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Circulating tumor cells (CTC) seed cancer metastases; however, the underlying cellular and molecular mechanisms remain unclear. CTC clusters were less frequently detected but more metastatic than single CTCs of patients with triple-negative breast cancer and representative patient-derived xenograft models. Using intravital multiphoton microscopic imaging, we found that clustered tumor cells in migration and circulation resulted from aggregation of individual tumor cells rather than collective migration and cohesive shedding. Aggregated tumor cells exhibited enriched expression of the breast cancer stem cell marker CD44 and promoted tumorigenesis and polyclonal metastasis. Depletion of CD44 effectively prevented tumor cell aggregation and decreased PAK2 levels. The intercellular CD44–CD44 homophilic interactions directed multicellular aggregation, requiring its N-terminal domain, and initiated CD44–PAK2 interactions for further activation of FAK signaling. Our studies highlight that CD44+ CTC clusters, whose presence is correlated with a poor prognosis of patients with breast cancer, can serve as novel therapeutic targets of polyclonal metastasis.Significance:CTCs not only serve as important biomarkers for liquid biopsies, but also mediate devastating metastases. CD44 homophilic interactions and subsequent CD44–PAK2 interactions mediate tumor cluster aggregation. This will lead to innovative biomarker applications to predict prognosis, facilitate development of new targeting strategies to block polyclonal metastasis, and improve clinical outcomes.See related commentary by Rodrigues and Vanharanta, p. 22.This article is highlighted in the In This Issue feature, p. 1

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List of Videos S1-8 Tables S1-7 and Methods

The redox-based modifications of cysteine residues in proteins regulate their function in many biological processes. The gas molecule H(2)S has been shown to persulfidate redox sensitive cysteine residues resulting in an H(2)S-modified proteome known as the sulfhydrome. Tandem Mass Tags (TMT) multiplexing strategies for large-scale proteomic analyses have become increasingly prevalent in detecting cysteine modifications. Here we developed a TMT-based proteomics approach for selectively trapping and tagging cysteine persulfides in the cellular proteomes. We revealed the natural protein sulfhydrome of two human cell lines, and identified insulin as a novel substrate in pancreatic beta cells. Moreover, we showed that under oxidative stress conditions, increased H(2)S can target enzymes involved in energy metabolism by switching specific cysteine modifications to persulfides. Specifically, we discovered a Redox Thiol Switch, from protein S-glutathioinylation to S-persulfidation (RTS(GS)). We propose that the RTS(GS) from S-glutathioinylation to S-persulfidation is a potential mechanism to fine tune cellular energy metabolism in response to different levels of oxidative stress.

Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.

The recognition and cleavage of gasdermin D (GSDMD) by inflammatory caspases-1, 4, 5, and 11 are essential steps in initiating pyroptosis after inflammasome activation. Previous work has identified cleavage site signatures in substrates such as GSDMD, but it is unclear whether these are the sole determinants for caspase engagement. Here we report the crystal structure of a complex between human caspase-1 and the full-length murine GSDMD. In addition to engagement of the GSDMD N- and C-domain linker by the caspase-1 active site, an anti-parallel β sheet at the caspase-1 L2 and L2' loops bound a hydrophobic pocket within the GSDMD C-terminal domain distal to its N-terminal domain. This "exosite" interface endows an additional function for the GSDMD C-terminal domain as a caspase-recruitment module besides its role in autoinhibition. Our study thus reveals dual-interface engagement of GSDMD by caspase-1, which may be applicable to other physiological substrates of caspases.

Exomic studies have demonstrated that innate immune genes exhibit an even higher degree of variation than the majority of other gene families. However, the phenotypic implications of this genetic variation are not well understood, with effects ranging from hypomorphic to silent to hyperfunctioning. In this work, we study the functional consequences of this variation by investigating polymorphisms in gasdermin D, the key pyroptotic effector protein. We find that, although SNPs affecting potential posttranslational modifications did not affect gasdermin D function or pyroptosis, polymorphisms disrupting sites predicted to be structurally important dramatically alter gasdermin D function. The manner in which these polymorphisms alter function varies from conserving normal pyroptotic function to inhibiting caspase cleavage to disrupting oligomerization and pore formation. Further, downstream of inflammasome activation, polymorphisms that cause loss of gasdermin D function convert inflammatory pyroptotic cell death into immunologically silent apoptotic cell death. These findings suggest that human genetic variation can alter mechanisms of cell death in inflammation.

Pyroptosis, a lytic form of programmed cell death, both stimulates effective immune responses and causes tissue damage. Gasdermin (GSDM) proteins are a family of pore-forming executors of pyroptosis. While the most-studied member, GSDMD, exerts critical functions in inflammasome biology, emerging evidence demonstrates potential broad relevance for GSDM-mediated pyroptosis across diverse pathologies. In this review, we describe GSDM biology, outline conditions where inflammasomes and GSDM-mediated pyroptosis represent rational therapeutic targets, and delineate strategies to manipulate these central immunologic processes for the treatment of human disease.

Gasdermins are a family of structurally-related proteins originally described for their role in pyroptosis. Gasdermin B (GSDMB) is currently the least studied, and while its association with genetic susceptibility to chronic, mucosal inflammatory disorders is well-established, little is known of its functional relevance during active disease states. Herein, we report increased GSDMB in inflammatory bowel disease, with single-cell analysis identifying epithelial specificity to inflamed colonocytes/crypt top colonocytes. Surprisingly, mechanistic experiments and transcriptome profiling reveal lack of inherent GSDMB-dependent pyroptosis in activated epithelial cells and organoids, but instead, point to increased proliferation and migration during in vitro wound closure, which arrests in GSDMB-deficient cells that display hyper-adhesiveness and enhanced formation of vinculin-based actomyosin stress fibers dependent on PDGF-A-mediated FAK phosphorylation. Importantly, carriage of disease-associated GSDMB SNPs confers functional defects disrupting epithelial restitution/repair, which altogether, establishes GSDMB as a critical factor for restoration of epithelial barrier function and the resolution of inflammation.

