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Neurogenic heterotopic ossifications (NHOs) form in periarticular muscles following severe spinal cord (SCI) and traumatic brain injuries. The pathogenesis of NHO is poorly understood with no effective preventive treatment. The only curative treatment remains surgical resection of pathological NHOs. In a mouse model of SCI-induced NHO that involves a transection of the spinal cord combined with a muscle injury, a differential gene expression analysis revealed that genes involved in inflammation such as interleukin-1β (IL-1β) were overexpressed in muscles developing NHO. Using mice knocked-out for the gene encoding IL-1 receptor (IL1R1) and neutralizing antibodies for IL-1α and IL-1β, we show that IL-1 signaling contributes to NHO development following SCI in mice. Interestingly, other proteins involved in inflammation that were also overexpressed in muscles developing NHO, such as colony-stimulating factor-1, tumor necrosis factor or C-C chemokine ligand-2 did not promote NHO development. Finally using NHO biopsies from SCI and TBI patients, we show that IL-1β is expressed by CD68+ macrophages. IL-1α and IL-1β produced by activated human monocytes promote calcium mineralization and RUNX2 expression in fibro-adipogenic progenitors isolated from muscles surrounding NHOs. Altogether these data suggest that interleukin-1 promotes NHO development in both humans and mice. This article is protected by copyright. All rights reserved.

To maintain homeostasis, an organism must detect and resolve sterile tissue damage. The NLRP3 inflammasome coordinates such processes to clear tissue damage and induce repair. Dysregulated NLRP3 inflammasome activity, however, drives many conditions including Alzheimer's disease (AD). Recent reports posit that β-amyloid and tau aggregates trigger destructive NLRP3 inflammasome signalling in the brain, leading to AD pathophysiology and cognitive decline. Other endogenous molecules (e.g. TNF, ATP, serum amyloid A), as well as dysbiosis, can induce peripheral or central inflammation and thereby promote microglial NLRP3 inflammasome signalling and resultant AD. The NLRP3 inflammasome is thus emerging as a critical driver of sterile neuroinflammation and the resultant pathogenesis and progression of AD.

Background:Myocardial infarction (MI) triggers myelopoiesis, resulting in heightened production of neutrophils. However, the mechanisms that sustain their production and recruitment to the injured heart are unclear.Methods:Using a mouse model of the permanent ligation of the left anterior descending artery and flow cytometry, we first characterized the temporal and spatial effects of MI on different myeloid cell types. We next performed global transcriptome analysis of different cardiac cell types within the infarct to identify the drivers of the acute inflammatory response and the underlying signaling pathways. Using a combination of genetic and pharmacological strategies, we identified the sequelae of events that led to MI-induced myelopoiesis. Cardiac function was assessed by echocardiography. The association of early indexes of neutrophilia with major adverse cardiovascular events was studied in a cohort of patients with acute MI.Results:Induction of MI results in rapid recruitment of neutrophils to the infarct, where they release specific alarmins, S100A8 and S100A9. These alarmins bind to the Toll-like receptor 4 and prime the nod-like receptor family pyrin domain-containing 3 inflammasome in naïve neutrophils and promote interleukin-1β secretion. The released interleukin-1β interacts with its receptor (interleukin 1 receptor type 1) on hematopoietic stem and progenitor cells in the bone marrow and stimulates granulopoiesis in a cell-autonomous manner. Genetic or pharmacological strategies aimed at disruption of S100A8/A9 and their downstream signaling cascade suppress MI-induced granulopoiesis and improve cardiac function. Furthermore, in patients with acute coronary syndrome, higher neutrophil count on admission and after revascularization correlates positively with major adverse cardiovascular disease outcomes.Conclusions:Our study provides novel evidence for the primary role of neutrophil-derived alarmins (S100A8/A9) in dictating the nature of the ensuing inflammatory response after myocardial injury. Therapeutic strategies aimed at disruption of S100A8/A9 signaling or their downstream mediators (eg, nod-like receptor family pyrin domain-containing 3 inflammasome, interleukin-1β) in neutrophils suppress granulopoiesis and may improve cardiac function in patients with acute coronary syndrome.

