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Inflammasomes are important sentinels of innate immune defence, sensing pathogens and inducing cell death in infected cells1. There are several inflammasome sensors that each detect and respond to a specific pathogen- or damage-associated molecular pattern (PAMP or DAMP, respectively)1. During infection, live pathogens can induce the release of multiple PAMPs and DAMPs, which can simultaneously engage multiple inflammasome sensors2–5. Here we found that AIM2 regulates the innate immune sensors pyrin and ZBP1 to drive inflammatory signalling and a form of inflammatory cell death known as PANoptosis, and provide host protection during infections with herpes simplex virus 1 and Francisella novicida. We also observed that AIM2, pyrin and ZBP1 were members of a large multi-protein complex along with ASC, caspase-1, caspase-8, RIPK3, RIPK1 and FADD, that drove inflammatory cell death (PANoptosis). Collectively, our findings define a previously unknown regulatory and molecular interaction between AIM2, pyrin and ZBP1 that drives assembly of an AIM2-mediated multi-protein complex that we term the AIM2 PANoptosome and comprising multiple inflammasome sensors and cell death regulators. These results advance the understanding of the functions of these molecules in innate immunity and inflammatory cell death, suggesting new therapeutic targets for AIM2-, ZBP1- and pyrin-mediated diseases. AIM2 responds to infection with herpes simplex virus 1 or Francisella novicida by driving assembly of a large multi-protein complex containing multiple inflammasome sensors and cell death regulators.

In response to infection or sterile insults, inflammatory programmed cell death is an essential component of the innate immune response to remove infected or damaged cells. PANoptosis is a unique innate immune inflammatory cell death pathway regulated by multifaceted macromolecular complexes called PANoptosomes, which integrate components from other cell death pathways. Growing evidence shows that PANoptosis can be triggered in many physiological conditions, including viral and bacterial infections, cytokine storms, and cancers. However, PANoptosomes at the single cell level have not yet been fully characterized. Initial investigations have suggested that key pyroptotic, apoptotic, and necroptotic molecules including the inflammasome adaptor protein ASC, apoptotic caspase-8 (CASP8), and necroptotic RIPK3 are conserved components of PANoptosomes. Here, we optimized an immunofluorescence procedure to probe the highly dynamic multiprotein PANoptosome complexes across various innate immune cell death-inducing conditions. We first identified and validated antibodies to stain endogenous mouse ASC, CASP8, and RIPK3, without residual staining in the respective knockout cells. We then assessed the formation of PANoptosomes across innate immune cell death-inducing conditions by monitoring the colocalization of ASC with CASP8 and/or RIPK3. Finally, we established an expansion microscopy procedure using these validated antibodies to image the organization of ASC, CASP8, and RIPK3 within the PANoptosome. This optimized protocol, which can be easily adapted to study other multiprotein complexes and other cell death triggers, provides confirmation of PANoptosome assembly in individual cells and forms the foundation for a deeper molecular understanding of the PANoptosome complex and PANoptosis to facilitate therapeutic targeting.

There is strong evidence that nicotine can enhance cognitive functions and growing evidence that this effect may be larger in young healthy APOE e4 carriers. However, the moderating effects of the APOE e4 allele on cognitive impairments caused by nicotine deprivation in chronic smokers have not yet been studied with brain indices. We sought to determine whether young female carriers of the APOE e4 allele, relative to noncarriers, would exhibit larger abstinence-induced decreases in P3b amplitude during a two-stimulus auditory oddball task. We compared parietal P3bs in female chronic smokers with either APOE e3/e3 (n = 54) or e3/e4 (n = 20) genotype under nicotine-sated conditions and after 12–17-h nicotine deprivation. Nicotine deprivation significantly reduced P3b amplitudes in APOE e4 carriers, but not in APOE-e3/e3 individuals, such that the difference seen prior to nicotine deprivation was eliminated. The results suggest that subjects with the APOE e4 allele are more sensitive to nicotine, which could influence smoking patterns, the risk for nicotine dependence, and the cognitive effects of nicotine use in these individuals.

