4. NOD2 and Mycobacterial infection
Different from other bacteria, mycobacteria produces a specific enzyme called N-acetyl muramic acid hydroxylase (NamH), which can convert the basic unit of the mycobacteria cell wall, N-acetyl MDP, to N-glycolyl MDP, with stronger nod2-stimulating activity[88]. Mtb -infected monocyte-derived macrophage (MDM) upregulate Nod2 transcripts up to 10-fold, mRNAs up to 2-3-fold and proteins up to 10-fold[89,90]. In this section, we will summaries the contribution of NOD2-mediated innate and adaptive immunity in response to mycobacteria (Figure 3) .
In human and mouse-derived cells infected with mycobacteria, NOD2 produces a large number of immune effectors through the innate immune response, including TNF, IL-1β, IL-6, IL-12, IL-32, type I IFNs, nitric oxide (NO). TNF- and IL-1R-deficient mice fail to control mycobacteria and BCG, with increased bacterial burden, exacerbated lung inflammation, reduced IL-23, and rapid death[91–94]. Mtb -infected BMDM activates IRF5 and induces IFNα/β transcription via Nod2-RIP2-TBK1 signaling pathway, and promotes cell bactericidal effect[95]. However, NOD2-mediated type I IFNs is controversial in its capacity to clear mycobacteria. It has been suggested that type I IFNs encourage cell-to-cell spread of mycobacteria by secreting CXCL chemokines and necrotic apoptosis, but it is not yet known how type I IFNs leads to necroptosis[96,97]. Whereas some in vivo and in vitro experiments in mice suggest that type I IFNs enhances the expression of Inducible Nitric Oxide Synthase (iNOS) and eliminates mycobacteria through NO production[98,99]. Genetic disorders of type I IFNs immune function underlie severe mycobacterial infections[100–102]. Therefore, future studies should determine whether IFN is beneficial or detrimental under certain circumstances.
Mtb escapes the immune system by inhibiting autophagosome and lysosome fusion[103]. During infection, NOD2-dependent autophagy serves as an antimicrobial mechanism for clearing intracellular pathogens and Mesenchymal Stem Cells (MSC), which often excrete anti-tuberculosis mediators, leading to prolonged intracellular viability and maintenance of Mtb[104,105]. The autophagy proteins IRGM, LC3, and ATG16L1 were recruited into Mtb vesicles in a Rip2/p38-dependent manner and prompted the conversion of LC3-I to LC3-II, which serves as an indication of autophagosome formation[105,106,74]. The Signaling pathways of NOD-2 and TLR-4 (N2T4) also co-localizes Mtb in the lysosomes and induced autophagy to reduce the survival of Mtb hidden in host MSCs[104].
The BCG Vaccine is the longest used global vaccine and the only approved vaccine against Mtb . Recently, the role of trained immunity has been identified as the mechanism of BCG-mediated immunity againstMtb , which relies on NOD2[107]. Stimulation of NOD2-deficient macrophages with BCG did not show an increase in cytokine production after heterologous stimulation, suggesting that NOD2 is essential in the establishment of trained immunity[76]. Comparison of PBMC isolated before and after 3 months of BCG vaccination in volunteers revealed that the vaccine caused epigenetic modification (H3K4me3) reprogramming of innate immune cells induced by NOD2. When cells were re-exposed to mycobacteria and non-mycobacteria stimuli IFN-γ, TNF-α, and IL-1β expression were significantly up-regulated, as well as an increase in the expression of CD11b and TLR4 activation markers[77,108]. The mortality rate of children infected with M. tuberculosisfollowing BCG vaccination at birth is almost three times lower than that of unvaccinated children[109]. Moreover, the trained immunity is not dependent on adaptive immunity, as reduced Mtb loads could be consistently observed 1 week to 1 month following advance vaccination of T-cell-deficient mice with BCG, highlighting the role of macrophages in BCG-induced killing of Mtb [110]. The NOD2 signaling pathway for the treatment of infectious diseases through innate immune memory deserves further from investigation in future studies, not only in the field of immune training, but also in immune tolerance[111]. It is unknown whether NOD2-mediated immune tolerance plays a role in chronic infectious disease caused by mycobacteria. Understanding the effective disease tolerance mechanisms present in 90-95% of asymptomaticMtb -infected patients could help in designing prevention and treatment of active Mycobacteria.
DCs are a bridge between innate and adaptive immunity. N2T4enhances the efficacy of DCs and the potency to kill Mtb in vitro and in vivo, mainly through the following aspects: (1) the secretion of effectors IL-6, IL-12, IFN-γ, TNF-α, NO, (2) the induced of autophagy, (3) increasing CCR7, which is known to improve migration of immune cells to the lymph nodes, (4) enlarging the number of effector memory CD4+ and CD8+ T-cells. N2T4 also lowed the dose of isoniazid (one-fifth of the recommended concentration), and significant reduced in lung bacterial load. N2T4-targeted DC cells may be a suitable immunotherapeutic approach to evoke host immunity to Mtb [112–114]. The role of NOD2 on adaptive immunity was also confirmed by in vivo experiments in mice, where Nod2-/- mice infected with Mtb and BCG produced less Th1-type cytokines and reduced recruitment of CD8+ and CD4+ T cells In the later phase of the infection, with a higher mortality rate in mice[115]. Although Th2-type cytokines limit tissue damage in the periphery of granulomas and facilitate tissue repair during Mtbinfection[111,116], there is no direct evidence indicating whether NOD2-dependent IL-4, IL-5 plays a role in it.
Gene variants in the NOD2-mediated signaling pathway associated with susceptibility to Mycobacterium leprae infection in a large-scale leprosy GWAS in a Chinese population[4]. It has later been successively validated in Vietnam, India, and ethnically mixed Amazonian populations[117–119]. A study shows that human NOD2 recognizes structurally unique MDPs from Mycobacterium leprae [120]. Most interestingly, however, M. leprae , the bacterium most associated with the NOD2 polymorphism, is the only one known not to produce an N-glycolyl MDP structure that enables NOD2 to be more readily recognized[121]. The role of NOD2 in the immune response in leprosy has also been supported based on cell function experiments. Compared to TLR2/1 ligands, MDP acting on NOD2-activated monocytes is more likely to induce rapid differentiation of monocytes into CD1+ DCs and to accomplish MHC I-associated antigen presentation by a IL-32-dependent mechanism[122], which was confirmed in leprosy lesions. Due to the harsh culture conditions of M. leprae and the absence of in vivo or in vitro leprosy models, the mechanism of action of NOD2 in M. leprae infection is unclear.