Group A Streptococcus (GAS) is a Gram-positive bacterial pathogen that causes an array of infectious diseases in humans. Accumulating clinical evidence suggests that proinflammatory interleukin (IL)-1β signaling plays an important role in GAS disease progression. The host regulates the production and secretion of IL-1β via the cytosolic inflammasome pathway. Activation of the NLR family pyrin domain-containing 3 (NLRP3) inflammasome complex requires two signals: a priming signal that stimulates increased transcription of genes encoding the components of the inflammasome pathway, and an activating signal that induces assembly of the inflammasome complex. Here we show that GAS-derived lipoteichoic acid can provide a priming signal for NLRP3 inflammasome activation. As only few GAS-derived proteins have been associated with inflammasome-dependent IL-1β signaling, we investigated novel candidates that might play a role in activating the inflammasome pathway by infecting mouse bone marrow-derived macrophages and human THP-1 macrophage-like cells with a panel of isogenic GAS mutant strains. We found that the cytolysins streptolysin O (SLO) and streptolysin S are the main drivers of IL-1β release in proliferating logarithmic phase GAS. Using a mutant form of recombinant SLO, we confirmed that bacterial pore formation on host cell membranes is a key mechanism required for inflammasome activation. Our results suggest that streptolysins are major determinants of GAS-induced inflammation and present an attractive target for therapeutic intervention.

Microglial NLRP3 inflammasome activation is emerging as a key contributor to neuroinflammation during neurodegeneration. Pathogenic protein aggregates such as β-amyloid and α-synuclein trigger microglial NLRP3 activation, leading to caspase-1 activation and IL-1β secretion. Both caspase-1 and IL-1β contribute to disease progression in the mouse SOD1G93A model of amyotrophic lateral sclerosis (ALS), suggesting a role for microglial NLRP3. Prior studies, however, suggested SOD1G93A mice microglia do not express NLRP3, and SOD1G93A protein generated IL-1β in microglia independent to NLRP3. Here, we demonstrate using Nlrp3-GFP gene knock-in mice that microglia express NLRP3 in SOD1G93A mice. We show that both aggregated and soluble SOD1G93A activates inflammasome in primary mouse microglia leading caspase-1 and IL-1β cleavage, ASC speck formation, and the secretion of IL-1β in a dose- and time-dependent manner. Importantly, SOD1G93A was unable to induce IL-1β secretion from microglia deficient for Nlrp3, or pretreated with the specific NLRP3 inhibitor MCC950, confirming NLRP3 as the key inflammasome complex mediating SOD1-induced microglial IL-1β secretion. Microglial NLRP3 upregulation was also observed in the TDP-43Q331K ALS mouse model, and TDP-43 wild-type and mutant proteins could also activate microglial inflammasomes in a NLRP3-dependent manner. Mechanistically, we identified the generation of reactive oxygen species and ATP as key events required for SOD1G93A -mediated NLRP3 activation. Taken together, our data demonstrate that ALS microglia express NLRP3, and that pathological ALS proteins activate the microglial NLRP3 inflammasome. NLRP3 inhibition may therefore be a potential therapeutic approach to arrest microglial neuroinflammation and ALS disease progression.

BACKGROUND AND PURPOSE: Chronic liver diseases feature excessive collagen and matrix protein deposition or crosslinking that characterises fibrosis, leads to scar tissue, and disrupts liver functions. There is no effective treatment. This study investigated whether treatment with selective histone deacetylase (HDAC) inhibitors might specifically reduce type 2 inflammation in the injured liver, thereby attenuating fibrogenesis in mice. EXPERIMENTAL APPROACH: Thioacetamide (TAA) was used to induce hepatic inflammation, fibrosis, and liver damage in female C57BL/6 mice, similar to the clinical features of chronic human liver disease. We used eight inhibitors of different human HDAC enzymes to probe histological (IHC and TUNEL), biochemical and immunological changes (flow cytometry, qPCR, Legendplex, and elisa) in pathology, fibrosis, hepatic immune cell flux, and inflammatory cytokine expression. KEY RESULTS: Inhibitors of class I, but not class II, HDAC enzymes potently suppressed chronic hepatic inflammation and fibrosis in mice, attenuating accumulation and activation of IL‐33‐dependent, but not IL‐25‐dependent, group 2 innate lymphoid cells (ILC2) and inhibiting type 2 inflammation that drives hepatic stellate cells to secrete excessive collagen and matrix proteins. CONCLUSIONS AND IMPLICATIONS: The results show that potent and selective inhibitors of class I only HDAC enzymes profoundly inhibit hepatocyte death and type 2 inflammation to prevent TAA‐induced liver fibrosis in mice. The specific HDAC enzymes identified here may be key promoters of inflammation in chronic liver fibrosis.

