Summary Intestinal epithelial cells (IECs) form a critical barrier against pathogen invasion. By generation of mice in which inflammasome expression is restricted to IECs, we describe a coordinated epithelium-intrinsic inflammasome response in vivo. This response was sufficient to protect against Salmonella tissue invasion, and involved a previously reported IEC expulsion that was coordinated with lipid mediator and cytokine production, and lytic IEC death. Excessive inflammasome activation in IECs was sufficient to result in diarrhea and pathology. Experiments with IEC organoids demonstrated that IEC expulsion did not require other cell types. IEC expulsion was accompanied by a major actin rearrangement in neighboring cells that maintained epithelium integrity, but, did not absolutely require Caspase-1 or Gasdermin D. Analysis of Casp1−/−Casp8−/− mice revealed a functional Caspase-8 inflammasome in vivo. Thus, a coordinated IEC-intrinsic, Caspase-1 and -8 inflammasome response plays a key role in intestinal immune defense and pathology.

Purpose: Early detection would significantly decrease the mortality rate of ovarian cancer. In this study, we characterize and validate the combination of six serum biomarkers that discriminate between disease-free and ovarian cancer patients with high efficiency. Experimental Design: We analyzed 362 healthy controls and 156 newly diagnosed ovarian cancer patients. Concentrations of leptin, prolactin, osteopontin, insulin-like growth factor II, macrophage inhibitory factor, and CA-125 were determined using a multiplex, bead-based, immunoassay system. All six markers were evaluated in a training set (181 samples from the control group and 113 samples from OC patients) and a test set (181sample control group and 43 ovarian cancer). Results: Multiplex and ELISA exhibited the same pattern of expression for all the biomarkers. None of the biomarkers by themselves were good enough to differentiate healthy versus cancer cells. However, the combination of the six markers provided a better differentiation than CA-125. Four models with <2% classification error in training sets all had significant improvement (sensitivity 84%-98% at specificity 95%) over CA-125 (sensitivity 72% at specificity 95%) in the test set. The chosen model correctly classified 221out of 224 specimens in the test set, with a classification accuracy of 98.7%. Conclusions: We describe the first blood biomarker test with a sensitivity of 95.3% and a specificity of 99.4% for the detection of ovarian cancer. Six markers provided a significant improvement over CA-125 alone for ovarian cancer detection. Validation was performed with a blinded cohort. This novel multiplex platform has the potential for efficient screening in patients who are at high risk for ovarian cancer.

Coordinated recombination of homologous antigen receptor loci is thought to be important for allelic exclusion. Here, we show that homologous Ig alleles pair in a stage-specific manner that mirrors the recombination patterns of these loci. The frequency of homologous Ig pairing was substantially reduced in the absence of the RAG1-RAG2 recombinase and was rescued in Rag1-/- developing B cells with a transgene expressing a RAG1 active site mutant that supports DNA binding but not cleavage. The introduction of DNA breaks on one Ig allele induced ATM-dependent repositioning of the other allele to pericentromeric heterochromatin. ATM activated by the cleaved allele acts in trans on the uncleaved allele to prevent bi-allelic recombination and chromosome breaks or translocations.

SUMMARY NLR (nucleotide-binding domain [NBD]- and leucine-rich repeat [LRR]-containing) proteins mediate innate immune sensing of pathogens in mammals and plants. How NLRs detect their cognate stimuli remains poorly understood. Here, we analyzed ligand recognition by NAIP (NLR Apoptosis Inhibitory Protein) inflammasomes. Mice express multiple highly related NAIP paralogs that recognize distinct bacterial proteins. We analyzed a panel of 43 chimeric NAIPs, allowing us to map the NAIP domain responsible for specific ligand detection. Surprisingly, ligand specificity was mediated not by the LRR domain, but by an internal region encompassing several NBD-associated α-helical domains. Interestingly, we find that the ligand specificity domain has evolved under positive selection in both rodents and primates. We further show that ligand binding is required for the subsequent co-oligomerization of NAIPs with the downstream signaling adaptor NLRC4 (NLR family, CARD-containing 4). These data provide a molecular basis for how NLRs detect ligands and assemble into inflammasomes.

Robust innate immune detection of rapidly evolving pathogens is critical for host defense. Nucleotide-binding domain leucine-rich repeat (NLR) proteins function as cytosolic innate immune sensors in plants and animals. However, the structural basis for ligand-induced NLR activation has so far remained unknown. NAIP5 (NLR family, apoptosis inhibitory protein 5) binds the bacterial protein flagellin and assembles with NLRC4 to form a multiprotein complex called an inflammasome. Here we report the cryo–electron microscopy structure of the assembled ~1.4-megadalton flagellin-NAIP5-NLRC4 inflammasome, revealing how a ligand activates an NLR. Six distinct NAIP5 domains contact multiple conserved regions of flagellin, prying NAIP5 into an open and active conformation. We show that innate immune recognition of multiple ligand surfaces is a generalizable strategy that limits pathogen evolution and immune escape.