Commensal enteric bacteria lipopolysaccharide impairs host defense against disseminated Candida albicans fungal infection.

Commensal enteric bacteria maintain systemic immune responsiveness that protects against disseminated or localized infection in extra-intestinal tissues caused by pathogenic microbes. However, as shifts in infection susceptibility after commensal bacteria eradication have primarily been probed using viruses, the broader applicability to other pathogen types remains undefined. In sharp contrast to diminished antiviral immunity, we show commensal bacteria eradication bolsters protection against disseminated Candida albicans fungal infection. Enhanced antifungal immunity reflects more robust systemic expansion of Ly6G(hi)Ly6C(int) neutrophils, and their mobilization into infected tissues among antibiotic-treated compared with commensal bacteria-replete control mice. Reciprocally, depletion of neutrophils from expanded levels or intestinal lipopolysaccharide reconstitution overrides the antifungal protective benefits conferred by commensal bacteria eradication. This discordance in antifungal compared with antiviral immunity highlights intrinsic differences in how commensal bacteria control responsiveness for specific immune cell subsets, because pathogen-specific CD8(+) T cells that protect against viruses were suppressed similarly after C. albicans and influenza A virus infection. Thus, positive calibration of antiviral immunity by commensal bacteria is counterbalanced by restrained activation of other immune components that confer antifungal immunity.

Thymic independence of adaptive immunity to the intracellular pathogen Shigella flexneri serotype 2a.

Shigella flexneri is a facultative intracellular pathogen. While immunity to several intracellular pathogens is mediated by T lymphocytes, it is unknown whether cellular immune responses are important to adaptive immunity to S. flexneri. We show that vaccination with S. flexneri serotype 2a confers protection to mice that lack T lymphocytes or gamma interferon (IFN-gamma), specific depletion of T lymphocytes does not alter the protection, and adoptive transfer of splenocytes from vaccinated mice does not confer protection to naive mice. In contrast, vaccination conferred no protection to mice that lack B lymphocytes and adoptive transfer of immune sera conferred partial protection to naive mice. These data demonstrate that in the mouse bronchopulmonary model, adaptive immunity to S. flexneri 2a is an antibody-mediated, B-lymphocyte-dependent process and can be generated in the absence of T lymphocytes or IFN-gamma.

Adaptive immune response to Shigella flexneri 2a cydC in immunocompetent mice and mice lacking immunoglobulin A.

Shigella flexneri cydC, which is deficient in cytochrome bd, was rapidly cleared from the lungs of intranasally inoculated mice and was Sereny negative, yet it induced 93% protection against challenge with wild-type S. flexneri. Mice that lack immunoglobulin A (IgA) were fully protected, suggesting that IgA may not be required for adaptive immunity in this model system.

Impact of either elevated or decreased levels of cytochrome bd expression on Shigella flexneri virulence.

Shigella spp. are the major cause of bacillary dysentery worldwide. The pathogenic process involves bacterial invasion and lysis of the phagocytic vacuole, followed by replication and movement within the cell cytoplasm and, ultimately, spread directly into adjacent cells. This study demonstrates that S. flexneri cytochrome bd expression is necessary for normal intracellular survival and virulence. Cytochrome bd is one of two terminal oxidases in the bacterial respiratory chain that reduce molecular oxygen to water, utilizing intermediates shuttled through the electron transport chain. S. flexneri mutants that contain a disruption in the cydC locus, which leads to defective cytochrome bd expression, or in the riboflavin (ribE) or ubiquinol-8 (ubiH) biosynthetic pathway, which leads to elevated cytochrome bd expression, were evaluated in intracellular survival and virulence assays. The cydC mutant formed significantly smaller plaques, had significantly decreased intracellular survival, and had a 100-fold increase in lethal dose for mice compared with the wild type. The ribE and ubiH mutants each formed significantly larger plaques and had a 10-fold decrease in lethal dose for mice compared with the wild type. The data indicate that expression of cytochrome bd is required for S. flexneri intracellular survival and virulence.

Clearance of Shigella flexneri infection occurs through a nitric oxide-independent mechanism.

Nitric oxide (NO) generated by gamma interferon (IFN-gamma) activation of macrophages mediates the killing of many intracellular pathogens. IFN-gamma is essential to innate resistance to Shigella flexneri infection. We demonstrate that NO is produced following S. flexneri infection both in mice and in activated cells in vitro and that while it is able to kill S. flexneri in a cell-free system, it is not required for clearance of S. flexneri in either infected mice or in activated cells in vitro.

An essential role for gamma interferon in innate resistance to Shigella flexneri infection.

Shigella spp. are the major cause of bacillary dysentery worldwide. To identify immune effectors associated with protection of the naive host during infection, the susceptibility to pulmonary Shigella infection of each of various mouse strains that have a targeted deletion in a specific aspect of the immune system was evaluated. Our results demonstrate that mice deficient in gamma interferon are 5 orders of magnitude more susceptible to Shigella than are wild-type mice, whereas mice deficient in B and T lymphocytes or in T lymphocytes alone exhibit no difference in susceptibility. Significantly lower numbers of shigellae were recovered from immunocompetent compared with gamma-interferon-deficient mice after infection. While immunocompetent mice were able to clear a sublethal Shigella inoculum by day 5 postinfection, progressively increasing numbers of shigellae were cultured from the lungs of gamma interferon-deficient mice over the same period. Histopathology of the lungs from immunocompetent mice infected with a sublethal Shigella inoculum showed mild inflammatory changes, whereas the lungs from gamma interferon-deficient mice demonstrated progressively worsening acute bronchiolitis with ulceration. Further, the time to death in gamma interferon-deficient mice correlates inversely with the size of the Shigella inoculum. To identify the cellular source of gamma interferon, we infected SCID mice, T-cell-receptor-deficient mice, beige mice (a mouse strain deficient in natural killer [NK] cell activity), and mice depleted of NK cells using anti-asialo-GM1. Each NK cell-deficient mouse strain exhibited a 10-fold-greater susceptibility to Shigella infection than immunocompetent mice. To test the protective effects of gamma interferon in vitro, survival of intracellular Shigella was examined in primary macrophages from wild-type mice, primary macrophages from gamma interferon-deficient mice, a macrophage cell line, and a fibroblast cell line. Following activation with gamma interferon, each cell type eradicated intracellular Shigella, while nonactivated macrophages fostered Shigella replication and nonactivated fibroblast cells fostered both Shigella replication and intercellular spread. Taken together, these data establish that NK cell-mediated gamma interferon is essential to resistance following primary Shigella infection.