Inhibition of the NLRP3 inflammasome is a promising strategy for the development of new treatments for inflammatory diseases. MCC950 is a potent and specific small-molecule inhibitor of the NLRP3 pathway, but its molecular target is not defined. Here, we show that MCC950 directly interacts with the Walker B motif within the NLRP3 NACHT domain, thereby blocking ATP hydrolysis and inhibiting NLRP3 activation and inflammasome formation.

Group A Streptococcus (GAS) is a Gram-positive bacterial pathogen that causes significant morbidity and mortality worldwide. Recent clinical evidence suggests that the inflammatory marker interleukin-1β (IL-1β) plays an important role in GAS disease progression, and presents a potential target for therapeutic intervention. Interaction with GAS activates the host inflammasome pathway to stimulate production and secretion of IL-1β, but GAS can also stimulate IL-1β production in an inflammasome-independent manner. This review highlights progress that has been made in understanding the importance of host cell inflammasomes and IL-1 signalling in GAS disease, and explores challenges and unsolved problems in this host-pathogen interaction. TAKE AWAY: Inflammasome signalling during GAS infection is an emerging field of research. GAS modulates the NLRP3 inflammasome pathway through multiple mechanisms. SpeB contributes to IL-1β production independently of the inflammasome pathway. IL-1β signalling can be host-protective, but also drive severe GAS disease.

Vincristine is an important component of many regimens used for pediatric and adult malignancies, but it causes a dose-limiting sensorimotor neuropathy for which there is no effective treatment. This study aimed to delineate the neuro-inflammatory mechanisms contributing to the development of mechanical allodynia and gait disturbances in a murine model of vincristine-induced neuropathy, as well as to identify novel treatment approaches. Here, we show that vincristine-induced peripheral neuropathy is driven by activation of the NLRP3 inflammasome and subsequent release of interleukin-1β from macrophages, with mechanical allodynia and gait disturbances significantly reduced in knockout mice lacking NLRP3 signaling pathway components, or after treatment with the NLRP3 inhibitor MCC950. Moreover, treatment with the IL-1 receptor antagonist anakinra prevented the development of vincristine-induced neuropathy without adversely affecting chemotherapy efficacy or tumor progression in patient-derived medulloblastoma xenograph models. These results detail the neuro-inflammatory mechanisms leading to vincristine-induced peripheral neuropathy and suggest that repurposing anakinra may be an effective co-treatment strategy to prevent vincristine-induced peripheral neuropathy.

ObjectivesThrombotic and microvascular complications are frequently seen in deceased COVID-19 patients. However, whether this is caused by direct viral infection of the endothelium or inflammation-induced endothelial activation remains highly contentious.MethodsHere, we use patient autopsy samples, primary human endothelial cells and an in vitro model of the pulmonary epithelial-endothelial cell barrier to show that primary human endothelial cells express very low levels the SARS-CoV-2 receptor ACE2 and the protease TMPRSS2.ResultsAccordingly, endothelial cells can only be infected when SARS-CoV-2 is present at very high concentrations. However, this is not a productive infection (i.e. no infectious virus is produced) and viral entry induces an inflammatory response. We also show that SARS-CoV-2 does not infect endothelial cells in 3D vessels under flow conditions. We further demonstrate that in a co-culture model endothelial cells are not infected with SARS-CoV-2. They do however sense and respond to infection in the adjacent epithelial cells, increasing ICAM-1 expression and releasing pro-inflammatory cytokines.ConclusionsTaken together, these data suggest that in vivo, endothelial cells are unlikely to be infected with SARS-CoV-2 and that infection is only likely to occur if the adjacent pulmonary epithelium is denuded (basolateral infection) or a high viral load is present in the blood (apical infection). In such a scenario, whilst SARS-CoV-2 infection of the endothelium can occur, it does not contribute to viral amplification. However, endothelial cells are still likely to play a key role in SARS-CoV-2 pathogenesis by sensing adjacent infection and mounting a pro-inflammatory response to SARS-CoV-2.