The inflammasomes are signaling platforms that promote the activation of inflammatory caspases such as caspases-1, -4, -5, and -11. Recent studies identified gasdermin D (GSDMD) as an effector for pyroptosis downstream of the inflammasome signaling pathways. Cleavage of GSDMD by inflammatory caspases allows its N-terminal domain to associate with membrane lipids and form pores that induce pyroptotic cell death. Despite the important role of GSDMD in pyroptosis, the molecular mechanisms of GSDMD recognition and cleavage by inflammatory caspases that trigger pyroptosis are poorly understood. Here, we demonstrate that the catalytic domains of inflammatory caspases can directly bind to both the full-length GSDMD and its cleavage site peptide, FLTD. A GSDMD-derived inhibitor, N-acetyl-Phe-Leu-Thr-Asp-chloromethylketone (Ac-FLTD-CMK), inhibits GSDMD cleavage by caspases-1, -4, -5, and -11 in vitro, suppresses pyroptosis downstream of both canonical and noncanonical inflammasomes, as well as reduces IL-1β release following activation of the NLRP3 inflammasome in macrophages. By contrast, the inhibitor does not target caspase-3 or apoptotic cell death, suggesting that Ac-FLTD-CMK is a specific inhibitor for inflammatory caspases. Crystal structure of caspase-1 in complex with Ac-FLTD-CMK reveals extensive enzyme–inhibitor interactions involving both hydrogen bonds and hydrophobic contacts. Comparison with other caspase-1 structures demonstrates drastic conformational changes at the four active-site loops that assemble the catalytic groove. The present study not only contributes to our understanding of GSDMD recognition by inflammatory caspases but also reports a specific inhibitor for these caspases that can serve as a tool for investigating inflammasome signaling.

Heightened and extended inflammation underlies the pathogenesis of many disorders, including inflammatory bowel disease, sepsis, and inflammatory arthritis. Ubiquitin networks help dictate the strength and duration of inflammatory signaling. In innate immunity, the itchy E3 ubiquitin protein ligase (ITCH)-A20 ubiquitin–editing complex inhibits receptor-interacting Ser/Thr kinase (RIPK) activation by removing Lys-63–linked polyubiquitinated chains from key proteins in the nuclear factor kappa B (NF-κB) signaling pathway. The complex then attaches polyubiquitinated chains to these proteins to target them for lysosomal or proteasomal destruction. ITCH is phosphorylated and thereby inhibited by inhibitor of nuclear factor kappa B kinase subunit beta (IKKβ) to fine-tune the inflammatory response to the strength of the offending signal. However, the biochemical mechanism by which E3 ubiquitination is impaired by IKK-driven phosphorylation remains unclear. Here, we report that this phosphorylation impedes ITCH binding to its cognate E2 ubiquitin ligase, UbcH7. Using CRISPR-Cas9 genetic knockout to mimic the ITCH-UbcH7–inhibited state, we further show that genetic UbcH7 deficiency phenocopies ITCH phosphorylation in regulating RIPK2 ubiquitination. We conclude that phosphorylation can disrupt the binding of an E3 ubiquitin ligase to an E2-conjugating enzyme, leading to prolonged inflammatory signaling. To our knowledge, this is the first report of E3 ubiquitin ligase phosphorylation inhibiting E3 ligase activity by impairing E2–E3 complex formation.

Ubiquitylation of the TNFR1 signalling complex (TNF-RSC) controls the activation of RIPK1, a kinase critically involved in mediating multiple TNFα-activated deleterious events. However, the molecular mechanism that coordinates different types of ubiquitylation modification to regulate the activation of RIPK1 kinase remains unclear. Here, we show that ABIN-1/NAF-1, a ubiquitin-binding protein, is recruited rapidly into TNF-RSC in a manner dependent on the Met1-ubiquitylating complex LUBAC to regulate the recruitment of A20 to control Lys63 deubiquitylation of RIPK1. ABIN-1 deficiency reduces the recruitment of A20 and licenses cells to die through necroptosis by promoting Lys63 ubiquitylation and activation of RIPK1 with TNFα stimulation under conditions that would otherwise exclusively activate apoptosis in wild-type cells. Inhibition of RIPK1 kinase and RIPK3 deficiency block the embryonic lethality of Abin-1 –/– mice. We propose that ABIN-1 provides a critical link between Met1 ubiquitylation mediated by the LUBAC complex and Lys63 deubiquitylation by phospho-A20 to modulate the activation of RIPK1.

