The intracellular bacterium Francisella tularensis ensures its survival and proliferation within phagocytes of the infected host through phagosomal escape and cytosolic replication, to cause the disease tularemia. The cytokine interferon-c (IFN-c) is important in controlling primary infections in vivo, and in vitro intracellular proliferation of Francisella in macrophages, but its actual effects on the intracellular cycle of the bacterium are ambiguous. Here, we have performed an extensive analysis of the intracellular fate of the virulent F. tularensis subsp. tularensis strain Schu S4 in primary IFN-c-activated murine and human macrophages to understand how this cytokine controls Francisella proliferation. In both murine bone marrow-derived macrophages (muBMMs) and human blood monocyte-derived macrophages (MDMs), IFN-c controlled bacterial proliferation. Schu S4 growth inhibition was not due to a defect in phagosomal escape, since bacteria disrupted their phagosomes with indistinguishable kinetics in both muBMMs and MDMs, regardless of their activation state. Rather, IFN-c activation restricted cytosolic replication of Schu S4 in a manner independent of reactive oxygen or nitrogen species. Hence, IFN-c induces phagocyte NADPH oxidase Phox-and inducible nitric oxide synthase (iNOS)-independent cytosolic effector mechanisms that restrict growth of virulent Francisella in macrophages. INTRODUCTIONMacrophages are an essential component of innate host defences against microbial pathogens, and can recognize, phagocytose and destroy invading organisms through a repertoire of antimicrobial effectors. These include a degradative endosomal system that culminates in the delivery of degradative hydrolases to the maturing phagosome, the phagocyte NADPH oxidase Phox, an inducible nitric oxide synthase (iNOS) and cationic antimicrobial peptides, all of which act in conjunction to kill phagocytosed microbes (Radtke & O'Riordan, 2006). While many of these effectors are constitutively functional in macrophages, innate immune signals such as cytokines can further induce their expression and/or enhance their activities, therefore increasing the microbicidal potential of macrophages.Francisella tularensis is a highly virulent, Gram-negative, facultative intracellular bacterium that causes tularemia in a wide variety of mammals, including humans (Oyston et al., 2004). F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B) are the prominent cause of human tularemia, while F. tularensis subsp. novicida is considered non-pathogenic to humans (Ellis et al., 2002). Essential to Francisella pathogenesis is its ability to invade, survive and proliferate within mamAbbreviations: FAC, ferric ammonium citrate; IFN-c, interferon-c; iNOS, inducible nitric oxide synthase; LVS, live vaccine strain; MDM, human blood monocyte-derived macrophage; muBMM, murine bone marrowderived macrophage; NAC, N-acetyl-L-cysteine; NMMLA, N5-[imino(-methylamino)methyl]-L-ornithine; PEC, peritoneal exudate cells; pi, postinfection; RNS...

Scrub typhus is a neglected tropical disease, caused by Orientia tsutsugamushi, a Gram-negative bacterium that is transmitted to mammalian hosts during feeding by Leptotrombidium mites and replicates predominantly within endothelial cells. Most studies of scrub typhus in animal models have utilized either intraperitoneal or intravenous inoculation; however, there is limited information on infection by the natural route in murine model skin or its related early host responses. Here, we developed an intradermal (i.d.) inoculation model of scrub typhus and focused on the kinetics of the host responses in the blood and major infected organs. Following ear inoculation with 6 x 104 O. tsutsugamushi, mice developed fever at 11–12 days post-infection (dpi), followed by marked hypothermia and body weight loss at 14–19 dpi. Bacteria in blood and tissues and histopathological changes were detected around 9 dpi and peaked around 14 dpi. Serum cytokine analyses revealed a mixed Th1/Th2 response, with marked elevations of MCP-1/CCL2, MIP-1α/CCL3 and IL-10 at 9 dpi, followed by increased concentrations of pro-inflammatory markers (IL-6, IL-12, IFN-γ, G-CSF, RANTES/CCL5, KC/CCL11, IL-1α/β, IL-2, TNF-α, GM-CSF), as well as modulatory cytokines (IL-9, IL-13). Cytokine levels in lungs had similar elevation patterns, except for a marked reduction of IL-9. The Orientia 47-kDa gene and infectious bacteria were detected in several organs for up to 84 dpi, indicating persistent infection. This is the first comprehensive report of acute scrub typhus and persistent infection in i.d.-inoculated C57BL/6 mice. This is a significant improvement over current murine models for Orientia infection and will permit detailed studies of host immune responses and infection control interventions.

A vaccine based on outer membrane vesicles of pertussis (omvpV) is protective in a mouse-challenge model and induces a broad antibody and mixed Th1/Th2/Th17 response against multiple antigens following subcutaneous immunization. However, this route did not result in mucosal immunity and did not prevent nasopharyngeal colonization. in this study, we explored the potential of intranasal immunization with omvpV. only intranasal immunization induced strong mucosal immune responses that encompasses enhanced pulmonary and nasal IgA antibody levels, mainly directed against Vag8 and LpS. furthermore, high numbers of igA-and igG-producing plasma cells were detected as well as lung-resident IgA memory B-cells. Finally, only intranasal immunization induced pulmonary Th1/ Th17-related cytokine responses. The magnitude and type of systemic immunity was comparable between both routes and included high systemic igG antibody levels, strong igG-producing plasma cell responses, memory B-cells residing in the spleen and systemic Th1/Th2/Th17-related cytokine responses. importantly, only intranasal immunization prevented colonization in both the lungs and the nasal cavity. In conclusion, intranasal omvPV immunization induces mucosal IgA and Th17-mediated responses without influencing the systemic immunity profile. These responses resulted in prevention of Bordetella pertussis colonization in the respiratory tract, including the nasal cavity, thereby potentially preventing transmission.

Rickettsiae actively escape from vacuoles and replicate free in the cytoplasm of host cells, where inflammasomes survey the invading pathogens. In the present study, we investigated the interactions of Rickettsia australis with the inflammasome in both mouse and human macrophages. R. australis induced a significant level of IL-1β secretion by human macrophages, which was significantly reduced upon treatment with an inhibitor of caspase-1 compared to untreated controls, suggesting caspase-1-dependent inflammasome activation. Rickettsia induced significant secretion of IL-1β and IL-18 in vitro by infected mouse bone marrow-derived macrophages (BMMs) as early as 8–12 h post infection (p.i.) in a dose-dependent manner. Secretion of these cytokines was accompanied by cleavage of caspase-1 and was completely abrogated in BMMs deficient in caspase-1/caspase-11 or apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), suggesting that R. australis activate the ASC-dependent inflammasome. Interestingly, in response to the same quantity of rickettsiae, NLRP3-/- BMMs significantly reduced the secretion level of IL-1β compared to wild type (WT) controls, suggesting that NLRP3 inflammasome contributes to cytosolic recognition of R. australis in vitro. Rickettsial load in spleen, but not liver and lung, of R. australis-infected NLRP3-/- mice was significantly greater compared to WT mice. These data suggest that NLRP3 inflammasome plays a role in host control of bacteria in vivo in a tissue-specific manner. Taken together, our data, for the first time, illustrate the activation of ASC-dependent inflammasome by R. australis in macrophages in which NLRP3 is involved.