Objectives Thrombotic and microvascular complications are frequently seen in deceased COVID‐19 patients. However, whether this is caused by direct viral infection of the endothelium or inflammation‐induced endothelial activation remains highly contentious. Methods Here, we use patient autopsy samples, primary human endothelial cells and an in vitro model of the pulmonary epithelial–endothelial cell barrier. Results We show that primary human endothelial cells express very low levels of the SARS‐CoV‐2 receptor ACE2 and the protease TMPRSS2, which blocks their capacity for productive viral infection, and limits their capacity to produce infectious virus. Accordingly, endothelial cells can only be infected when they overexpress ACE2, or are exposed to very high concentrations of SARS‐CoV‐2. We also show that SARS‐CoV‐2 does not infect endothelial cells in 3D vessels under flow conditions. We further demonstrate that in a co‐culture model endothelial cells are not infected with SARS‐CoV‐2. Endothelial cells do however sense and respond to infection in the adjacent epithelial cells, increasing ICAM‐1 expression and releasing pro‐inflammatory cytokines. Conclusions Taken together, these data suggest that in vivo, endothelial cells are unlikely to be infected with SARS‐CoV‐2 and that infection may only occur if the adjacent pulmonary epithelium is denuded (basolateral infection) or a high viral load is present in the blood (apical infection). In such a scenario, whilst SARS‐CoV‐2 infection of the endothelium can occur, it does not contribute to viral amplification. However, endothelial cells may still play a key role in SARS‐CoV‐2 pathogenesis by sensing adjacent infection and mounting a pro‐inflammatory response to SARS‐CoV‐2., We show that endothelial cells are not productively infected with SARS‐CoV‐2 but mount a pro‐inflammatory response to the virus.

Immune profiling of secreted factors from human placentas across gestation identifies inflammasome hyperactivation in placental trophoblasts resulting in constitutive IL-1β and IL-18 secretion. Trophoblast-secreted IL-1β primes circulating monocytes for inflammasome activation and is important for control of placental Listeria monocytogenes infection., The human placenta is a dynamic organ that modulates physiological adaptations to pregnancy. To define the immunological signature of the human placenta, we performed unbiased profiling of secreted immune factors from human chorionic villi isolated from placentas at mid and late stages of pregnancy. We show that placental trophoblasts constitutively secrete the inflammasome-associated cytokines IL-1β and IL-18, which is blocked by NLRP3 inflammasome inhibitors and occurs without detectable gasdermin D cleavage. We further show that placenta-derived IL-1β primes monocytes for inflammasome induction to protect against Listeria monocytogenes infection. Last, we show that the human placenta responds to L. monocytogenes infection through additional inflammasome activation and that inhibition of this pathway sensitizes villi to infection. Our results thus identify the inflammasome as an important mechanism by which the human placenta regulates systemic and local immunity during pregnancy to defend against L. monocytogenes infection., Graphical Abstract

Inflammation is the result of a complex network of cellular and molecular interactions and mechanisms that facilitate immune protection against intrinsic and extrinsic stimuli, particularly pathogens, to maintain homeostasis and promote tissue healing. However, dysregulation in the immune system elicits excess/abnormal inflammation resulting in unintended tissue damage and causes major inflammatory diseases including asthma, chronic obstructive pulmonary disease, atherosclerosis, inflammatory bowel diseases, sarcoidosis and rheumatoid arthritis. It is now widely accepted that both endoplasmic reticulum (ER) stress and inflammasomes play critical roles in activating inflammatory signalling cascades. Notably, evidence is mounting for the involvement of ER stress in exacerbating inflammasome‐induced inflammatory cascades, which may provide a new axis for therapeutic targeting in a range of inflammatory disorders. Here, we comprehensively review the roles, mechanisms and interactions of both ER stress and inflammasomes, as well as their interconnected relationships in inflammatory signalling cascades. We also discuss novel therapeutic strategies that are being developed to treat ER stress‐ and inflammasome‐related inflammatory disorders., In this Review, we discuss the state‐of‐the‐art understanding of the pathways and factors involved in ER stress and inflammasome activation. We describe how these pathways induce inflammatory responses and are involved in chronic inflammatory diseases. We discuss new links between ER stress inflammasome activity and inflammation, and potential new therapeutic approaches to suppress ER stress and inflammasome‐induced inflammation.