Inflammasomes are multimeric heterogeneous mega-Dalton protein complexes that play key roles in the host innate immune response to infection and sterile insults. Assembly of the inflammasome complex following infection or injury begins with the oligomerization of the upstream inflammasome-forming sensor and proceeds through a multistep process of well-coordinated events and downstream effector functions. Together, these steps enable elegant experimental readouts with which to reliably assess the successful activation of the inflammasome complex and cell death. Here, we describe a comprehensive protocol that details several in vitro (in bone marrow–derived macrophages) and in vivo (in mice) strategies for activating the inflammasome and explain how to subsequently assess multiple downstream effects in parallel to unequivocally establish the activation status of the inflammasome and cell death pathways. Our workflow assesses inflammasome activation via the formation of the apoptosis-associated speck-like protein containing a CARD (ASC) speck; cleavage of caspase-1 and gasdermin D; release of IL-1β, IL-18, caspase-1, and lactate dehydrogenase from the cell; and real-time analysis of cell death by imaging. Analyses take up to ~24 h to complete. Overall, our multifaceted approach provides a comprehensive and consistent protocol for assessing inflammasome activation and cell death. This protocol describes a toolbox for comprehensive characterization of inflammasome activation and cell death in response to both in vivo (in mice) and in vitro (using bone marrow–derived macrophages) models of infection, sterile insults, and cancer.

Chronic inflammation during many diseases is associated with bone loss. While interferons (IFNs) are often inhibitory to osteoclast formation, the complex role that IFN and interferon-stimulated genes (ISGs) play in osteoimmunology during inflammatory diseases is still poorly understood. We show that mice deficient in IFN signaling components including IFN alpha and beta receptor 1 (IFNAR1), interferon regulatory factor 1 (IRF1), IRF9, and STAT1 each have reduced bone density and increased osteoclastogenesis compared to wild type mice. The IFN-inducible guanylate-binding proteins (GBPs) on mouse chromosome 3 (GBP1, GBP2, GBP3, GBP5, GBP7) are required to negatively regulate age-associated bone loss and osteoclastogenesis. Mechanistically, GBP2 and GBP5 both negatively regulate in vitro osteoclast differentiation, and loss of GBP5, but not GBP2, results in greater age-associated bone loss in mice. Moreover, mice deficient in GBP5 or chromosome 3 GBPs have greater LPS-mediated inflammatory bone loss compared to wild type mice. Overall, we find that GBP5 contributes to restricting age-associated and inflammation-induced bone loss by negatively regulating osteoclastogenesis., The innate immune system and inflammation modulate bone homeostasis through complex regulation of bone remodelling cells including osteoblasts and osteoclasts. Here, the authors show that the type I interferon pathway and guanylate binding proteins functionally limit bone loss by inhibiting osteoclast functions.

Mice deficient in SHANK-associated RH domain-interacting protein (SHARPIN), a component of the linear ubiquitin chain assembly complex (LUBAC), develop a spontaneous inflammatory disorder with pathologic hallmarks similar to atopic dermatitis and psoriasis in humans. Previous studies identified the crucial role of components of the TNF and IL-1 signaling pathways in the progression of disease in SHARPIN-deficient mice. However, an innate immune adaptor or sensor that relates to the disease progression has remained unknown. In this study, we found that the genetic ablation of myeloid differentiation primary response 88 (MyD88) completely rescued skin inflammation in SHARPIN-deficient (Sharpin(cpdm)) mice. Systemic inflammation and immune cell dysregulation were partially rescued. Fibroblasts derived from Sharpin(cpdm)Myd88(−⁄−) mice failed to provide protection against TNF-induced cell death. Sharpin(cpdm)Myd88(−⁄−) mice had reduced TNF production in their skin. Furthermore, depletion of the microbiota through the oral administration of antibiotics (ABX) partially rescued both the skin inflammation and systemic inflammation, demonstrating a role for the gut microbiota in SHARPIN-deficient mice. Our findings suggest a detrimental role for the innate immune adaptor MyD88 in instigating skin inflammation in Sharpin(cpdm) mice.

The colon cancer survival rate is significantly affected by location, stage, and size of the cancer. Polypectomy was shown be as equally effective as surgery in early-stage colon cancer, but there have been no established clinical guidelines in the management of colon cancer based on the size of the polyp or the tumor location. The aim of our study was to assess the early-stage colon cancer-specific survival rate in patients who underwent endoscopic polypectomy versus surgery, based on size and location of tumor in early-stage colon cancer.This is a population-based nationwide study in the USA.Of 13,157 patients, 15.5% underwent endoscopic treatment and 84.5% underwent surgical therapy. For early cancer tumors located in the left colon, polypectomy yielded comparable 5-year survivals to surgery irrespective of size of the tumors. Five-year early cancer-specific survivals were similar for tumors located in the right colon that were 20 mm in size (94.5 vs 94.3%, p value = 0.94). However, tumors 20 mm in size that were located in the right colon had better survivals when treated surgically compared to those treated with polypectomy (20-39 mm: 91.8 vs 74.2%; ≥ 40 mm: 92.4 vs 60%, both p values 0.01). Similar results were obtained on propensity score analysis.Polypectomy was as effective as surgical therapy for small tumors. For larger tumors, surgical therapy is better than polypectomy for right-sided tumors, but both are equally effective for left-sided tumors.

