IL-32 Is a Host Protective Cytokine against Mycobacterium tuberculosis in Differentiated THP-1 Human Macrophages

Abstract

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-α by ∼60%, IL-1β 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-32γ 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.

Paradoxical effects of constitutive human IL-32γ in transgenic mice during experimental colitis

“…For example, in stable clones of IL-32 shRNA in the human macrophage cell line THP-1, both LPS and IL-1β-induced production of TNFα and IL-8 were markedly reduced compared with clones of scrambled shRNA (15). Suppression of endogenous IL-32 in THP-1 cells also resulted in decreased cytokines induced by live infection (8). When endogenous IL-32 was silenced in freshly isolated human peripheral blood mononuclear cells (PBMC) with IL-32 siRNA, IL-6, IFNγ, and TNFα were reduced by 57, 51, and 36%, respectively (12).…”

“…Vector-mediated overexpression of IL-32 results in increased TNFα and IL-1β production (13,14). On the other hand, silencing of endogenous IL-32 by either siRNA or shRNA results in decreased IL-1β, IL-8, and TNFα production (8,12,15) or IL-6 induced by IL-1β (16). Furthermore, IL-32 is present in the intestinal tissue of patients with CD (11) or UC (17).…”

“…IL-32 also induces IL-1β, IL-6, and MIP-2 in cell lines and primary cells (6,(8)(9)(10)(11)(12). Vector-mediated overexpression of IL-32 results in increased TNFα and IL-1β production (13,14).…”

IL-32γ Induces the Maturation of Dendritic Cells with Th1- and Th17-Polarizing Ability through Enhanced IL-12 and IL-6 Production

“…The quantities of IFN-g, IL-4, IL-17, IL-6, IL-12p40, and IL-12p70 in the culture supernatants were determined by a sandwich ELISA using mAbs specific for each cytokine, as previously described (27). The mAbs used to coat the plates and the biotinylated second mAbs were as following: for IFN-g, HB170 and XMG1.2; for IL-4, 11B11 and BVD6; for IL-17, eBio17CK15A5 and eBio17B7; for IL-6, 20F3.11 and 32C11.4; for IL12p40, C17.8 and C15.6; for IL-12p70, C18.2 and C17.8.…”

“…The knockdown of endogenous IL-32 by small interfering RNA in HIV-1-infected PBMCs has been demonstrated to reduce the expression of Th1 cytokines and chemokines as well as CD40L and C5a (26). IL-32 has also been shown to induce the generation of host protective cytokines against Mycobacterium tuberculosis in differentiated THP-1 human macrophages (27). Furthermore, IL-32 overexpression in mouse bone marrow transplantation results in increased levels of proinflammatory cytokines, exacerbates collagen-induced arthritis, and induces more profound inflammation in sulfuric acid-induced colitis (28).…”