Gasdermin-D (GSDMD) in inflammasome-activated macrophages is cleaved by caspase-1 to generate N-GSDMD fragments. N-GSDMD then oligomerizes in the plasma membrane (PM) to form pores that increase membrane permeability, leading to pyroptosis and IL-1β release. In contrast, we report that although N-GSDMD is required for IL-1β secretion in NLRP3-activated human and murine neutrophils, N-GSDMD does not localize to the PM or increase PM permeability or pyroptosis. Instead, biochemical and microscopy studies reveal that N-GSDMD in neutrophils predominantly associates with azurophilic granules and LC3+ autophagosomes. N-GSDMD trafficking to azurophilic granules causes leakage of neutrophil elastase into the cytosol, resulting in secondary cleavage of GSDMD to an alternatively cleaved N-GSDMD product. Genetic analyses using ATG7-deficient cells indicate that neutrophils secrete IL-1β via an autophagy-dependent mechanism. These findings reveal fundamental differences in GSDMD trafficking between neutrophils and macrophages that underlie neutrophil-specific functions during inflammasome activation., In macrophages, IL-1β secretion is mediated by N-GSDMD pores in the plasma membrane (PM). Here the authors show that in neutrophils, IL-1β secretion occurs in the absence of PM pores, via autophagosomes; N-GSDMD does not traffic to PM but to azurophilic granules, thereby releasing neutrophil elastase which cleaves further N-GSDMD into alternative fragments.

Resisting or tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here, genomic analyses of anatomically modern humans, extinct Denisovan hominins, and mice revealed a series of missense variants in the immune response inhibitor A20 (encoded byTNFAIP3), substituting non-catalytic residues of the ubiquitin protease domain to diminish IκB-dependent phosphorylation and activation of A20. Two A20 variants with partial phosphorylation deficits appeared beneficial: one originating in Denisovans and introgressed in modern humans throughout Oceania, and another in a mouse strain resistant to Coxsackievirus. By contrast, a variant with 95% loss of phosphorylation caused spontaneous inflammatory disease in humans and mice. Analysis of the partial phosphorylation variant in mice revealed diminished tolerance of bacterial lipopolysaccharide or to poxvirus inoculation as trade-offs for enhanced immunity.One Sentence SummaryModern and ancient variants reveal a genetically tunable element for balancing immunity and microbial tolerance.

Previously, we reported that increased synthesis of the gas hydrogen sulfide (H2S) during the Integrated Stress Response (ISR) induced proteome-wide cysteine-sulfhydration with the predominant modified pathway being enzymes of cellular energy metabolism (Gao, et al. 2015). Using pancreatic beta cells and quantitative proteomics in this study, we identified a Redox Thiol Switch from S-glutathionylation to S-sulfhydration and we named it, RTSGS. About half of the identified proteins are involved in energy metabolism, and one novel target was the mitochondrial phosphoenolpyruvate carboxykinase 2 (PCK2) whose catalytic Cys306was targeted by both modifications. The enzymatic activity of PCK2 was inhibited by S-glutathionylation, and this inhibition was largely reversed by S-sulfhydration. S-sulfhydration also reversed the S-glutathionylation-mediated inhibition of glucose flux, indicating a broad metabolic significance. We propose that a Redox Thiol Switch from S-glutathionylation to S-sulfhydration is a key mechanism to fine tune cellular energy metabolism in response to different levels of oxidative stress.

Cyclin suppresses antitumor immunity Despite the dramatic success of cancer immunotherapy, many types of cancer do not respond. Understanding why could help us to find ways to enhance the overall responsiveness of tumors to immunotherapies. Dorand et al. report that cyclin-dependent kinase 5 (Cdk5), an enzyme that is highly expressed by neurons in many brain cancers, may dampen the ability of T cells to reject tumors. In a mouse model of medulloblastoma, if tumors were Cdk5 deficient, T cells were able to remove them. This heightened antitumor immunity correlated with reduced expression of the inhibitory molecule programmed cell death ligand 1 (PD-L1), a target of current cancer immunotherapies. Science , this issue p. 399

Resolution of inflammation at mucosal surfaces is a process critical for patients with IBD. GWAS revealed variant SNPs within the gene encoding gasdermin-B (GSDMB) that associates with an increased susceptibility to IBD and elevated GSDMB expression. The aim of this study was to determine the expression pattern and function significance of GSDMB in the pathogenesis of IBD. Primary intestinal biopsy and isolated intestinal epithelial specimens from IBD patients and non-IBD controls were evaluated for mRNA and protein expression. Using the human intestinal epithelial cell line, HT-29, CRISPR-cas9 knockout cells lacking GSDMB were generated, and used for in vitro XTT, cell-death and cell migration assays. Additionally, human epithelial cells transfected with GSDMB constructs and/or stimulated with methotrexate (MTX) were employed for relevant functional assays. We report for the first time a dramatic increase in full-length epithelial-specific GSDMB in IBD patients compared to healthy controls, which unlike other gasdermin family members, does not partake in program-mediated cell death. Additionally, we describe a functional decrease in in vitro proliferative activity, wound repair and an increase in focal adhesion proteins in epithelial cells lacking GSDMB, phenotypically rescued via transfection or MTX treatment. Importantly, GSDMB expression is decreased in IBD patients in remission, to that of levels of non-IBD controls. Taken together, our data suggests that increased epithelial-derived GSDMB during IBD is protective in function and promotes epithelial-specific adhesion and migration, providing rationale for the potential therapeutic use of GSDMB to optimize gut mucosal healing and restoration to homeostasis.

