Raw data for each of the conditions showed that wild type cells secreted a mean of 6.34, 3.51 and 1.8 ng/ml of TNF-α at MOIs of 100, 10 and 1 respectively, while Nod2 deficient cells secreted a mean of 3.52, 2.35 and 1.3 ng/ml at the same MOIs. For IL-6, wild type cells secreted 1.6 ng/ml of IL-6 for MOI 1, while Nod2 deficient cells secreted a mean of 0.58 ng/ml for IL-6 for MOI 1.

Mentions: NLR family members that activate pathways other than the inflammasome include Nod1 and Nod2, both of which signal through the kinase Rip2 for the activation of NF-κB and MAPKs [19], [38]. To determine whether Nod1 or Nod2 might be involved in responses to B. burgdorferi, we stimulated wild type and Rip2 deficient BMDM with B. burgdorferi. Rip2 deficient cells showed a 34% decrease in TNF-α secretion by ELISA compared to wild-type cells (data not shown). Because both Nod1 and Nod2 utilize Rip2, we next sought to determine whether either Nod1 or Nod2 (or both) were involved in cellular responses to B. burgdorferi. Nod1 and Nod2 deficient macrophages were stimulated with B. burgdorferi. Examination of mRNA transcripts for pro-IL1β, IL-6 and TNF-α after 6 hrs of incubation with B. burgdorferi showed decreases of 60–75% in Nod1 and Nod2 deficient macrophages compared to wild type (Figure 4). However, when cell culture supernatants were examined by ELISA, only Nod2 deficient cells showed a significant decrease in TNF-α protein levels (Figure 4) or IL-6 protein levels (Figure 5 and data not shown) in comparison with the wild type. Thus, it seems unlikely that Nod1 plays an important role in B. burgdorferi signaling, which is consistent with previous observations that Nod1 is not involved in immune response activation downstream of B. burgdorferi [12].


Nod2 suppresses Borrelia burgdorferi mediated murine Lyme arthritis and carditis through the induction of tolerance.

Petnicki-Ocwieja T, DeFrancesco AS, Chung E, Darcy CT, Bronson RT, Kobayashi KS, Hu LT - PLoS ONE (2011)

Nod2 mediates the activation of cytokines in response to B. burgdorferi.BMDM from wild type (B6) and Nod2 deficient mice were stimulated with B. burgdorferi at the designated MOIs for 6 hours. Cytokine levels in supernatants were measures by ELISA; bars represent mean percent change ± s.e.m. of three independent experiments. Values for wild type (B6) cells for each experiment were normalized to 100% and other values are shown relative to B6. Because of the normalization, the s.e.m. for B6 is zero and no error bars are visible, * p<0.05. Raw data for each of the conditions showed that wild type cells secreted a mean of 6.34, 3.51 and 1.8 ng/ml of TNF-α at MOIs of 100, 10 and 1 respectively, while Nod2 deficient cells secreted a mean of 3.52, 2.35 and 1.3 ng/ml at the same MOIs. For IL-6, wild type cells secreted 1.6 ng/ml of IL-6 for MOI 1, while Nod2 deficient cells secreted a mean of 0.58 ng/ml for IL-6 for MOI 1.
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Related In: Results  -  Collection

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pone-0017414-g005: Nod2 mediates the activation of cytokines in response to B. burgdorferi.BMDM from wild type (B6) and Nod2 deficient mice were stimulated with B. burgdorferi at the designated MOIs for 6 hours. Cytokine levels in supernatants were measures by ELISA; bars represent mean percent change ± s.e.m. of three independent experiments. Values for wild type (B6) cells for each experiment were normalized to 100% and other values are shown relative to B6. Because of the normalization, the s.e.m. for B6 is zero and no error bars are visible, * p<0.05. Raw data for each of the conditions showed that wild type cells secreted a mean of 6.34, 3.51 and 1.8 ng/ml of TNF-α at MOIs of 100, 10 and 1 respectively, while Nod2 deficient cells secreted a mean of 3.52, 2.35 and 1.3 ng/ml at the same MOIs. For IL-6, wild type cells secreted 1.6 ng/ml of IL-6 for MOI 1, while Nod2 deficient cells secreted a mean of 0.58 ng/ml for IL-6 for MOI 1.
Mentions: NLR family members that activate pathways other than the inflammasome include Nod1 and Nod2, both of which signal through the kinase Rip2 for the activation of NF-κB and MAPKs [19], [38]. To determine whether Nod1 or Nod2 might be involved in responses to B. burgdorferi, we stimulated wild type and Rip2 deficient BMDM with B. burgdorferi. Rip2 deficient cells showed a 34% decrease in TNF-α secretion by ELISA compared to wild-type cells (data not shown). Because both Nod1 and Nod2 utilize Rip2, we next sought to determine whether either Nod1 or Nod2 (or both) were involved in cellular responses to B. burgdorferi. Nod1 and Nod2 deficient macrophages were stimulated with B. burgdorferi. Examination of mRNA transcripts for pro-IL1β, IL-6 and TNF-α after 6 hrs of incubation with B. burgdorferi showed decreases of 60–75% in Nod1 and Nod2 deficient macrophages compared to wild type (Figure 4). However, when cell culture supernatants were examined by ELISA, only Nod2 deficient cells showed a significant decrease in TNF-α protein levels (Figure 4) or IL-6 protein levels (Figure 5 and data not shown) in comparison with the wild type. Thus, it seems unlikely that Nod1 plays an important role in B. burgdorferi signaling, which is consistent with previous observations that Nod1 is not involved in immune response activation downstream of B. burgdorferi [12].

Bottom Line: The internalization of Borrelia burgdorferi, the causative agent of Lyme disease, by phagocytes is essential for an effective activation of the immune response to this pathogen.In vitro stimulation of Nod2 deficient bone marrow derived macrophages (BMDM) resulted in decreased induction of multiple cytokines, interferons and interferon regulated genes compared with wild-type cells.However, B. burgdorferi infection of Nod2 deficient mice resulted in increased rather than decreased arthritis and carditis compared to control mice.

View Article: PubMed Central - PubMed

Affiliation: Division of Geographic Medicine and Infectious Diseases, Tufts Medical Center, Boston, Massachusetts, United States of America.

ABSTRACT
The internalization of Borrelia burgdorferi, the causative agent of Lyme disease, by phagocytes is essential for an effective activation of the immune response to this pathogen. The intracellular, cytosolic receptor Nod2 has been shown to play varying roles in either enhancing or attenuating inflammation in response to different infectious agents. We examined the role of Nod2 in responses to B. burgdorferi. In vitro stimulation of Nod2 deficient bone marrow derived macrophages (BMDM) resulted in decreased induction of multiple cytokines, interferons and interferon regulated genes compared with wild-type cells. However, B. burgdorferi infection of Nod2 deficient mice resulted in increased rather than decreased arthritis and carditis compared to control mice. We explored multiple potential mechanisms for the paradoxical response in in vivo versus in vitro systems and found that prolonged stimulation with a Nod2 ligand, muramyl dipeptide (MDP), resulted in tolerance to stimulation by B. burgdorferi. This tolerance was lost with stimulation of Nod2 deficient cells that cannot respond to MDP. Cytokine patterns in the tolerance model closely paralleled cytokine profiles in infected Nod2 deficient mice. We propose a model where Nod2 has an enhancing role in activating inflammation in early infection, but moderates inflammation after prolonged exposure to the organism through induction of tolerance.

Show MeSH
Related in: MedlinePlus