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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-mediated tolerance affects ifn-α, cxcl10, but not ifit1 and igtp mRNA.Activated BMDM from wild type (B6) and Nod2 deficient mice were treated with MDP at 100 µg/ml 24 hours prior to stimulation to B. burgdorferi at an MOI 10. Just prior to infection, cells were washed in DMEM containing 10% FBS and fresh media was added. Cells were collected 16 hours post-infection for inf-α (A) and 6 hours post-infection for cxcl10 (B), igtp (C) and ifit1 (D). mRNA expression was measured by RT-qPCR. Bars represent mean percent change ± s.e.m. of three independent experiments, * p<0.05.
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pone-0017414-g011: Nod2-mediated tolerance affects ifn-α, cxcl10, but not ifit1 and igtp mRNA.Activated BMDM from wild type (B6) and Nod2 deficient mice were treated with MDP at 100 µg/ml 24 hours prior to stimulation to B. burgdorferi at an MOI 10. Just prior to infection, cells were washed in DMEM containing 10% FBS and fresh media was added. Cells were collected 16 hours post-infection for inf-α (A) and 6 hours post-infection for cxcl10 (B), igtp (C) and ifit1 (D). mRNA expression was measured by RT-qPCR. Bars represent mean percent change ± s.e.m. of three independent experiments, * p<0.05.

Mentions: Previous studies have shown that cells that undergo prolonged stimulation of Nod2, mimicking prolonged infection, no longer respond to subsequent stimulation by bacterial ligands, such as LPS, thus becoming tolerant to those stimuli [32], [54]. We wanted to investigate whether sustained stimulation of Nod2 in vitro would decrease the pro-inflammatory response to B. burgdorferi through the induction of tolerance. We stimulated activated BMDM with the Nod2 ligand, MDP, at 100 µg/ml or control for 24 hours. We selected this time point because it has previously been established that cells treated with MDP for 24 hours become tolerant to further MDP or TLR stimulus [32]. In our experiments, sustained exposure to MDP resulted in a 40% and 75% decrease in secretion of TNF-α and IL-6 respectively in response to B. burgdorferi compared to cells not treated with MDP (p = 0.037, Figure 10A, B). However, in Nod2 deficient cells, prolonged exposure to MDP did not significantly affect levels of protein secretion in response to B. burgdorferi when compared to wild type cells. Of note, the levels of il-10 transcripts in Nod2 deficient cells were elevated in comparison to wild type levels in MDP pre-treated samples, again suggesting that a decrease in IL-10 is not the mechanism for increased inflammation in Nod2 deficient cells (Figure 10C). We also observed greater expression of ifn-α and cxcl10 transcripts by RT-qPCR in Nod2 deficient cells treated with MDP compared with MDP treated wild type cells (Figure 11A, B). However, ifit1 and igtp transcripts, which were not affected by Nod2 deficiency (Figure 6), did not show a decrease in expression upon MDP stimulation of wild type cells. Rather, we observed a synergistic effect of B. burgdorferi and MDP stimulation on ifit1 and igtp induction in wild type cells that was, as expected, absent in Nod2 deficient cells (Figure 11C, D). These data suggest that prolonged exposure to the Nod2 ligand MDP in vitro, results in suppression of pro-inflammatory responses to B. burgdorferi and reverses the phenotype seen in cells immediately infected with B. burgdorferi where Nod2 appears to play a stimulatory role. The results of these in vitro tolerance experiments parallel the IL-6 responses seen in the in vivo experiments, where levels are increased in Nod2 deficient mice compared with levels in wild type mice.


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-mediated tolerance affects ifn-α, cxcl10, but not ifit1 and igtp mRNA.Activated BMDM from wild type (B6) and Nod2 deficient mice were treated with MDP at 100 µg/ml 24 hours prior to stimulation to B. burgdorferi at an MOI 10. Just prior to infection, cells were washed in DMEM containing 10% FBS and fresh media was added. Cells were collected 16 hours post-infection for inf-α (A) and 6 hours post-infection for cxcl10 (B), igtp (C) and ifit1 (D). mRNA expression was measured by RT-qPCR. Bars represent mean percent change ± s.e.m. of three independent experiments, * p<0.05.
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Related In: Results  -  Collection

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pone-0017414-g011: Nod2-mediated tolerance affects ifn-α, cxcl10, but not ifit1 and igtp mRNA.Activated BMDM from wild type (B6) and Nod2 deficient mice were treated with MDP at 100 µg/ml 24 hours prior to stimulation to B. burgdorferi at an MOI 10. Just prior to infection, cells were washed in DMEM containing 10% FBS and fresh media was added. Cells were collected 16 hours post-infection for inf-α (A) and 6 hours post-infection for cxcl10 (B), igtp (C) and ifit1 (D). mRNA expression was measured by RT-qPCR. Bars represent mean percent change ± s.e.m. of three independent experiments, * p<0.05.
Mentions: Previous studies have shown that cells that undergo prolonged stimulation of Nod2, mimicking prolonged infection, no longer respond to subsequent stimulation by bacterial ligands, such as LPS, thus becoming tolerant to those stimuli [32], [54]. We wanted to investigate whether sustained stimulation of Nod2 in vitro would decrease the pro-inflammatory response to B. burgdorferi through the induction of tolerance. We stimulated activated BMDM with the Nod2 ligand, MDP, at 100 µg/ml or control for 24 hours. We selected this time point because it has previously been established that cells treated with MDP for 24 hours become tolerant to further MDP or TLR stimulus [32]. In our experiments, sustained exposure to MDP resulted in a 40% and 75% decrease in secretion of TNF-α and IL-6 respectively in response to B. burgdorferi compared to cells not treated with MDP (p = 0.037, Figure 10A, B). However, in Nod2 deficient cells, prolonged exposure to MDP did not significantly affect levels of protein secretion in response to B. burgdorferi when compared to wild type cells. Of note, the levels of il-10 transcripts in Nod2 deficient cells were elevated in comparison to wild type levels in MDP pre-treated samples, again suggesting that a decrease in IL-10 is not the mechanism for increased inflammation in Nod2 deficient cells (Figure 10C). We also observed greater expression of ifn-α and cxcl10 transcripts by RT-qPCR in Nod2 deficient cells treated with MDP compared with MDP treated wild type cells (Figure 11A, B). However, ifit1 and igtp transcripts, which were not affected by Nod2 deficiency (Figure 6), did not show a decrease in expression upon MDP stimulation of wild type cells. Rather, we observed a synergistic effect of B. burgdorferi and MDP stimulation on ifit1 and igtp induction in wild type cells that was, as expected, absent in Nod2 deficient cells (Figure 11C, D). These data suggest that prolonged exposure to the Nod2 ligand MDP in vitro, results in suppression of pro-inflammatory responses to B. burgdorferi and reverses the phenotype seen in cells immediately infected with B. burgdorferi where Nod2 appears to play a stimulatory role. The results of these in vitro tolerance experiments parallel the IL-6 responses seen in the in vivo experiments, where levels are increased in Nod2 deficient mice compared with levels in wild type mice.

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