Circulating tumor cells (CTC) seed cancer metastases; however, the underlying cellular and molecular mechanisms remain unclear. CTC clusters were less frequently detected but more metastatic than single CTCs of patients with triple-negative breast cancer and representative patient-derived xenograft models. Using intravital multiphoton microscopic imaging, we found that clustered tumor cells in migration and circulation resulted from aggregation of individual tumor cells rather than collective migration and cohesive shedding. Aggregated tumor cells exhibited enriched expression of the breast cancer stem cell marker CD44 and promoted tumorigenesis and polyclonal metastasis. Depletion of CD44 effectively prevented tumor cell aggregation and decreased PAK2 levels. The intercellular CD44–CD44 homophilic interactions directed multicellular aggregation, requiring its N-terminal domain, and initiated CD44–PAK2 interactions for further activation of FAK signaling. Our studies highlight that CD44+ CTC clusters, whose presence is correlated with a poor prognosis of patients with breast cancer, can serve as novel therapeutic targets of polyclonal metastasis. Significance: CTCs not only serve as important biomarkers for liquid biopsies, but also mediate devastating metastases. CD44 homophilic interactions and subsequent CD44–PAK2 interactions mediate tumor cluster aggregation. This will lead to innovative biomarker applications to predict prognosis, facilitate development of new targeting strategies to block polyclonal metastasis, and improve clinical outcomes. See related commentary by Rodrigues and Vanharanta, p. 22. This article is highlighted in the In This Issue feature, p. 1

Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.

Dysregulation of inflammatory cell death is a key driver of many inflammatory diseases. Pyroptosis, a highly inflammatory form of cell death, uses intracellularly generated pores to disrupt electrolyte homeostasis and execute cell death. Gasdermin D, the pore-forming effector protein of pyroptosis, coordinates membrane lysis and the release of highly inflammatory molecules, such as interleukin-1β, which potentiate the overactivation of the innate immune response. However, to date, there is no pharmacologic mechanism to disrupt pyroptosis. Here, we identify necrosulfonamide as a direct chemical inhibitor of gasdermin D, the pyroptotic pore-forming protein, which binds directly to gasdermin D to inhibit pyroptosis. Pharmacologic inhibition of pyroptotic cell death by necrosulfonamide is efficacious in sepsis models and suggests that gasdermin D inhibitors may be efficacious clinically in inflammatory diseases.

The mammalian IAPs, X-linked inhibitor of apoptosis protein (XIAP) and cellular inhibitor of apoptosis protein 1 and 2 (cIAP1 and cIAP2), play pivotal roles in innate immune signaling and inflammatory homeostasis, often working in parallel or in conjunction at a signaling complex. IAPs direct both nucleotide-binding oligomerization domain-containing 2 (NOD2) signaling complexes and cell death mechanisms to appropriately regulate inflammation. Although it is known that XIAP is critical for NOD2 signaling and that the loss of cIAP1 and cIAP2 blunts NOD2 activity, it is unclear whether these three highly related proteins can compensate for one another in NOD2 signaling or in mechanisms governing apoptosis or necroptosis. This potential redundancy is critically important, given that genetic loss of XIAP causes both very early onset inflammatory bowel disease and X-linked lymphoproliferative syndrome 2 (XLP-2) and that the overexpression of cIAP1 and cIAP2 is linked to both carcinogenesis and chemotherapeutic resistance. Given the therapeutic interest in IAP inhibition and the potential toxicities associated with disruption of inflammatory homeostasis, we used synthetic biology techniques to examine the functional redundancies of key domains in the IAPs. From this analysis, we defined the features of the IAPs that enable them to function at overlapping signaling complexes but remain independent and functionally exclusive in their roles as E3 ubiquitin ligases in innate immune and inflammatory signaling.

NLRP3 inflammasome plays a critical spatiotemporal role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). This study reports a mechanistic insight into noncanonical NLRP3 inflammasome activation in microglia for the effector stage of EAE. Microglia-specific deficiency of ASC (apoptosis-associated speck-like protein containing a C-terminal caspase-activation and recruitment [CARD] domain) attenuated T cell expansion and neutrophil recruitment during EAE pathogenesis. Mechanistically, TLR stimulation led to IRAKM-caspase-8-ASC complex formation, resulting in the activation of caspase-8 and IL-1β release in microglia. Noncanonical inflammasome-derived IL-1β produced by microglia in the CNS helped to expand the microglia population in an autocrine manner and amplified the production of inflammatory cytokines/chemokines. Furthermore, active caspase-8 was markedly increased in the microglia in the brain tissue from patients with multiple sclerosis. Taken together, our study suggests that microglia-derived IL-1β via noncanonical caspase-8-dependent inflammasome is necessary for microglia to exert their pathogenic role during CNS inflammation.

SummaryLoss of NF-κB signaling causes immunodeficiency, whereas inhibition of NF-κB can be efficacious in treating chronic inflammatory disease. Inflammatory NF-κB signaling must therefore be tightly regulated, and although many mechanisms to downregulate NF-κB have been elucidated, there have only been limited studies demonstrating positive feedforward regulation of NF-κB signaling. In this work, we use a bioinformatic and proteomic approach to discover that the IKK family of proteins can phosphorylate the E3 ubiquitin ligase ITCH, a critical downregulator of TNF-mediated NF-κB activation. Phosphorylation of ITCH by IKKs leads to impaired ITCH E3 ubiquitin ligase activity and prolongs NF-κB signaling and pro-inflammatory cytokine release. Since genetic loss of ITCH mirrors IKK-induced ITCH phosphorylation, we further show that the ITCH−/− mouse’s spontaneous lung inflammation and subsequent death can be delayed when TNF signaling is genetically deleted. This work identifies a new positive feedforward regulation of NF-κB activation that drives inflammatory disease.

