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Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a human pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome (HUS). Shiga toxin (Stx), the primary virulence factor of EHEC, mediates much of the morbidity and mortality associated with EHEC infection. Although Stx is well characterized in terms of its toxic activity, it is not clear if Stx has a specific role in immune cells during EHEC infection. In this study, we identified an inflammasome inhibitory role for Stx during EHEC infection. Stx specifically suppressed the activation of caspase-11-mediated cytosolic LPS sensing pathway in macrophages and mice. Stx did not inhibit the production of inflammasome-independent cytokines, TNF and IL-6. Similarly, Stx did not impair the activation of AIM2, NLRC4, or the canonical NLRP3 inflammasomes. Multiples subtypes of stx were able to suppress the caspase-11-mediated inflammasome responses. Stx was also capable of suppressing inflammasome responses induced by intraperitoneal injection of LPS as well as by Citrobacter rodentium infection in mice. Together, these data reveal that Stx plays an important role in overcoming inflammasome responses during EHEC infection.

Interferon-inducible human oligoadenylate synthetase-like (OASL) and its mouse ortholog, Oasl2, enhance RNA-sensor RIG-I-mediated type I interferon (IFN) induction and inhibit RNA virus replication. Here, we show that OASL and Oasl2 have the opposite effect in the context of DNA virus infection. In Oasl2-/- mice and OASL-deficient human cells, DNA viruses such as vaccinia, herpes simplex, and adenovirus induced increased IFN production, which resulted in reduced virus replication and pathology. Correspondingly, ectopic expression of OASL in human cells inhibited IFN induction through the cGAS-STING DNA-sensing pathway. cGAS was necessary for the reduced DNA virus replication observed in OASL-deficient cells. OASL directly and specifically bound to cGAS independently of double-stranded DNA, resulting in a non-competitive inhibition of the second messenger cyclic GMP-AMP production. Our findings define distinct mechanisms by which OASL differentially regulates host IFN responses during RNA and DNA virus infection and identify OASL as a negative-feedback regulator of cGAS.

Inflammasomes are cytosolic multi-molecular complexes that sense intracellular microbial danger signals and metabolic perturbations. Inflammasome activation leads to the activation of caspase-1 and the release of pro-inflammatory cytokines IL-1β and IL-18 accompanied by cell death. An inflammasome-based surveillance machinery for Gram-negative bacterial infections has been recently discovered. This noncanonical inflammasome relies on sensing the cytosolic presence of lipopolysaccharide (LPS) of Gram-negative bacteria via inflammatory caspases such as caspase-4, -5, and -11. This Review discusses the recent findings related to the mechanism of activation of the noncanonical inflammasome and its biological functions.

The introduction of metal-on-metal total disc replacements motivated studies to evaluate the effects of cobalt-chromium (CoCr) nanoparticles on cells of the dura mater. Porcine fibroblasts and epithelial cells isolated from the dura mater were cultured with clinically-relevant CoCr nanoparticles and the ions, generated by the particles over 24 h, at doses up to 121 μm(3)per cell. Cell viability and production of proinflammatory cytokines was assessed over 4 days. The capacity of the particles to induce oxidative stress in the cells was evaluated at 24 h. The CoCr particles and their ions significantly reduced the viability of the dural epithelial cells in a dose-dependent manner but not the fibroblasts. Both cell types secreted IL-8 in response to particle exposure at doses of 60.5 μm(3) (epithelial cells) and 121 μm(3) (fibroblasts, epithelial cells) per cell. No significant release of IL-6 was observed in both cell types at any dose. Reactive oxygen species were induced in both cell types at 50 μm(3) per cell after 24 h exposure. The data suggested novel differences in the resistance of the dural epithelial cells and fibroblasts to CoCr nanoparticle/ion toxicity and demonstrated the inflammatory potential of the particles. The data contributes to a greater understanding of the potential biological consequences of the use of metal-on-metal total disc prostheses.

Sensing of lipopolysaccharide (LPS) in the cytosol triggers caspase-11 activation and is central to host defense against Gram-negative bacterial infections and to the pathogenesis of sepsis. Most Gram-negative bacteria that activate caspase-11, however, are not cytosolic, and the mechanism by which LPS from these bacteria gains access to caspase-11 in the cytosol remains elusive. Here, we identify outer membrane vesicles (OMVs) produced by Gram-negative bacteria as a vehicle that delivers LPS into the cytosol triggering caspase-11-dependent effector responses in vitro and in vivo. OMVs are internalized via endocytosis, and LPS is released into the cytosol from early endosomes. The use of hypovesiculating bacterial mutants, compromised in their ability to generate OMVs, reveals the importance of OMVs in mediating the cytosolic localization of LPS. Collectively, these findings demonstrate a critical role for OMVs in enabling the cytosolic entry of LPS and, consequently, caspase-11 activation during Gram-negative bacterial infections.

