Nod2 Suppresses Borrelia burgdorferi Mediated Murine Lyme Arthritis and Carditis through the Induction of Tolerance

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.

MyD88- and TRIF-Independent Induction of Type I Interferon Drives Naive B Cell Accumulation but Not Loss of Lymph Node Architecture in Lyme Disease

“…5). This is consistent with previous studies showing that type I IFN-responsive gene induction following stimulation of bone marrow-derived macrophages with B. burgdorferi was MyD88, TRIF, and Nod2 independent but IRF3 dependent (27), although a role for Nod2 was suggested by others (41). Furthermore, studies with human cells have suggested a role for various TLRs in B. burgdorferi-induced IFN production (42,43).…”

TRIF Mediates Toll-Like Receptor 2-Dependent Inflammatory Responses to Borrelia burgdorferi

“…Bone marrow-derived macrophages (BMDM) were generated as previously described (33). Bone marrow cells were flushed from mouse femurs and tibiae with sterile Dulbecco modified Eagle medium (DMEM) and cultured on plastic petri dishes (100 mm by 15 mm) for 5 to 7 days in DMEM supplemented with 30% L929 cell conditioned medium, 20% fetal bovine serum (FBS), and 1% penicillinstreptomycin.…”

“…Melting curve analysis for purity was performed on each sample by performing 80 cycles of increasing temperature for 10 s, each beginning at 55°C. Primers used in qPCR analysis of murine irf7, il-6, il-10, and ifn-␣ universal primers; cxcl10, ifit1, and ␤-actin were as previously described (33,34). All samples for each biological replicate were run in duplicates and checked for intra-run variation and, if needed, analyzed again.…”

Crohn’s Disease: a Role of Gut Microbiota and Nod2 Gene Polymorphisms in Disease Pathogenesis

“…In vitro, Nod2 stimulation by MDP lead to release of pro-inflammatory NF-κB-dependent cytokines (such as IL-1β, TNF-α and IL-6), as well as secretion of IL-23 (which promotes Th17 differentiation) after costimulation with MDP and TLR-2 ligands and deletion of Nod2 in murine cells or loss of Nod2 function mutations in the LRR ligand recognition domains of Nod2 from human donors lead to a decrease in NF-κB activation, MAPK signaling and pro-inflammatory cytokine secretion (33,41). Whereas in in vivo animal models of inflammation the role of NOD2 deficiency has varied, with some models showing deficient mice having increased inflammation (60,62) and other models showing deficient mice having decreased inflammation (33,(63)(64)(65). These differences are related to the "chronicity" of the infection: during the early stages of infection Nod2 is a positive regulator of inflammation, however, after a period of sustained stimulation, the role of Nod2 switches and it becomes a negative regulator of inflammation, potentially via the induction of tolerance to further microbial stimulation through either Nod2 itself or other microbial receptors, such as TLR2 and TLR4 (65)(66)(67).…”

“…Whereas in in vivo animal models of inflammation the role of NOD2 deficiency has varied, with some models showing deficient mice having increased inflammation (60,62) and other models showing deficient mice having decreased inflammation (33,(63)(64)(65). These differences are related to the "chronicity" of the infection: during the early stages of infection Nod2 is a positive regulator of inflammation, however, after a period of sustained stimulation, the role of Nod2 switches and it becomes a negative regulator of inflammation, potentially via the induction of tolerance to further microbial stimulation through either Nod2 itself or other microbial receptors, such as TLR2 and TLR4 (65)(66)(67). This is dependent on IRF4 in mice and humans and downregulation of the IRAK-1 kinase and perhaps upregulation of IL-1R-associated kinase M (IRAK-M) in humans and also on early secretion of IL-10, TGF-β, IL-1Ra (10).…”