Neutrophils are produced in the bone marrow and then patrol blood vessels from which they can be rapidly recruited to a site of infection. Neutrophils bind, engulf, and efficiently kill invading microbes via a suite of defense mechanisms. Diverse extracellular and intracellular microbes induce neutrophils to extrude neutrophil extracellular traps (NETs) through the process of NETosis. Here, we review the signaling mechanisms and cell biology underpinning the key NETosis pathways during infection and the antimicrobial functions of NETs in host defense.

The nod‐like receptor protein 3 (NLRP3) inflammasome drives inflammation in response to mitochondrial dysfunction. As metabolic powerhouses with prokaryotic ancestry, mitochondria are a cache for danger‐associated molecular patterns and pathogen‐associated molecular pattern‐like molecules that elicit potent innate immune responses. Persistent mitochondrial damage caused by infection, or genetic or environmental factors, can lead to inappropriate or sustained inflammasome signalling. Here, we review the features of mitochondria that drive inflammatory signalling, with a particular focus on mitochondrial activation of the NLRP3 inflammasome. Given that mitochondrial network dynamics, metabolic activity and redox state are all intricately linked to each other and to NLRP3 inflammasome activity, we highlight the importance of a holistic approach to investigations of NLRP3 activation by dysfunctional mitochondria., The NLRP3 inflammasome drives inflammation in response to mitochondrial dysfunction. Here, we review the features of mitochondria that drive inflammatory signalling, with a particular focus on mitochondrial activation of the NLRP3 inflammasome. Given that mitochondrial network dynamics, metabolic activity and redox state are all intricately linked to each other and to NLRP3 inflammasome activity, we highlight the importance of a holistic approach to investigations of NLRP3 activation by dysfunctional mitochondria.

Inflammasomes are signaling hubs that activate inflammatory caspases to drive cytokine maturation and cell lysis. Inflammasome activation by Salmonella Typhimurium infection or Salmonella-derived molecules is extensively studied in murine myeloid cells. Salmonella-induced inflammasome signaling in human innate immune cells, is however, poorly characterized. Here, we show that Salmonella mutation to inactivate the Salmonella pathogenicity island-2 type III secretion system (SPI2 T3SS) potentiates S. Typhimurium-induced inflammasome responses from primary human macrophages, resulting in strong IL-1β production and macrophage death. Inactivation of the SPI1 T3SS diminished human macrophage responses to WT and ΔSPI2 Salmonella. Salmonella ΔSPI2 elicited a mixed inflammasome response from human myeloid cells, in which NLR family CARD-domain containing protein 4 (NLRC4) and NLR family PYRIN-domain containing protein 3 (NLRP3) perform somewhat redundant functions in generating IL-1β and inducing pyroptosis. Our data suggest that Salmonella employs the SPI2 T3SS to subvert SPI1-induced NLRP3 and NLRC4 inflammasome responses in human primary macrophages, in a species-specific immune evasion mechanism. The Gram-negative bacterium Salmonella Typhimurium suppresses human macrophage inflammasome responses via the Salmonella Pathogenicity Island 2.