Summary Inflammasome-activated caspase-1 cleaves gasdermin D to unmask its pore-forming activity, the predominant consequence of which is pyroptosis. Here, we report an additional biological role for gasdermin D in limiting cytosolic DNA surveillance. Cytosolic DNA is sensed by Aim2 and cyclic GMP-AMP synthase (cGAS) leading to inflammasome and type I interferon responses, respectively. We found that gasdermin D activated by the Aim2 inflammasome suppressed cGAS-driven type I interferon response to cytosolic DNA and Francisella novicida in macrophages. Similarly, interferon-β (IFN-β) response to F. novicida infection was elevated in gasdermin D-deficient mice. Gasdermin D-mediated negative regulation of IFN-β occurred in a pyroptosis-, interleukin-1 (IL-1)-, and IL-18-independent manner. Mechanistically, gasdermin D depleted intracellular potassium (K+) via membrane pores, and this K+ efflux was necessary and sufficient to inhibit cGAS-dependent IFN-β response. Thus, our findings have uncovered an additional interferon regulatory module involving gasdermin D and K+ efflux.

The innate immune system is essential for detection and elimination of pathogenic microbes. Francisella is an intracellular pathogen that replicates in the cytosol of macrophages. In the cytosol, it can be recognized by host defense sensors. Francisella is sensed by cyclic GMP-AMP synthase (c-GAS) leading to the production of type I Interferons (IFN). It can also be recognized by the PYHIN DNA sensor absent in melanoma (AIM2) resulting in the activation of inflammasome responses i.e. production of pro-inflammatory cytokines IL-1b and IL-18 as well as pyroptosis. While mice deficient in inflammasome components are more susceptible to Francisella infection, type I interferon signaling deficient mice are resistant to Francisella bacterial infection. Inflammasome activation results in gasdermin-D cleavage leading to pyroptotic cell death. Our results show that gasdermin-D activation plays an important role in the nature of cytokine responses to Francisella infection, therefore influencing the outcome of the infection. These findings have major implications in understanding the balance of cytokine production in response to cytosolic immune surveillance.

Septic shock is the leading cause of morbidity and mortality in intensive care units worldwide. Gram-negative bacteria constitute one of the most common causes of sepsis that results in high fatality by initiating an excessive and uncontrolled host inflammatory response. At the epicenter of this response is the innate immune detection of lipopolysaccharides (LPS). In addition to TLR4 recognition of LPS, recent studies revealed a new LPS sensing mechanism in the cytosol. Inflammatory caspases, such as caspase-11, detect LPS to execute pyroptosis, an inflammatory form of cell death, and caspase-1 activation. A prerequisite for the activation of noncanonical inflammasome is the transcriptional induction of caspase-11, which is mediated by TLR4-TRIF, type I interferon, and complement signaling. Additionally, guanylate binding proteins (GBPs), interferon regulatory factor 1, and IRGB10 orchestrate the release of LPS from the vacuolar bacterial pathogens into the cytosol. While the host factors involved in LPS activation of caspase-11 are fairly characterized, the role of bacterial factors in this process is not clear. Our new findings from this study delineate the bacterial components necessary for the optimal engagement of the cytosolic LPS sensing pathway and eliciting noncanonical inflammasome responses.

Sensing of lipopolysaccharide (LPS) in the cytosol triggers caspase-11 activation and is central to host defense against Gram-negative bacterial infections and to the pathogenesis of sepsis. Most Gram-negative bacteria that activate caspase-11 however are not cytosolic and the underlying mechanism by which LPS from these bacteria gains access to caspase-11 in the cytosol remains elusive. Here we identify outer membrane vesicles (OMV) produced by Gram-negative bacteria as a vehicle that delivers LPS into the cytosol triggering caspase-11-dependent effector responses in vitro and in vivo. OMV are internalized by macrophages and LPS is released into the cytosol from early and late endosomes. The use of hypovesiculating bacterial mutants, compromised in their ability to generate OMV, reveal the importance of OMV in mediating the cytosolic localization of LPS. Collectively, these findings demonstrate a critical role for OMV in enabling the cytosolic entry of LPS and consequently caspase-11 activation during Gram-negative bacterial infections. In broader terms, our data reveal a fundamental mechanism that serves as a conduit to deliver pathogen-associated molecular patterns (PAMP) to the cytosol to alert the immune system. Our data suggest that OMV with the cargo of immunostimulatory PAMP represent a cardinal sign of infection with not only viable but also multiplying bacteria consistent with the “patterns of pathogenesis” hypothesis.

Pathogen and cellular by-products released during infection or trauma are critical for initiating mucosal inflammation. This was studied in mice undergoing pulmonary inflammation after Staphylococcal enterotoxin A (SEA) inhalation. A potent pro-inflammatory factor was detected using a sensitive bioassay suggesting the presence of an alarmin, a danger-associated molecular pattern (DAMP), and/or a cytokine. Highly purified bronchoalveolar lavage fluid (BALF) fractions obtained by sequential chromatography were screened for bioactivity and subjected to mass spectrometry. SEA was the only identified protein with known inflammatory potential, and unexpectedly, it co-purified with immunosuppressive proteins. Among them was lactoferrin, which inhibited SEA and anti-CD3/-CD28 stimulation by promoting T cell death and reducing TNF synthesis. Higher doses of lactoferrin were required to inhibit effector compared to resting T cells. This inhibition relied on the continual presence of lactoferrin rather than a programming event. The data show that bioactive SEA is constituted in a mucosal niche within BALF even after the initiation of inflammation. These results may have clinical value in human diagnostic settings since traces levels of SEA can be detected using a sensitive bioassay, and may help pinpoint potential mediators of lung inflammation when molecular approaches fail. Submitted to American Journal of Respiratory Cell and Molecular Biology.