The PPE18 protein of Mycobacterium tuberculosis Inhibits NF-κ B/rel-mediated proinflammatory cytokine production by upregulating and phosphorylating suppressor of cytokine signaling 3 protein

Mycobacterium tuberculosis bacteria are known to suppress proinflammatory cytokines like IL-12 and TNF-α for a biased Th2 response that favors a successful infection and its subsequent intracellular survival. However, the signaling pathways targeted by the bacilli to inhibit production of these cytokines are not fully understood. In this study, we demonstrate that the PPE18 protein of M. tuberculosis inhibits LPS-induced IL-12 and TNF-α production by blocking nuclear translocation of p50, p65 NF-κB, and c-rel transcription factors. We found that PPE18 upregulates the expression as well as tyrosine phosphorylation of suppressor of cytokine signaling 3 (SOCS3) and the phosphorylated SOCS3 physically interacts with IκBα–NF-κB/rel complex, inhibiting phosphorylation of IκBα at the serine 32/36 residues by IκB kinase-β and thereby prevents nuclear translocation of the NF-κB/rel subunits in LPS-activated macrophages. Specific knockdown of SOCS3 by small interfering RNA enhanced IκBα phosphorylation, leading to increased nuclear levels of NF-κB/rel transcription factors vis-a-vis IL-12 p40 and TNF-α production in macrophages cotreated with PPE18 and LPS. The PPE18 protein did not affect the IκB kinase-β activity. Our study describes a novel mechanism by which phosphorylated SOCS3 inhibits NF-κB activation by masking the phosphorylation site of IκBα. Also, this study highlights the possible mechanisms by which the M. tuberculosis suppresses production of proinflammatory cytokines using PPE18. The macrophages elicit various antimycobacterial mechanisms during the innate phase of activation that play crucial roles in deciding the outcome of Mycobacterium tuberculosis infection (1). Among these, production of proinflammatory cytokines like IL-12 and TNF-α is critical for mounting an optimal defense against mycobacterial infection (2–5). The TNF-α is important for granuloma formation and induction of cytotoxicity against the bacilli (6, 7). In contrast, IL-12 plays a crucial role in activating the protective Th1 immune response (5, 8, 9). Regulation of proinflammatory responses often involves diverse signaling cascades, and the pathogenic M. tuberculosis bacteria have evolved several mechanisms to modulate these signaling pathways to suppress IL-12 and TNF-α production to favor its long-term survival and persistence inside the host (10–12). Interestingly, virulence of pathogenic mycobacteria is found to be inversely correlated with the levels of IL-12 and TNF-α secreted (11–14). Human Monocyte-Derived Macrophages (MDMs) are shown to produce a significantly lower amount of IL-12 after exposure to M. tuberculosis than other bacteria (9), indicating that M. tuberculosis specifically targets the signaling cascades that lead to production of this cytokine. However, the molecular mechanisms by which the pathogenic mycobacteria can downregulate induction of these proinflammatory cytokines are not fully understood. The mycobacteria are characterized by the presence of two unique gene families, the proline-glutamic acid and the proline-proline-glutamic acid (PPE) (15), which are highly expanded in pathogenic species like M. tuberculosis (16). Although the proteins are predicted to play a role in generating antigenic variation (17–20), the functional role of these proteins in mycobacterial pathogenesis is not clearly understood. Some of these genes are predicted to play an important role in mycobacterial persistence inside the host and are crucial in M. tuberculosis pathogenesis (21–23). Recently, we reported that a PPE family of proteins of M. tuberculosis, PPE18, inhibits the Th1-type response by specifically downregulating IL-12 p40 and other proinflammatory indicators (24). We found that PPE18 binds to the TLR2 leucine-rich repeat (LRR) 11–15 region and causes an inhibition of IL-12 p40 (24). It appears that PPE18 probably targets the proinflammatory signaling downstream of TLR2 by activating certain negative regulators of this pathway. Interestingly, suppressor of cytokine signaling 3 (SOCS3) downstream of TLR2 (25) acts as negative regulator of proinflammatory signaling (26–29). Because several pathogenic mycobacterial species induce expression of the SOCS3 protein (30–32), we speculated that probably SOCS3 protein is involved in the suppression of induction of IL-12 p40 in macrophages treated with PPE18. The SOCS3 protein is known to play important roles in the regulation of proinflammatory responses during infection (25–28, 33, 34). Constitutively expressed SOCS3 was found to favor development of Th2-type response and correlate well with the severity of Th2-mediated disease like atopic asthma (35). Further, SOCS3-transduced dendritic cells are found to be effective inducers of Th2-type T cells in vitro and in vivo through reduced production of IL-12, IFN-γ and IL-23 p19 cytokines (36). Although the mechanisms are not clear, SOCS3-transduced dendritic cells are shown to be effective inducers of Th2-type T cells in vitro and in vivo (36). Various studies have indicated a direct role of SOCS3 in downregulating the IFN-γ signaling pathway (37, 38). SOCS3 is known to predominantly target the STAT family of proteins to attenuate IFN-γ–specific proinflammatory signaling (31, 39, 40). But it could be possible that the effect of SOCS3 is not restricted to STAT signaling alone but also to other signaling pathways to downregulate IL-12 and TNF-α cytokines during tuberculosis. Although mycobacteria are known to increase SOCS3 expression in macrophages (31, 32), their significance in the macrophage innate-effector response in tuberculosis is not well understood. It is possible that the bacilli exploit the SOCS3 signaling pathway to inhibit proinflammatory cytokine induction in macrophages. In this study, we describe a unique signaling pathway responsible for the SOCS3-mediated downregulation of proinflammatory cytokines that involves the IκBα–NF-κB/rel signaling and conclude that PPE18 targets this signaling pathway to suppress induction of IL-12 p40 and TNF-α cytokines in activated macrophages to favor a Th2-type response (24) that helps its replication and persistence of the bacilli inside the hosts