Pyroptosis is a form of cell death important in defenses against pathogens, but which can also result in a potent and sometimes pathological inflammatory response. During pyroptosis, gasdermin D (GSDMD), the pore-forming effector protein, is cleaved, forms oligomers, and inserts into the membranes of the cell, resulting in rapid cell death. However, the potent cell death induction caused by GSDMD has complicated our ability to understand the biology of this protein. Studies aimed at visualizing GSDMD have relied on expression of GSDMD fragments in epithelial cell lines that naturally lack GSDMD expression and also lack the proteases necessary to cleave GSDMD. In this work, we performed mutagenesis and molecular modeling to strategically place tags and fluorescent proteins within GSDMD that support native pyroptosis and facilitate live-cell imaging of pyroptotic cell death. Here, we demonstrate that these fusion proteins are cleaved by caspases-1 and 11 at Asp-276. Mutations that disrupted the predicted p30-p20 autoinhibitory interface resulted in GSDMD aggregation, supporting the oligomerizing activity of these mutations. Furthermore, we show that these novel GSDMD fusions execute inflammasome-dependent pyroptotic cell death in response to multiple stimuli and allow for visualization of the morphological changes associated with pyroptotic cell death in real time. This work therefore provides new tools that not only expand the molecular understanding of pyroptosis, but also enable its direct visualization.

The RIPK2 kinase transduces signaling downstream of the intracellular peptidoglycan sensors NOD1 and NOD2 to promote a productive inflammatory response. However, excessive NOD2 signaling has been associated with numerous diseases, including inflammatory bowel disease (IBD), sarcoidosis and inflammatory arthritis, making pharmacologic inhibition of RIPK2 an appealing strategy. In this work, we report the generation, identification, and evaluation of novel RIPK2 specific inhibitors. These compounds potently inhibit the RIPK2 tyrosine kinase activity in in vitro biochemical assays and cellular assays, as well as effectively reduce RIPK2-mediated effects in an in vivo peritonitis model. In conjunction with the development of these inhibitors, we have also defined a panel of genes whose expression is regulated by RIPK2 kinase activity. Such RIPK2 activation markers may serve as a useful tool for predicting settings likely to benefit from RIPK2 inhibition. Using these markers and the FDA-approved RIPK2 inhibitor Gefitinib, we show that pharmacologic RIPK2 inhibition drastically improves disease in a spontaneous model of Crohn Disease-like ileitis. Furthermore, using novel RIPK2-specific inhibitors, we show that cellular recruitment is inhibited in an in vivo peritonitis model. Altogether, the data presented in this work provides a strong rationale for further development and optimization of RIPK2-targeted pharmaceuticals and diagnostics.

Common variable immunodeficiency (CVID), the most frequent symptomatic primary immune deficiency in humans, is a heterogeneous group of immunologic disorders estimated to affect 1:10,000–1:50,000. Although a clear disease etiology remains elusive, a common characteristic of CVID is deficient IgG Ab production in response to infection or vaccination. Patients often also exhibit autoimmune cytopenias with symptoms of abnormal T cell function, including reductions in naive T cells, which correlate with clinical severity. In this study, we discovered that targeted alterations in the glycome of the myeloid lineage lead to spontaneous immunodeficiency characteristic of both humoral and T cell dysfunction regularly found in human CVID. Mice carrying a myeloid-specific knockout of the Mgat2 gene encoding UDP-GlcNAc:α-6-d-mannoside β-1,2-N-acetylglucosaminyltransferase II enzyme exhibit deficiencies in IgG responses to both protein and polysaccharide conjugate vaccines. Interestingly, the immunodeficiency is associated with decreased T cell activity because of a persistent autoimmune-mediated depletion of naive T cells, which is induced by changes in erythrocyte surface glycosylation. The N-glycosylation dependent autoepitopes that emerge on erythrocytes lead to autoimmune hemolytic anemia, and the causative auto-IgM cross-reacts with naive T cells despite the lack of glycan change on T cells. These findings demonstrate that alterations in erythrocyte glycosylation trigger the development of autoantibodies directed at both erythrocytes and naive T cells, revealing a possible mechanistic link between the induction of autoimmune hemolytic anemia, the reduction in naive T cells, and poor Ab responses to vaccine in severe CVID patients.

Summary Gasdermin D (GSDMD) is an effector molecule for pyroptosis downstream of canonical and noncanonical inflammasome signaling pathways. Cleavage of GSDMD by inflammatory caspases triggers the oligomerization and lipid binding by its N-terminal domain, which assembles membrane pores, whereas its C-terminal domain binds the N-terminal domain to inhibit pyroptosis. Despite recent progress in our understanding of the structure and function of the murine gasdermin A3 (mGSDMA3), the molecular mechanisms of GSDMD activation and regulation remain poorly characterized. Here, we report the crystal structures of the full-length murine and human GSDMDs, which reveal the architecture of the GSDMD N-terminal domains and demonstrate distinct and common features of autoinhibition among gasdermin family members utilizing their β1-β2 loops. Disruption of the intramolecular domain interface enhanced pyroptosis, whereas mutations at the predicted lipid-binding or oligomerization surface reduced cytolysis. Our study provides a framework for understanding the autoinhibition, lipid binding, and oligomerization of GSDMD by using overlapping interfaces.

