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Nuclear factor-kappa B (NFκB) is a ubiquitous transcription factor that mediates pro-inflammatory responses required for host control of many microbial pathogens; on the other hand, NFκB has been implicated in the pathogenesis of other inflammatory and infectious diseases. Mice with genetic disruption of the p50 subunit of NFκB are more likely to succumb to Mycobacterium tuberculosis (MTB). However, the role of NFκB in host defense in humans is not fully understood. We sought to examine the role of NFκB activation in the immune response of human macrophages to MTB. Targeted pharmacologic inhibition of NFκB activation using BAY 11-7082 (BAY, an inhibitor of IκBα kinase) or an adenovirus construct with a dominant-negative IκBα significantly decreased the number of viable intracellular mycobacteria recovered from THP-1 macrophages four and eight days after infection. The results with BAY were confirmed in primary human monocyte-derived macrophages and alveolar macrophages. NFκB inhibition was associated with increased macrophage apoptosis and autophagy, which are well-established killing mechanisms of intracellular MTB. Inhibition of the executioner protease caspase-3 or of the autophagic pathway significantly abrogated the effects of BAY. We conclude that NFκB inhibition decreases viability of intracellular MTB in human macrophages via induction of apoptosis and autophagy.

Macrophages provide a first line of defense against Mycobacterium tuberculosis. However, in instances where macrophage activation for killing is suboptimal, M. tuberculosis is capable of surviving intracellularly. IL-32 is a recently described cytokine induced by M. tuberculosis in a variety of cell types including human monocytes and macrophages. In this study, we investigated the biological significance of IL-32 in an in vitro model of M. tuberculosis infection in differentiated THP-1 human macrophages in which IL-32 expression was silenced using stable expression of short hairpin RNA (shRNA). Inhibition of endogenous IL-32 production in THP-1 cells that express one of three distinct shRNA-IL-32 constructs significantly decreased M. tuberculosis induction of TNF-alpha by approximately 60%, IL-1beta by 30-60%, and IL-8 by 40-50% and concomitantly increased the number of cell-associated M. tuberculosis bacteria compared with THP-1 cells stably expressing a scrambled shRNA. In THP-1 cells infected with M. tuberculosis and stimulated with rIL-32, a greater level of apoptosis was observed compared with that with M. tuberculosis infection alone. Obversely, there was significant abrogation of apoptosis induced by M. tuberculosis and a concomitant decrease in caspase-3 activation in cells depleted of endogenous IL-32. rIL-32gamma significantly reduced the number of viable intracellular M. tuberculosis bacteria, which was modestly but significantly abrogated with a caspase-3 inhibitor. We conclude that IL-32 plays a host defense role against M. tuberculosis in differentiated THP-1 human macrophages.

In mice, and possibly in humans, nitric oxide (NO) is an important host-defense molecule against Mycobacterium tuberculosis. Inducible nitric oxide synthase (iNOS) and NO are upregulated in murine macrophages stimulated with interferon-gamma (IFNgamma) and mannose-capped lipoarabinomannan (ManLAM), a major lipoglycan in the cell wall of M. tuberculosis. Interleukin-4 (IL-4) can inhibit NO expression and may impair host immune response to M. tuberculosis. Therefore, we sought to determine the mechanism by which IL-4 inhibits IFNgamma+ManLAM-induced NO production. Since l-arginine is the substrate for both iNOS and arginase, and IL-4 increases arginase activity by inducing its production, a plausible mechanism of IL-4 inhibition of NO expression is via depletion of l-arginine through increased arginase activity. Herein, we show that IL-4 inhibited iNOS gene expression at the transcriptional level, suggesting an inhibitory mechanism that is independent of the competition for l-arginine between iNOS and arginase. Furthermore, pharmacologic inhibition of IL-4-induced arginase activity did not abrogate IL-4 inhibition of IFNgamma+ManLAM-induced NO expression. Instead, inhibition by IL-4 was mediated principally by the ability of IL-4 to inhibit the production of IFNgamma-induced interferon-gamma response factor-1 (IRF-1) protein, a critically important transcriptional element that enhances expression of IFNgamma-inducible genes such as iNOS.