Summary: IL-1β requires processing by caspase-1 to generate the active, pro-inflammatory cytokine. Acute IL-1β secretion from inflammasome-activated macrophages requires caspase-1-dependent GSDMD cleavage, which also induces pyroptosis. Mechanisms of IL-1β secretion by pyroptotic and non-pyroptotic cells, and the precise functions of caspase-1 and GSDMD therein, are unresolved. Here, we show that, while efficient early secretion of endogenous IL-1β from primary non-pyroptotic myeloid cells in vitro requires GSDMD, later IL-1β release in vitro and in vivo proceeds independently of GSDMD. IL-1β maturation is sufficient for slow, caspase-1/GSDMD-independent secretion of ectopic IL-1β from resting, non-pyroptotic macrophages, but the speed of IL-1β release is boosted by inflammasome activation, via caspase-1 and GSDMD. IL-1β cleavage induces IL-1β enrichment at PIP2-enriched plasma membrane ruffles, and this is a prerequisite for IL-1β secretion and is mediated by a polybasic motif within the cytokine. We thus reveal a mechanism in which maturation-induced IL-1β trafficking facilitates its unconventional secretion. : Interleukin-1β is a potent pro-inflammatory cytokine whose dysregulated production drives a myriad of human diseases. Monteleone et al. uncover the trafficking mechanisms driving the unconventional secretion of mature interleukin-1β in non-pyroptotic and pyroptotic myeloid cells and reveal functions for caspase-1 and GSDMD therein. Keywords: interleukin-1, unconventional protein secretion, inflammasome, caspase-1, macrophage, neutrophil, gasdermin, pyroptosis, trafficking, phosphoinositides

The mammalian inhibitor of apoptosis proteins (IAPs) are key regulators of cell death and inflammation. A major function of IAPs is to block the formation of a cell death–inducing complex, termed the ripoptosome, which can trigger caspase-8–dependent apoptosis or caspase-independent necroptosis. Recent studies report that upon TLR4 or TNF receptor 1 (TNFR1) signaling in macrophages, the ripoptosome can also induce NLRP3 inflammasome formation and IL-1β maturation. Whether neutrophils have the capacity to assemble a ripoptosome to induce cell death and inflammasome activation during TLR4 and TNFR1 signaling is unclear. In this study, we demonstrate that murine neutrophils can signal via TNFR1-driven ripoptosome assembly to induce both cell death and IL-1β maturation. However, unlike macrophages, neutrophils suppress TLR4-dependent cell death and NLRP3 inflammasome activation during IAP inhibition via deficiencies in the CD14/TRIF arm of TLR4 signaling.

Differentiation of CD4+ Th cells is critical for immunity to malaria. Several innate immune signaling pathways have been implicated in the detection of blood-stage Plasmodium parasites, yet their influence over Th cell immunity remains unclear. In this study, we used Plasmodium-reactive TCR transgenic CD4+ T cells, termed PbTII cells, during nonlethal P. chabaudi chabaudi AS and P. yoelii 17XNL infection in mice, to examine Th cell development in vivo. We found no role for caspase1/11, stimulator of IFN genes, or mitochondrial antiviral-signaling protein, and only modest roles for MyD88 and TRIF-dependent signaling in controlling PbTII cell expansion. In contrast, IFN regulatory factor 3 (IRF3) was important for supporting PbTII expansion, promoting Th1 over T follicular helper (Tfh) differentiation, and controlling parasites during the first week of infection. IRF3 was not required for early priming by conventional dendritic cells, but was essential for promoting CXCL9 and MHC class II expression by inflammatory monocytes that supported PbTII responses in the spleen. Thereafter, IRF3-deficiency boosted Tfh responses, germinal center B cell and memory B cell development, parasite-specific Ab production, and resolution of infection. We also noted a B cell–intrinsic role for IRF3 in regulating humoral immune responses. Thus, we revealed roles for IRF3 in balancing Th1- and Tfh-dependent immunity during nonlethal infection with blood-stage Plasmodium parasites.

Immune cells are armed with Toll-like receptors (TLRs) for sensing and responding to pathogens and other danger cues. The role of extracellular-signal-regulated kinases 1/2 (Erk1/2) in TLR signaling remains enigmatic, with both pro- and anti-inflammatory functions described. We reveal here that the immune-specific transmembrane adaptor SCIMP is a direct scaffold for Erk1/2 in TLR pathways, with high-resolution, live-cell imaging revealing that SCIMP guides the spatial and temporal recruitment of Erk2 to membrane ruffles and macropinosomes for pro-inflammatory TLR4 signaling. SCIMP-deficient mice display defects in Erk1/2 recruitment to TLR4, c-Fos activation, and pro-inflammatory cytokine production, with these effects being phenocopied by Erk1/2 signaling inhibition. Our findings thus delineate a selective role for SCIMP as a key scaffold for the membrane recruitment of Erk1/2 kinase to initiate TLR-mediated pro-inflammatory responses in macrophages.

