WASH-driven actin polymerization is required for efficient mycobacterial phagosome maturation arrest.

Pathogenic mycobacteria survive in phagocytic host cells primarily as a result of their ability to prevent fusion of their vacuole with lysosomes, thereby avoiding a bactericidal environment. The molecular mechanisms to establish and maintain this replication compartment are not well understood. By combining molecular and microscopical approaches we show here that after phagocytosis the actin nucleation-promoting factor WASH associates and generates F-actin on the mycobacterial vacuole. Disruption of WASH or depolymerization of F-actin leads to the accumulation of the proton-pumping V-ATPase around the mycobacterial vacuole, its acidification and reduces the viability of intracellular mycobacteria. This effect is observed for M. marinum in the model phagocyte Dictyostelium but also for M. marinum and M. tuberculosis in mammalian phagocytes. This demonstrates an evolutionarily conserved mechanism by which pathogenic mycobacteria subvert the actin-polymerization activity of WASH to prevent phagosome acidification and maturation, as a prerequisite to generate and maintain a replicative niche.

Suppressor of Cytokine Signaling 3 in Macrophages Prevents Exacerbated Interleukin-6-Dependent Arginase-1 Activity and Early Permissiveness to Experimental Tuberculosis.

Suppressor of cytokine signaling 3 (SOCS3) is a feedback inhibitor of interleukin (IL)-6 signaling in macrophages. In the absence of this molecule, macrophages become extremely prone to an IL-6-dependent expression of arginase-1 (Arg1) and nitric oxide synthase (NOS)2, the prototype markers for alternative or classical macrophage activation, respectively. Because both enzymes are antipodean macrophage effector molecules in Mycobacterium tuberculosis (Mtb) infection, we assessed the relevance of SOCS3 for macrophage activation during experimental tuberculosis using macrophage-specific SOCS3-deficient (LysMcreSOCS3loxP/loxP) mice. Aerosol infection of LysMcreSOCS3loxP/loxP mice resulted in remarkably higher bacterial loads in infected lungs and exacerbated pulmonary inflammation. This increased susceptibility to Mtb infection was accompanied by enhanced levels of both classical and alternative macrophage activation. However, high Arg1 expression preceded the increased induction of NOS2 and at early time points of infection mycobacteria were mostly found in cells positive for Arg1. This sequential activation of Arg1 and NOS2 expression in LysMcreSOCS3loxP/loxP mice appears to favor the initial replication of Mtb particularly in Arg1-positive cells. Neutralization of IL-6 in Mtb-infected LysMcreSOCS3loxP/loxP mice reduced arginase activity and restored control of mycobacterial replication in LysMcreSOCS3loxP/loxP mice. Our data reveal an unexpected role of SOCS3 during experimental TB: macrophage SOCS3 restrains early expression of Arg1 and helps limit Mtb replication in resident lung macrophages, thereby limiting the growth of mycobacteria. Together, SOCS3 keeps IL-6-dependent divergent macrophage responses such as Nos2 and Arg1 expression under control and safeguard protective macrophage effector mechanisms.