N-linked glycosylation is a central regulatory factor that influences the immune system in varied and profound ways, including leukocyte homing, T cell receptor signaling and others. Moreover, N-glycan branching has been demonstrated to change as a function of infection and inflammation. Our previous findings suggest that complex-type N-glycans on the class II major histocompatibility complex play an important role in antigen selection within antigen presenting cells (APCs) such that highly branched N-glycans promote polysaccharide (glycoantigen, GlyAg) presentation following Toll-like receptor 2 (TLR2)-dependent antigen processing. In order to explore the impact of N-glycan branching on the myeloid-derived APC population without the confounding problems of altering the branching of lymphocytes and non-hematopoietic cells, we created a novel myeloid-specific knockout of the β-1,2-N-acetylglucosaminyltransferase II (Mgat2) enzyme. Using this novel mouse, we found that the reduction in multi-antennary N-glycans characteristic of Mgat2 ablation had no impact on GlyAg-mediated TLR2 signaling. Likewise, no deficits in antigen uptake or cellular homing to lymph nodes were found. However, we discovered that Mgat2 ablation prevented GlyAg presentation and T cell activation in vitro and in vivo without apparent alterations in protein antigen response or myeloid-mediated protection from infection. These findings demonstrate that GlyAg presentation can be regulated by the N-glycan branching pattern of APCs, thereby establishing an in vivo model where the T cell-dependent activity of GlyAgs can be experimentally distinguished from GlyAg-mediated stimulation of the innate response through TLR2.

Nucleotide-binding oligomerization domain-containing 2 (NOD2) is an intracellular receptor that plays an essential role in innate immunity as a sensor of a component of the bacterial cell wall, muramyl dipeptide (MDP). Crohn's disease (CD)-associated NOD2 variants lead to defective innate immune responses, including decreased NF-κB activation and cytokine production. We report herein that SAMP1/YitFc (SAMP) mice, which develop spontaneous CD-like ileitis in the absence of NOD2 genetic mutations, fail to respond to MDP administration by displaying decreased innate cytokine production and dysregulated NOD2 signaling compared with parental AKR control mice. We show that, unlike in other mouse strains, in vivo administration of MDP does not prevent dextran sodium sulfate-induced colitis in SAMP mice and that the abnormal NOD2 response is specific to the hematopoietic cellular component. Moreover, we demonstrate that MDP fails to enhance intracellular bacterial killing in SAMP mice. These findings shed important light on the initiating molecular events underlying CD-like ileitis.

Summary While the I kappa kinase (IKK) scaffolding protein NF-κB essential modulator (NEMO) binds to polyubiquitin chains to transmit inflammatory signals, NEMO itself is also ubiquitinated in response to a variety of inflammatory agonists. Although there have been hints that polyubiquitination of NEMO is essential for avoiding inflammatory disorders, the in vivo physiologic role of NEMO ubiquitination is unknown. In this work, we knock in a NEMO allele in which two major inflammatory agonist-induced ubiquitination sites cannot be ubiquitinated. We show that mice with a nonubiquitinatable NEMO allele display embryonic lethality. Heterozygous females develop inflammatory skin lesions, decreased B cell numbers, and hypercellular spleens. Embryonic lethality can be complemented by mating onto a TNFR1 −/− background, at the cost of severe steatohepatitis and early mortality, and we also show that NEMO ubiquitination is required for optimal innate immune signaling responses. These findings suggest that NEMO ubiquitination is crucial for NF-κB activity in response to innate immune agonists.

Dysregulation of NOD2 signaling is implicated in the pathology of various inflammatory diseases, including Crohn's disease, asthma, and sarcoidosis, making signaling proteins downstream of NOD2 potential therapeutic targets. Inhibitor-of-apoptosis (IAP) proteins, particularly cIAP1, are essential mediators of NOD2 signaling, and in this work, we describe a molecular mechanism for cIAP1's regulation in the NOD2 signaling pathway. While cIAP1 promotes RIP2's tyrosine phosphorylation and subsequent NOD2 signaling, this positive regulation is countered by another E3 ubiquitin ligase, ITCH, through direct ubiquitination of cIAP1. This ITCH-mediated ubiquitination leads to cIAP1's lysosomal degradation. Pharmacologic inhibition of cIAP1 expression in ITCH(-/-) macrophages attenuates heightened ITCH(-/-) macrophage muramyl dipeptide-induced responses. Transcriptome analysis, combined with pharmacologic inhibition of cIAP1, further defines specific pathways within the NOD2 signaling pathway that are targeted by cIAP1. This information provides genetic signatures that may be useful in repurposing cIAP1-targeted therapies to correct NOD2-hyperactive states and identifies a ubiquitin-regulated signaling network centered on ITCH and cIAP1 that controls the strength of NOD2 signaling.

Canonical inflammasome activation induces a caspase-1/gasdermin D (Gsdmd)–dependent lytic cell death called pyroptosis that promotes antimicrobial host defense but may contribute to sepsis. The nature of the caspase-1–dependent change in plasma membrane (PM) permeability during pyroptotic progression remains incompletely defined. We assayed propidium2+ (Pro2+) influx kinetics during NLRP3 or Pyrin inflammasome activation in murine bone marrow–derived macrophages (BMDMs) as an indicator of this PM permeabilization. BMDMs were characterized by rapid Pro2+ influx after initiation of NLRP3 or Pyrin inflammasomes by nigericin (NG) or Clostridium difficile toxin B (TcdB), respectively. No Pro2+ uptake in response to NG or TcdB was observed in Casp1−/− or Asc−/− BMDMs. The cytoprotectant glycine profoundly suppressed NG and TcdB-induced lysis but not Pro2+ influx. The absence of Gsdmd expression resulted in suppression of NG-stimulated Pro2+ influx and pyroptotic lysis. Extracellular La3+ and Gd3+ rapidly and reversibly blocked the induced Pro2+ influx and markedly delayed pyroptotic lysis without limiting upstream inflammasome assembly and caspase-1 activation. Thus, caspase-1–driven pyroptosis requires induction of initial prelytic pores in the PM that are dependent on Gsdmd expression. These PM pores also facilitated the efflux of cytosolic ATP and influx of extracellular Ca2+. Although lanthanides and Gsdmd deletion both suppressed PM pore activity and pyroptotic lysis, robust IL-1β release was observed in lanthanide-treated BMDMs but not in Gsdmd-deficient cells. This suggests roles for Gsdmd in both passive IL-1β release secondary to pyroptotic lysis and in nonlytic/nonclassical IL-1β export.

