3. NOD2 and immunity
3.1 Innate immunity
Innate immunity is a host response that exerts a direct or nonspecific effect on pathogens by activating immune cells and inflammatory mediators. NOD2 drives the inflammatory response to bacteria through activation of the NF-κB, and MAPK pathways, both of which inspire the increased expression of pro-inflammatory factors including IL-1β, TNF-α, IL-6, IL-8, IL-12p40, chemokine CCL2, CXCL2[51–54], which is also observed in neutrophils[55–57].
In addition to the activation of each of the PRRs, multiple signaling pathways of NOD1, NOD2, and TLR act synergistically in reaching intracellular signaling cascades to produce more pro-inflammatory factors involved in the early innate immune response to infection. Stimulation of mouse-derived peritoneal macrophages or THP-1 by TOLL-like receptor and NOD-like receptor agonist coupling not only enhances the release of the downstream NF-κB activation and pro-inflammatory factor, but also accelerates bacterial clearance from circulation and internal organs through enhanced phagocytosis by macrophages, protecting mice from microbial sepsis-induced lethality[58,59].
Excessive production of pro-inflammatory cytokines causes physiological and immunopathological damage to tissues, and therefore macrophage tolerance induced by MDP, LPS, or other bacterial ligands is considered a protective mechanism[60]. In line with this observation, it was found that there is a lack of cross-tolerance between Toll-like receptors and NOD2 signaling and that macrophages are heavily dependent on NOD1 and NOD2 when TLR signaling is tolerance inhibited[61]. Compared with wild mice, Nod1-/-Nod2-/- or RIPK2-/- mice showed significantly impaired survival and bacterial clearance after exposure to LPS or E. coli in vivo, with a decreased expression of the promotional cytokine IL-6[61].
3.2 Adaptive immunity
Once pathogenic microorganisms have breached the various barriers of innate immunity, adaptive immunity is required to activate antigen-specific T and B lymphocytes, which is heavily dependent on DCs. The failure of NOD2 to drive Th2-regulatory signaling leads to intestinal mucosal disorders, which is one of the reasons for the development of CD[62,63]. MDP induces Th2 cell-dependent IL-4 and IL-5 secretion, which requires up-regulation of non-hematopoietic thymic stromal lymphopoietin protein (TSLP) and expression of OX40 ligand (OX40L) on DCs. The TSLP-OX40L axis is essential for DCs to act as antigen-presenting cells (APC) to regulate the induction and functional alterations of Th2 cells [63]. Meanwhile, NOD2 enhances IL-23 secretion in DCs, thereby promoting the differentiation of CD4+ T cells to TH17 and the secretion of IL-17, which may be responsible for bacterial clearance, as demonstrated in NOD2-mutant DCs from some CD patients[64].
Coordination of NOD2 with other signaling pathways also plays an important role in influencing NOD2-mediated adaptive immunity. Under combined treatment with monophospholipid lipid A (MPLA, TLR4 agonist) and MDP (NOD2 agonist), more intense cellular and humoral immune responses were observed in BMDCs, such as higher expression of the maturation/activation markers MHCII, CD80, and CD86, proliferation of splenic CD4+ and CD8+ T-cells, and elevation of IFN-γ and IgG, compared to the use of single stimulants[58]. TLR2 synergizes with NOD2 to signal through TBK1 to PI31, which stabilizes the immunoproteasome by interacting with Sec16A on the ER, promoting major histocompatibility complex (MHC) I antigen presentation in DCs and initiating the CD8+ T-cell response[65].It is similarly obtained that co-stimulation of NOD2 with TLR2 ligand also promotes the production of Th1-polarized cytokine IL-12 by macrophages, which initiates antigen-specific T and B cell immunity in vivo[62]. In a recent study, a synthesized dual NOD2/TLR7 agonist adjuvant has been shown to exhibit strong Th1-biased adjuvant activity upon activation of PBMC and BMDC, which cannot be compared to that of a single receptor agonist[66]. The chemical coupling of PRR agonists provides a piece of strong evidence for cross-talk in signaling pathways and may also facilitate the development of novel vaccines in the future.