Bezbradica and Schroder preview new work from Kuri et al. that images endogenous ASC dynamics in zebrafish., Assembly of the ASC speck is critical for signaling by the inflammasome. In this issue, Kuri et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201703103) use live microscopy to track fluorescently tagged endogenous ASC in the zebrafish, describing the molecular domains driving ASC speck assembly and identifying a key role for macrophages in ASC speck removal in vivo.

X-linked Inhibitor of Apoptosis (XIAP) deficiency predisposes people to pathogen-associated hyperinflammation. Upon XIAP loss, Toll-like receptor (TLR) ligation triggers RIPK3-caspase-8-mediated IL-1β activation and death in myeloid cells. How XIAP suppresses these events remains unclear. Here, we show that TLR-MyD88 causes the proteasomal degradation of the related IAP, cIAP1, and its adaptor, TRAF2, by inducing TNF and TNF Receptor 2 (TNFR2) signaling. Genetically, we define that myeloid-specific cIAP1 loss promotes TLR-induced RIPK3-caspase-8 and IL-1β activity in the absence of XIAP. Importantly, deletion of TNFR2 in XIAP-deficient cells limited TLR-MyD88-induced cIAP1-TRAF2 degradation, cell death, and IL-1β activation. In contrast to TLR-MyD88, TLR-TRIF-induced interferon (IFN)β inhibited cIAP1 loss and consequent cell death. These data reveal how, upon XIAP deficiency, a TLR-TNF-TNFR2 axis drives cIAP1-TRAF2 degradation to allow TLR or TNFR1 activation of RIPK3-caspase-8 and IL-1β. This mechanism may explain why XIAP-deficient patients can exhibit symptoms reminiscent of patients with activating inflammasome mutations. Refereed/Peer-reviewed

The NLRP3 inflammasome is a multi-protein complex that assembles in response to tissue damage or infection, triggering activation of caspase-1, an enzyme that converts interleukin (IL)-1β into its active form. A role for the NLRP3 inflammasome is emerging in inflammatory pain, but its influence in other pain types is largely unexamined. Therefore the aim of this study was to assess the role of the NLRP3 inflammasome and its downstream product caspase-1 in a model of acute burn-induced pain in male mice. A superficial burn was induced on the plantar surface of the left hind paw using a hot plate set at 52.5°C for 25s. Development of burn-induced mechanical allodynia, thermal allodynia, edema and weight bearing changes was assessed in Nlrp3 −/− and caspase-1-deficient ( Ice −/− ) mice, and in mice administered the selective NLRP3 inflammasome inhibitor MCC950. Burn-induced mechanical and thermal allodynia developed normally in Nlrp3 −/− and Ice −/− mice and mice administered MCC950. Burn-induced edema was significantly reduced in Ice −/− mice only. Burn-induced weight bearing changes were attenuated in Nlrp3 −/− mice and mice administered MCC950 72h after burn only. This study suggests that NLRP3 and its downstream product caspase-1 have a limited role in the development of burn-induced pain.

The NOD-like receptor protein NLRC3 attenuates myeloid cell inflammatory responses. In this issue of Immunity, Uchimura et al. (2018) reveal additional T-cell-intrinsic functions for NLRC3 in restricting T cell metabolism, T helper 1 and T helper 17 cell responses, and antiviral and autoimmune responses.

Specific IL-1 family cytokines are initially expressed as inactive, cytosolic pro-forms. Chan and Schroder review inflammasome signaling and cell death decisions, mechanisms underpinning IL-1α, IL-1β, IL-18, and IL-37 maturation and release, and the functions of these cytokines in protective and pathological inflammation., Specific IL-1 family cytokines are expressed by cells as cytosolic pro-forms that require cleavage for their activity and cellular release. IL-1β, IL-18, and IL-37 maturation and secretion is governed by inflammatory caspases within signaling platforms called inflammasomes. By inducing pyroptosis, inflammasomes can also drive the release of the alarmin IL-1α. Recent advances have transformed our mechanistic understanding of inflammasome signaling, cell death decisions, and cytokine activation and secretion. Here, we provide an updated view of inflammasome signaling; mechanisms underpinning IL-1α, IL-1β, IL-18, and IL-37 maturation and release; and the functions of these cytokines in protective and pathological inflammation.