Although a clear association has been established between IL-33 and inflammatory bowel disease, mechanistic studies to date, primarily using acute murine models of colitis, have yielded contradicting results, demonstrating both pathogenic and protective roles. We used a well-characterized, spontaneous model of inflammatory bowel disease [ie, SAMP1/YitFc (SAMP) mice] to investigate the role of IL-33 during chronic intestinal inflammation. Our results showed marked eosinophil infiltration into the gut mucosa with increased levels of eotaxins and type 2 helper T-cell (Th2) cytokines as disease progressed and became more severe, which could be reversed upon either eosinophil depletion or blockade of IL-33 signaling. Exogenous IL-33 administration recapitulated these effects in ilea of uninflamed (parental) control AKR/J mice. Human data supported these findings, showing colocalization and up-regulation of IL-33 and eosinophils in the colonic mucosa of inflammatory bowel disease patients versus noninflamed controls. Finally, colonization of commensal flora by fecal material transplantation into germ-free SAMP and the presence of the gut microbiome induced IL-33, subsequent eosinophil infiltration, and mounting of Th2 immune responses, leading to exacerbation of chronic intestinal inflammation characteristic of SAMP mice. These data demonstrate a pathogenic role for IL-33-mediated eosinophilia and activation of Th2 immunity in chronic intestinal inflammation that is dependent on the gut microbiome. Targeting IL-33 may represent a novel therapeutic approach to treat patients with inflammatory bowel disease.

Tumor progression locus 2 (TPL-2) functions as a MEK-1/2 kinase, which is essential for Toll-like receptor 4 (TLR4) activation of extracellular signal-regulated kinase 1 and 2 (ERK-1/2) mitogen-activated protein (MAP) kinases in lipopolysaccharide (LPS)-stimulated macrophages and for inducing the production of the proinflammatory cytokines tumor necrosis factor and interleukin-1β. In unstimulated cells, association of TPL-2 with NF-κB1 p105 prevents TPL-2 phosphorylation of MEK-1/2. LPS stimulation of TPL-2 MEK-1/2 kinase activity requires TPL-2 release from p105. This is triggered by IκB kinase 2 (IKK-2) phosphorylation of the p105 PEST region, which promotes p105 ubiquitination and degradation by the proteasome. LPS activation of ERK-1/2 additionally requires transphosphorylation of TPL-2 on serine 400 in its C terminus, which controls TPL-2 signaling to ERK-1/2 independently of p105. However, the identity of the protein kinase responsible for TPL-2 serine 400 phosphorylation remained unknown. In the present study, we show that TPL-2 serine 400 phosphorylation is mediated by IKK2. The IKK complex therefore regulates two of the key regulatory steps required for TPL-2 activation of ERK-1/2, underlining the close linkage of ERK-1/2 MAP kinase activation to upregulation of NF-κB-dependent transcription.

Despite their homology, IκB kinase α (IKKα) and IKKβ have divergent roles in NF-κB signaling. IKKβ strongly activates NF-κB while IKKα can downregulate NF-κB under certain circumstances. Given this, identifying independent substrates for these kinases could help delineate their divergent roles. Peptide substrate array technology followed by bioinformatic screening identified TRAF4 as a substrate for IKKα. Like IKKα, TRAF4 is atypical within its family because it is the only TRAF family member to negatively regulate innate immune signaling. IKKα's phosphorylation of serine-426 on TRAF4 was required for this negative regulation. Binding to the Crohn's disease susceptibility protein, NOD2, is required for TRAF4 phosphorylation and subsequent inhibition of NOD2 signaling. Structurally, serine-426 resides within an exaggerated β-bulge in TRAF4 that is not present in the other TRAF proteins, and phosphorylation of this site provides a structural basis for the atypical function of TRAF4 and its atypical role in NOD2 signaling.

The RIP kinases (RIPKs) play an essential role in inflammatory signaling and inflammatory cell death. However, the function of their kinase activity has been enigmatic, and only recently has kinase domain activity been shown to be crucial for their signal transduction capacity. Despite this uncertainty, the RIPKs have been the subject of intense pharmaceutical development with a number of compounds currently in preclinical testing. In this work, we seek to determine the functional redundancy between the kinase domains of the four major RIPK family members. We find that although RIPK1, RIPK2, and RIPK4 are similar in that they can all activate NF-κB and induce NF-κB essential modulator ubiquitination, only RIPK2 is a dual-specificity kinase. Domain swapping experiments showed that the RIPK4 kinase domain could be converted to a dual-specificity kinase and is essentially indistinct from RIPK2 in biochemical and molecular activity. Surprisingly, however, replacement of RIPK2’s kinase domain with RIPK4’s did not complement a nucleotide-binding oligomerization domain 2 signaling or gene expression induction defect in RIPK2−/− macrophages. These findings suggest that RIPK2’s kinase domain is functionally unique compared with other RIPK family members and that pharmacologic targeting of RIPK2 can be separated from the other RIPKs.