3.3 NOD2 and autophagy
Autophagy was known as a highly conserved process in eukaryotic cells to maintain homeostasis degrades cellular proteins and organelles. NOD2 and the autophagy protein ATG16L1 are two of the most important genes associated with CD. Accumulating studies have confirmed that ATG16L1 plays important roles in the NOD2 pathway, including pathogen targeting, autophagy induction, antigen presentation and cytokine production. Atg16Ll promotes the local conversion of LC3-I to LC3-II through binding to Atg5-Atg12, and is recruited to the plasma membrane of bacterial entry sites by NOD2. Direct binding of NOD2 and ATG16L1 leads to the rapid formation of autophagosomes around invading bacteria[67]. The NOD2 ligand MDP induces the formation of autophagosomes in human DCs, thereby enhancing the clearance of bacteria and MHC II-associated antigen presentation, which requires the involvement of the autophagy proteins Atg5, Atg7, Atg16L1, and RIP2[68]. In addition, NOD2 promotes the expression of cytokines IL-1β, IL-6, IL-8, TNF-α through ATG16L1-associated autophagy[69–71]. The number of intracellularE. coli was increased in Atg16L1-/- and NOD2-/- in vitro and in vivo models[70,72].
However, ATG16L1 can also interact with IKK, an important link in the NOD2 pathway that activates the transcription factor NF-κB, to protect cells from excessive anti-inflammatory effects. IKK is involved in stabilizing ATG16L1 at Ser278, inhibiting endoplasmic reticulum stress and secreting cytoprotective IL-18, which may be involved in counteracting the pro-inflammatory effects triggered by classical IKK/NF-κB activation[73]. NOD2 also clears excess ROS through ATG16L1-mediated mitochondrial autophagy and exerts its cytoprotective mechanism[74].
3.4 Trained immunity and tolerant immunity
In recent years, there has been a growing realization that immune memory is not an exclusive characteristic of adaptive immunity. The innate immune cell appears capable of acquiring memory characteristics following a transient stimulus, resulting in a more powerful response when challenged twice, which is known as trained immunity. Trained immunity explains some of the heterologous effects of vaccines, thus increase protection against secondary infections[75]. Mouse- or human-derived cells pre-stimulated by Bacillus Calmette-Guérin (BCG) underwent NOD2-induced trained immunity and showed non-specific protection against infection and increase of pro-inflammatory cytokines IFN-γ, TNF-α, and IL-1β[76]. In severe combined immune-deficiency (SCID) mice lacking T and B cells injected with lethal Candida albicans 2 weeks after BCG or MDP, survival was significantly higher in BCG-vaccinated mice than in saline-injected mice[77]. Likewise, activation of NOD2 by MDP in macrophages/monocytes drives a strong innate immune memory and adaptive response that can strengthen antiviral defenses to prevent SARS-CoV2 epidemics in part[78].
NOD2 is also involved in a state of hypo-responsiveness to secondary stimuli called immune tolerance. This is a feedback negative regulatory mechanism that limits the health costs from excessive immune damage and avoids immunopathology[79]. NOD2-induced immune tolerance is relevant to CD, which is characterized by a dysregulation of immune homeostasis due to excessive inflammation of the intestinal flora[80]. Compared with macrophages derived from normal volunteers, a higher TNF-α, IL-8, and IL-1β were observed in macrophages from volunteers at risk for CD harboring the Leu1007insC Nod2 variant[81].
It has been suggested that immune tolerance caused by chronic stimulation with MDP is due to the destabilization of NOD2 or its pathway. Following the action of MDP, the rapid dissociation of the HSP90 complex from NOD2 and the binding of the SOCS-3 protein to NOD2 accelerate the process of NOD2 degradation, resulting in a weakened host response when confronted with either MDP orC.rodentium [82,83]. Consistently, transmembrane proteins ZNRF4-mediated degradation of RIP2 is a negative regulatory mechanism for NOD2-induced NF-κB, cytokine, and antimicrobial responses. MDP-tolerant mice lacking ZNRF4 exhibit enhanced control ofLactobacillus monocytogenes infection[84]. NOD2 tolerance may also be caused by powerful negative feedback. Acute NOD2 stimulation promotes inflammation by inducing IRAK-1 activation, while chronic NOD2 stimulation inhibits IRAK-1 activity, induces the expression of IRAK-1 inhibitory protein IRAK-M, and significantly reduces cytokines TNF-α, IL-8, and IL-1, which promote tolerance to the luminal flora and maintains the balance of intestinal immune tolerance[81]. NOD2-mediated early secretion of IL-1Rα, IL-10, and TGF-β is important for their decline during prolonged NOD2 stimulation, and inhibition of the NOD2 mTOR pathway and selective blockade of autocrine secretion of these cytokines reverses NOD2 tolerance[85].
A Borrelia burgdorferi disease model has also been proposed, in which Nod2 plays a potentiating role in activating inflammation in early infection, but reduces tissue damage by inducing tolerance after prolonged exposure to the organism [86]. Although the inflammatory response of tolerant macrophages to bacterial infection was relatively weakened, this was partially offset by accelerated maturation of NF-κB-dependent phagosomes, enhanced bactericidal activity, and up-regulation of expression of lysosomal enzymes and membrane transport regulators (Rab10 and Acp5)[87].