NOD2, a member of the phylogenetically conserved NLR (NACHT-leucine-rich repeat) family, is an essential pattern recognition sensor for MTB-derived antigens. Mycobacterial antigens fail to induce an appropriate increase in TNF-α synthesis in human PBMC that express mutant NOD2 proteins and in murine macrophages lacking NOD2 . The gene mutations that encode mutant NOD2 proteins, however, appear to be rare in patients with tuberculosis. NOD2 agonists may also modulate innate responses to MTB by inducing resistance to apoptosis that facilitates the survival of MTB in infected macrophages . Thus, NOD2 may play a role in attenuating two key putative mycobactericidal pathways. To further define a role for NOD2 in disease pathogenesis, we analysed NOD2 transcriptional responses in pulmonary leucocytes and mononuclear cells harvested from patients with pulmonary tuberculosis (PTB).
Our gene expression studies revealed that there are no characteristic NOD2 transcriptional responses in pulmonary leucocytes obtained from patients with tuberculosis. NOD2 mRNA levels in patients generally compared with those in control donors. Nonetheless, increased NOD2 levels, which correlate with TLR2 and TLR4 expression, were noted in some patients with severe infection. This observation, coupled with the increases in NOD2 expression in peripheral leucocytes following treatment, suggest further study in a larger group of patients to confirm a role for NOD2 in PTB. In the present study, we only measured total leucocyte and lymphocyte counts in bronchoalveolar lavage fluid. As NOD2 is most prominently expressed in monocytes, with very little expression in neutrophils and lymphocytes, it is most likely that monocytes account for the overwhelming majority of NOD2 expression .
We did not quantify the number of epithelial cells present in broncho-alveolar fluid and we cannot determine the effect of mycobacterial infection on NOD2 expression in respiratory epithelial cells. Baseline NOD2 expression levels in primary respiratory epithelial cells is low  although NOD2 mRNA expression is enhanced in immortalised human bronchial epithelial cells that are infected with Streptococcus pneumoniae. It would therefore be important to determine the relative contribution of respiratory epithelial cells and monocytes to the increases in NOD2 expression seen in some patients.
In the present study, NOD2 mRNA expression levels were similar in patients (who often have active disease for many weeks prior to diagnosis) and controls. This does not exclude a role for NOD2 during the early stages of MTB infection, when M. tuberculosis encounters the alveolar macrophage and innate immune pathways are first activated. It is also possible, however, that the absolute levels of NOD2 expression may not play a role in determining susceptibility to MTB infection. Rather, structural variants of NOD2 proteins may modulate host immune responses as suggested by in vitro studies . Thus, studies are required to confirm whether individuals with gene mutations encoding for mutant NOD2 proteins are predisposed to MTB-infection. The lack of an association between Crohn's disease-associated NOD2 gene mutations and tuberculosis in African patients  does not exclude a role for NOD2 in MTB-infection because NOD2 gene mutations are probably rare in African populations . It would be instructive, therefore, to determine whether NOD2 gene mutations are associated with MTB-infection in Caucasian populations, where these mutations occur with much greater frequency.
We were surprised to find significantly higher levels of NOD2 mRNA expression in peripheral leucocytes obtained from patients who completed anti-tuberculosis therapy. Firstly, we hypothesise that this could have been due to translocation of antigen-specific leucocytes predominantly to the site of disease (lungs) with few NOD2 expressing leucocytes in the peripheral compartment, and reversal of this profile after treatment. However, the lack of preferential NOD2 upregulation in the lung makes this unlikely. Secondly, we speculate that MTB infection may subvert protective innate responses by downregulating NOD2 expression, whose levels therefore increase after successful chemotherapy. We might expect this to occur in parallel with TNF-α as this cytokine up-regulates NOD2 mRNA expression in various cell lineages including PBMC [26, 27]. However, in keeping with the observations of other investigators [28, 29], we did not observe increased TNF-α mRNA expression after treatment completion. We did not investigate the relationship between NOD2 and soluble TNF-α receptors, which may modulate TNF-α levels. Thirdly, it is intriguing to speculate that increased levels of NOD2 mRNA, which occur with completion of TB treatment, is a correlate of protective immunity. Similar longitudinal changes may occur with IFN-γ [17, 29, 30] and the Th1-like splice variant IL-4δ2 , which both increase significantly with anti-TB treatment. In keeping with these observations IL-4δ2 mRNA levels are also increased in healthy subjects with latent MTB infection who contain the disease [31, 32]. Longitudinal studies in TB infected patients, however, would be required to address the role of NOD2 in this context. Interestingly, preliminary data indicate that mycobacterial antigens regulate the expression of NOD2 splice variants  and further studies are required to clarify their role in tuberculosis.
A significant limitation of this study is that we did not study MTB-specific responses in subjects with known NOD2 gene mutations. However, we found these patients difficult to recruit in our clinical setting. We also acknowledge that real time PCR measures steady state mRNA levels only and not the activity of NOD2 protein, which is physiologically active at concentrations too low to detect by immunoassay. This study was powered to detect a 0.5 log change in NOD2 mRNA levels although smaller changes may be biologically meaningful. However, mRNA levels in patients and controls were similar and we found no trends suggesting that inter-group difference might be present.