Upon intracellular bacterial exposure, the Crohn's disease and sarcoidosis susceptibility protein NOD2 (nucleotide oligomerization domain protein 2) binds to the protein kinase RIP2 (receptor-interacting protein 2) to coordinate NF-κB (nuclear factor κ B)-mediated cytokine responses. While RIP2 clearly has kinase activity, the function of its kinase domain has been enigmatic. Although originally classified as a serine–threonine kinase based on homology scans, we find that RIP2 also has tyrosine kinase activity. RIP2 undergoes autophosphorylation on Tyr 474 (Y474). This phosphorylation event is necessary for effective NOD2 signaling and does not occur in the presence of the most common Crohn's disease-associated NOD2 allele. Given this tyrosine kinase activity, a small-molecule inhibitor screen designed to identify pharmacologic agents that inhibit RIP2's tyrosine kinase activity was performed. At nanomolar concentrations, the EGFR (epidermal growth factor receptor) tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) were found to inhibit both RIP2 tyrosine phosphorylation and MDP (muramyl dipeptide)-induced cytokine release in a variety of NOD2 hyperactivation states. This effect is specific for RIP2 and does not depend on EGFR. The finding that RIP2 has tyrosine kinase activity and the finding that gefitinib and erlotinib, two agents already used clinically for cancer chemotherapy, can inhibit this activity suggest that RIP2's tyrosine kinase activity could be targeted specifically in the treatment of inflammatory diseases.

Pyroptosis is an inflammatory form of programmed cell death that plays important roles in immune protection against infections and in inflammatory disorders. Gasdermin D (GSDMD) is an executor of pyroptosis upon cleavage by caspases-1/4/5/11 following canonical and noncanonical inflammasome activation. GSDMD N-terminal domain assembles membrane pores to induce cytolysis, whereas its C-terminal domain inhibits cell death through intramolecular association with the N domain. The molecular mechanisms of autoinhibition for GSDMD are poorly characterized. Here we report the crystal structures of the human and murine GSDMD C-terminal domains, which differ from those of the full-length murine GSDMA3 and the human GSDMB C-terminal domain. Mutations of GSDMD C-domain residues predicted to locate at its interface with the N-domain enhanced pyroptosis. Our results suggest that GSDMDs may employ a distinct mode of intramolecular domain interaction and autoinhibition, which may be relevant to its unique role in pyroptosis downstream of inflammasome activation.

CpG oligodeoxynucleotides (ODNs) signal through TLR9 to induce type I IFN (IFN-alphabeta) in dendritic cells (DCs). CpG-A ODNs are more efficacious than CpG-B ODNs for induction of IFN-alphabeta. Because IFN-alphabeta may contribute to autoimmunity, it is important to identify mechanisms to inhibit induction of IFN-alphabeta. In our studies, CpG-B ODN inhibited induction of IFN-alphabeta by CpG-A ODN, whereas induction of TNF-alpha and IL-12p40 by CpG-A ODN was not affected. CpG-B inhibition of IFN-alphabeta was observed in FLT3 ligand-induced murine DCs, purified murine myeloid DCs, plasmacytoid DCs, and human PBMCs. CpG-B ODN inhibited induction of IFN-alphabeta by agonists of multiple receptors, including MyD88-dependent TLRs (CpG-A ODN signaling via TLR9, or R837 or Sendai virus signaling via TLR7) and MyD88-independent receptors (polyinosinic:polycytidylic acid signaling via TLR3 or ds break-DNA signaling via a cytosolic pathway). CpG-B ODN did not inhibit the IFN-alphabeta positive feedback loop second-wave IFN-alphabeta, because IFN-alphabeta-induced expression of IFN-alphabeta was unaffected, and CpG-B inhibition of IFN-alphabeta was manifested in IFN-alphabetaR(-/-) DCs, which lack the positive feedback mechanism. Rather, CpG-B ODN inhibited early TLR-induced first wave IFN-alpha4 and IFN-beta. Chromatin immunoprecipitation revealed that association of IFN regulatory factor 1 with the IFN-alpha4 and IFN-beta promoters was induced by CpG-A ODN but not CpG-B ODN. Moreover, CpG-A-induced association of IFN regulatory factor 1 with these promoters was inhibited by CpG-B ODN. Our studies demonstrate a novel mechanism of transcriptional regulation of first-wave IFN-alphabeta that selectively inhibits induction of IFN-alphabeta downstream of multiple receptors and may provide targets for future therapeutic inhibition of IFN-alphabeta expression in vivo.

The cellular prion protein (PrP) is a highly conserved, widely expressed, glycosylphosphatidylinositol-anchored (GPI-anchored) cell surface glycoprotein. Since its discovery, most studies on PrP have focused on its role in neurodegenerative prion diseases, whereas its function outside the nervous system remains unclear. Here, we report that human pancreatic ductal adenocarcinoma (PDAC) cell lines expressed PrP. However, the PrP was neither glycosylated nor GPI-anchored, existing as pro-PrP and retaining its GPI anchor peptide signal sequence (GPI-PSS). We also showed that the PrP GPI-PSS has a filamin A-binding (FLNa-binding) motif and interacted with FLNa, an actin-associated protein that integrates cell mechanics and signaling. Binding of pro-PrP to FLNa disrupted cytoskeletal organization. Inhibition of PrP expression by shRNA in the PDAC cell lines altered the cytoskeleton and expression of multiple signaling proteins; it also reduced cellular proliferation and invasiveness in vitro as well as tumor growth in vivo. A subgroup of human patients with pancreatic cancer was found to have tumors that expressed pro-PrP. Most importantly, PrP expression in tumors correlated with a marked decrease in patient survival. We propose that binding of pro-PrP to FLNa perturbs FLNa function, thus contributing to the aggressiveness of PDAC. Prevention of this interaction could provide an attractive target for therapeutic intervention in human PDAC.