Interleukin-1β (IL-1β) represents one of the most important mediators of inflammation and host responses to infection. Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, induces IL-1β secretion at the site of infection, but the underlying mechanism(s) are poorly understood. In this work we show that Mtb infection of macrophages stimulates caspase-1 activity and promotes the secretion of IL-1β. This stimulation requires live intracellular bacteria expressing a functional ESX-1 secretion system. ESAT-6, an ESX-1 substrate implicated in membrane damage, is both necessary and sufficient for caspase-1 activation and IL-1β secretion. ESAT-6 promotes the access of other immunostimulatory agents such as AG85 into the macrophage cytosol, indicating that this protein may contribute to caspase-1 activation largely by perturbing host cell membranes. Using a high-throughput shRNA-based screen we found that numerous NOD-like receptors (NLRs) and CARD domain-containing proteins (CARDs) were important for IL-1β secretion upon Mtb infection. Most importantly, NLRP3, ASC and caspase-1 form an infection-inducible inflammasome complex that is essential for IL-1β secretion. In summary, we show that recognition of Mtb infection by the NLRP3 inflammasome requires the activity of the bacterial virulence factor ESAT-6, and the subsequent IL-1β response is regulated by a number of NLR/CARD proteins.

We recently showed that mucus from patients with ulcerative colitis, a chronic inflammatory disorder of the colon, is characterized by a low level of phosphatidylcholine (PC) while clinical studies reveal that therapeutic addition of PC using slow release preparations is beneficial. The positive role of PC in this disease is still elusive. Here we tested the hypothesis that exogenous application of PC has anti-inflammatory properties using three model systems. First, human Caco-2 cells were treated with tumor necrosis factor-α (TNF-α) to induce a pro-inflammatory response via activation of NF-κB. Second, latex bead phagosomes were analyzed for their ability to assemble actin in vitro, a process linked to pro-inflammatory signaling and correlating with the growth versus killing of mycobacteria in macrophages. The third system used was the rapid assembly of plasma membrane actin in macrophages in response to sphingosine 1-phosphate. TNF-α induced a pro-inflammatory response in Caco-2 cells, including 1) assembly of plasma membrane actin; 2) activation of both MAPKs ERK and p38; 3) transport of NF-κB subunits to the nucleus; and 4) subsequent up-regulation of the synthesis of pro-inflammatory gene products. Exogenous addition of most PCs tested significantly inhibited these processes. Other phospholipids like sphingomyelin or phosphatidylethanolamine showed no effects in these assays. PC also inhibited latex bead phagosome actin assembly, the killing of Mycobacterium tuberculosis in macrophages, and the sphingosine 1-phosphate-induced actin assembly in macrophages. TNF-α induces the activation of signaling molecules and the reorganization of the actin cytoskeleton in human intestinal cells. Exogenous application of PC blocks pro-inflammatory signaling in Caco-2 cells, in phagosomes in vitro and facilitates intracellular survival of mycobacteria. We provide further evidence that actin assembly by membranes is part of the pro-inflammatory response. Collectively, these results provide a molecular foundation for the clinical studies showing a beneficial effect of PC therapy in ulcerative colitis.

The design of antimicrobial peptide-based materials for biomedical applications such as fighting infections in various organs, cancer therapy, gene delivery and detection of bacteria.

Macrophages can potentially kill all mycobacteria by poorly understood mechanisms. In this study, we explore the role of NF-κB in the innate immune response of macrophages against Mycobacterium smegmatis, a nonpathogenic mycobacterium efficiently killed by macrophages, and Mycobacterium avium which survives within macrophages. We show that infection of macrophages with M. smegmatis induces an activation of NF-κB that is essential for maturation of mycobacterial phagosomes and bacterial killing. In contrast, the pathogenic M. avium partially represses NF-κB activation. Using microarray analysis, we identified many lysosomal enzymes and membrane-trafficking regulators, including cathepsins, LAMP-2 and Rab34, were regulated by NF-κB during infection. Our results argue that NF-κB activation increases the synthesis of membrane trafficking molecules, which may be rate limiting for regulating phagolysosome fusion during infection. The direct consequence of NF-κB inhibition is the impaired delivery of lysosomal enzymes to M. smegmatis phagosomes and reduced killing. Thus, the established role of NF-κB in the innate immune response can now be expanded to include regulation of membrane trafficking during infection.

Exosomes are endosome-derived vesicles that are released when multi-vesicular bodies (MVBs) fuse with the plasma membrane. Exosomes released from mycobacteria-infected cells have recently been shown to be pro-inflammatory. A prominent host molecule that is found within these exosomes is Hsp70, a member of the heat-shock family of proteins. We first characterized the exosomes purified from control and mycobacteria-infected cells. We found that relative to uninfected cells, macrophages infected with M. smegmatis and M. avium release more exosomes and the exosomes they released had more Hsp70 on their surface. Both exosomes and exogenous Hsp70 treatment of macrophages led to NF-κB activation and TNFα release in uninfected macrophages; Hsp70 levels were elevated in mycobacteria-infected cells. Macrophage treatment with Hsp70 also led to increase in the phagocytosis and maturation of latex-bead phagosomes. Finally, Hsp70 pre-incubation of M. smegmatis- and M. avium-infected cells led to increased phago-lysosome fusion, as well as more killing of mycobacteria within macrophages. Our results fit into an emerging concept whereby exosomes-containing Hsp70 are effective inducers of inflammation, also in response to mycobacterial infection.