Rapunzel syndrome is a rare entity with less than hundred case reports cited in the literature. In this, there is presence of a trichobezoar in the stomach which extends into the small intestine or beyond. It can typically cause abdominal pain and nausea, but can also present as an asymptomatic abdominal mass, progressing to abdominal obstruction and perforation. Many of these patients have associated psychiatric disorder. The gold standard for diagnosis is upper gastrointestinal endoscopy and treatment is surgical removal. Herein, we present surgical images of Rapunzel syndrome in a seven-year-old girl.

NLRs (nucleotide-binding domain and leucine-rich repeats) belong to a large family of cytoplasmic sensors that regulate an extraordinarily diverse range of biological functions. One of these functions is to contribute to immunity against infectious diseases, but dysregulation of their functional activity leads to the development of inflammatory and autoimmune diseases(1). Cytoplasmic innate immune sensors, including NLRs, are central regulators of intestinal homeostasis(2-9). NLRC3 (also known as CLR16.2 or NOD3) is a poorly characterized member of the NLR family and was identified in a genomic screen for genes encoding proteins bearing leucine-rich repeats (LRRs) and nucleotide-binding domains(10,1)1. Expression of NLRC3 is drastically reduced in the tumour tissue of patients with colorectal cancer compared to healthy tissues(12), highlighting an undefined potential function for this sensor in the development of cancer. Here we show that mice lacking NLRC3 are hyper-susceptible to colitis and colorectal tumorigenesis. The effect of NLRC3 is most dominant in enterocytes, in which it suppresses activation of the mTOR signalling pathways and inhibits cellular proliferation and stem-cell-derived organoid formation. NLRC3 associates with PI3Ks and blocks activation of the PI3K-dependent kinase AKT following binding of growth factor receptors or Toll-like receptor 4. These findings reveal a key role for NLRC3 as an inhibitor of the mTOR pathways, mediating protection against colorectal cancer.

Detrimental levels of intracellular reactive oxygen species engage a caspase-independent and non-inflammatory cell death called ‘oxeiptosis’ that serves as an important mechanism in diminishing inflammation.

Nod-like receptors (NLRs) have gained attention in recent years because of the ability of some family members to assemble into a multimeric protein complex known as the inflammasome. The role of NLRs and the inflammasome in regulating innate immunity against bacterial pathogens has been well studied. However, recent studies show that NLRs and inflammasomes also play a role during infections caused by protozoan parasites, which pose a significant global health burden. Herein, we review the diseases caused by the most common protozoan parasites in the world and discuss the roles of NLRs and inflammasomes in host immunity against these parasites.

Detection of bacterial ligands is a pre-requisite for inflammasome activation. During Pseudomonas aeruginosa infection, flagellin which is secreted through the T3SS is detected by the NLRC4 inflammasome. Activation of the NLRC4 inflammasome is believed to contribute to high IL-1β production and pathogenicity in cystic fibrosis patients with chronic P. aeruginosa infection. Interestingly, the majority of P. aeruginosa isolated from cystic fibrosis patients with chronic airway infection are non-motile and T3SS-negative, suggesting that yet un-characterized inflammasome pathways regulate IL-1β production in cystic fibrosis patients. Here we demonstrate the role of guanylate-binding proteins (GBPs) in regulating bacterial proliferation and inflammasome activation in response to T3SS-negative P. aeruginosa. Bacterial ligands liberated by the action of GBP2 and IRGB10 activate caspase-11 and regulate non-canonical NLRP3 inflammasome activation and IL-1β release. Overall, our results reveal the role of caspase-11 in inhibiting bacterial proliferation and promoting IL-1β secretion during T3SS-negative P. aeruginosa infection. This study suggests that non canonical inflammasomes might have co-evolved to detect Gram-negative bacterial pathogens that have evolved to bypass detection by canonical NLRs.

The field of innate immunity has been rapidly evolving and expanding its horizons during the past few years. 2018 was no exception, with the publication of several ground-breaking studies that bring into light new activators, regulators and signalling networks that drive innate immune responses and inflammation. The field of innate immunity has been rapidly evolving and expanding its horizons during the past few years. 2018 was no exception, with the publication of several ground-breaking studies that bring into light new activators, regulators and signalling networks that drive innate immune responses and inflammation.