Rapid induction of inflammatory lipid mediators by the inflammasome in vivo.

Detection of microbial products by host inflammasomes is an important mechanism of innate immune surveillance. Inflammasomes activate the caspase-1 (CASP1) protease, which processes the cytokines interleukin (IL)-1ß and IL-18, and initiates a lytic host cell death called pyroptosis. To identify novel CASP1 functions in vivo, we devised a strategy for cytosolic delivery of bacterial flagellin, a specific ligand for the NAIP5 (NLR family, apoptosis inhibitory protein 5)/NLRC4 (NLR family, CARD-domain-containing 4) inflammasome. Here we show that systemic inflammasome activation by flagellin leads to a loss of vascular fluid into the intestine and peritoneal cavity, resulting in rapid (less than 30 min) death in mice. This unexpected response depends on the inflammasome components NAIP5, NLRC4 and CASP1, but is independent of the production of IL-1ß or IL-18. Instead, inflammasome activation results, within minutes, in an 'eicosanoid storm'--a pathological release of signalling lipids, including prostaglandins and leukotrienes, that rapidly initiate inflammation and vascular fluid loss. Mice deficient in cyclooxygenase-1, a critical enzyme in prostaglandin biosynthesis, are resistant to these rapid pathological effects of systemic inflammasome activation by either flagellin or anthrax lethal toxin. Inflammasome-dependent biosynthesis of eicosanoids is mediated by the activation of cytosolic phospholipase A(2) in resident peritoneal macrophages, which are specifically primed for the production of eicosanoids by high expression of eicosanoid biosynthetic enzymes. Our results therefore identify eicosanoids as a previously unrecognized cell-type-specific signalling output of the inflammasome with marked physiological consequences in vivo.

The Action Research Program: Experiential Learning in Systems-Based Practice for First-Year Medical Students.

PROBLEM: Systems-based practice focuses on the organization, financing, and delivery of medical services. The American Association of Medical Colleges has recommended that systems-based practice be incorporated into medical schools' curricula. However, experiential learning in systems-based practice, including practical strategies to improve the quality and efficiency of clinical care, is often absent from or inconsistently included in medical education. INTERVENTION: A multidisciplinary clinician and nonclinician faculty team partnered with a cardiology outpatient clinic to design a 9-month clerkship for 1st-year medical students focused on systems-based practice, delivery of clinical care, and strategies to improve the quality and efficiency of clinical operations. The clerkship was called the Action Research Program. In 2013-2014, 8 trainees participated in educational seminars, research activities, and 9-week clinic rotations. A qualitative process and outcome evaluation drew on interviews with students, clinic staff, and supervising physicians, as well as students' detailed field notes. CONTEXT: The Action Research Program was developed and implemented at the University of California, San Francisco, an academic medical center in the United States. All educational activities took place at the university's medical school and at the medical center's cardiology outpatient clinic. OUTCOME: Students reported and demonstrated increased understanding of how care delivery systems work, improved clinical skills, growing confidence in interactions with patients, and appreciation for patients' experiences. Clinicians reported increased efficiency at the clinic level and improved performance and job satisfaction among medical assistants as a result of their unprecedented mentoring role with students. Some clinicians felt burdened when students shadowed them and asked questions during interactions with patients. Most student-led improvement projects were not fully implemented. LESSONS LEARNED: The Action Research Program is a small pilot project that demonstrates an innovative pairing of experiential and didactic training in systems-based practice. Lessons learned include the need for dedicated time and faculty support for students' improvement projects, which were the least successful aspect of the program. We recommend that future projects aiming to combine clinical training and quality improvement projects designate distinct blocks of time for trainees to pursue each of these activities independently. In 2014-2015, the University of California, San Francisco School of Medicine incorporated key features of the Action Research Program into the standard curriculum, with plans to build upon this foundation in future curricular innovations.

Differential requirements for NAIP5 in activation of the NLRC4 inflammasome.

Inflammasomes are cytosolic multiprotein complexes that assemble in response to infectious or noxious stimuli and activate the CASPASE-1 protease. The inflammasome containing the nucleotide binding domain-leucine-rich repeat (NBD-LRR) protein NLRC4 (interleukin-converting enzyme protease-activating factor [IPAF]) responds to the cytosolic presence of bacterial proteins such as flagellin or the inner rod component of bacterial type III secretion systems (e.g., Salmonella PrgJ). In some instances, such as infection with Legionella pneumophila, the activation of the NLRC4 inflammasome requires the presence of a second NBD-LRR protein, NAIP5. NAIP5 also is required for NLRC4 activation by the minimal C-terminal flagellin peptide, which is sufficient to activate NLRC4. However, NLRC4 activation is not always dependent upon NAIP5. In this report, we define the molecular requirements for NAIP5 in the activation of the NLRC4 inflammasome. We demonstrate that the N terminus of flagellin can relieve the requirement for NAIP5 during the activation of the NLRC4 inflammasome. We also demonstrate that NLRC4 responds to the Salmonella protein PrgJ independently of NAIP5. Our results indicate that NAIP5 regulates the apparent specificity of the NLRC4 inflammasome for distinct bacterial ligands.

Lethal inflammasome activation by a multidrug-resistant pathobiont upon antibiotic disruption of the microbiota.

The mammalian intestine harbors a complex microbial community that provides numerous benefits to its host. However, the microbiota can also include potentially virulent species, termed pathobiont, which can cause disease when intestinal homeostasis is disrupted. The molecular mechanisms by which pathobionts cause disease remain poorly understood. Here we describe a sepsis-like disease that occurs upon gut injury in antibiotic-treated mice. Sepsis was associated with the systemic spread of a specific multidrug-resistant Escherichia coli pathobiont that expanded markedly in the microbiota of antibiotic-treated mice. Rapid sepsis-like death required a component of the innate immune system, the Naip5-Nlrc4 inflammasome. In accordance with Koch's postulates, we found the E. coli pathobiont was sufficient to activate Naip5-Nlrc4 and cause disease when injected intravenously into unmanipulated mice. These findings reveal how sepsis-like disease can result from recognition of pathobionts by the innate immune system.