Macrophages are important innate immune regulators in the recognition and clearance of invading pathogens. Mycobacterium tuberculosis (M.tb), which causes tuberculosis, is a host-adapted intracellular pathogen of macrophages. Intracellular pattern recognition receptors in macrophages such as nucleotide-binding oligomerization domain (NOD) proteins regulate pro-inflammatory cytokine production. NOD2-mediated signaling pathways in response to M.tb have been studied primarily in mouse models and cell lines but not in primary human macrophages. Work in this dissertation examined whether NOD2 is expressed in human macrophages, controls the growth of M.tb, and functions in inducible nitric oxide synthase (iNOS) and novel protein kinase c (PKC) signaling pathways.
We examined NOD2 expression during monocyte differentiation and observed a marked increase in NOD2 transcript and protein following 2-3 days in culture. Pre-treatment of human monocyte-derived and alveolar macrophages with the NOD2 agonist muramyl dipeptide enhanced production of TNF-α and IL-1β in response to M.tb and M. bovis BCG (BCG) in a receptor interacting protein 2 (RIP2)-dependent fashion. The NOD2-mediated cytokine response was significantly reduced following knockdown of NOD2 expression by using small interfering RNA (siRNA) in human macrophages. Finally, NOD2 controlled the growth of both M.tb and BCG in human macrophages, whereas controlling only BCG growth in murine macrophages. Together, our results provide evidence that NOD2 is an important intracellular receptor in regulating the host response to M.tb and BCG infection in human macrophages.
Our observation of enhanced growth in NOD2 deficient cells at 24 hours (h) is of interest because during this time M.tb is becoming acclimated to the macrophage. This led us to examine the involvement of NOD2 in early innate immune responses. In order to study new biological functions and molecular mechanisms of NOD2 during early M.tb infection of human macrophages, we performed expression profiling of M.tb-infected NOD2 deficient macrophages using a microarray. Notably, we identified that inducible nitric oxide synthase (iNOS) expression is regulated by NOD2 during M.tb infection. iNOS expression is enhanced at 24 and 48 h after M.tb and BCG incubation in human macrophages. Our data show iNOS expression is decreased in NOD2 deficient cells stimulated with M.tb and the M.tb-specific NOD2 agonist N-glycolylated muramyl dipeptide (GMDP), indicating that NOD2 is involved in iNOS protein expression. These data provide evidence for a novel pathway involving NOD2 that controls M.tb growth in human macrophages through the regulation of iNOS expression.
We examined other potential signaling partners for NOD2 in human macrophages. Data show that the NOD2 agonist, GMDP, stimulates phosphorylation and activation of PKCδ. Corroborating these data, NOD2 deficient cells stimulated with PMA are significantly reduced in PKCδ phosphorylation. PMA is a known stimulus for classical and novel PKC isoforms. Additionally, the kinase known to interact with NOD2, RIP2, is activated by PMA. This activation is deficient upon knockdown of NOD2 in human macrophages. Thus, our findings strongly support a role for NOD2 in human macrophages during M.tb infection as well as in early innate immune responses that include iNOS and PKC pathways.