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].