INTRODUCTION Hypoxemia is a life-threatening condition and is commonly seen in children with severe pneumonia. A government-led, NGO-supported, multifaceted oxygen improvement program was implemented to increase access to oxygen therapy in 29 hospitals in Kaduna, Kano, and Niger states. The program installed pulse oximeters and oxygen concentrators, trained health care workers, and biomedical engineers (BMEs), and provided regular feedback to health care staff through quality improvement teams. OBJECTIVE The aim of this study is to evaluate whether the program increased screening for hypoxemia with pulse oximetry and prescription of oxygen for patients with hypoxemia. METHODOLOGY The study is an uncontrolled before-after interventional study implemented at the hospital level. Medical charts of patients under 5 admitted for pneumonia between January 2017 and August 2018 were reviewed and information on patient care was extracted using a standardized form. The preintervention period of this study was defined as 1 January to 31 October 2017 and the postintervention period as 1 February to 31 August 2018. The primary outcomes of the study were whether blood-oxygen saturation measurements (SpO2 ) were documented and whether children with hypoxemia were prescribed oxygen. RESULTS A total of 3418 patient charts were reviewed (1601 during the preintervention period and 1817 during the postintervention period). There was a significant increase in the proportion of patients with SpO2 measurements after the interventions were conducted (adjusted odds ratio [aOR] 5.0; 4.3-5.7, P

Background Hypoxemia is a severe condition associated with high rates of mortality, particularly in low- and middle-income countries (LMICs) with poor access to oxygen therapy. Despite its clinical significance, there have been few studies to describe the burden of hypoxemia. Thus, the primary objective of this study is to systematically describe the prevalence of hypoxemia among pediatric and adult patients in low- and middle-income countries. Methods/design Standard systematic review methods will be used. Bibliographic databases (MEDLINE, EMBASE, CINAHL) will be searched from 1998 onwards. The search strategy aims to identify studies that have measured peripheral blood oxygen saturation (SpO2) in children and adults presenting to health facilities in LMICs. Studies will be included if oxygen saturation measurements by pulse oximetry were measured. No studies will be excluded based on study design though patients recruited from intensive care units and post-operative care will be excluded. The primary outcome is the prevalence of hypoxemia on presentation to the healthcare facility. We define hypoxemia on the basis of SpO2 measurements, and use a threshold of SpO2 less than 90% at sea level though allow for a lower threshold for studies conducted at higher altitude and where justified. Standardized tools will be used to extract data on number of patients with SpO2 measurements, number of patients with hypoxemia, patient population characteristics, and study characteristics. Quality of the included studies will be assessed using the “Checklist for Prevalence Studies” developed by the Joanna Briggs Institute. If there are enough studies to do so, we will conduct meta-analysis using a random effects model to estimate prevalence of hypoxemia and conduct subgroup analyses by age and disease groups. Discussion Hypoxemia is a critical condition and understanding the burden of hypoxemia may support decision-making in LMICs to deploy pulse oximeters and oxygen treatments more efficiently to address diseases and patient populations with the highest burden. Previous studies on hypoxemia prevalence have focused too narrowly on a few diseases or specific patient populations (e.g., pneumonia in children under five) whereas any effort to improve access to oxygen requires understanding of the potential demand for oxygen for all diseases and population groups. Governments, UN agencies, donors, and NGOs are investing strongly to improve oxygen systems in LMICs. Effective oxygen system planning requires estimation of oxygen need, informed by robust data on hypoxemia prevalence and admission patterns at all the levels of the health system. This study aims to fill that gap by providing comprehensive estimates of hypoxemia prevalence. Systematic review registration PROSPERO CRD42019136622.

Background In Uganda, diarrhoea was the third leading cause of mortality among children under the age of five in 2010. To address this issue, the Ministry of Health (MOH) formed a national Diarrhoea and Pneumonia Coordination Committee (DPCC) in 2011. One of the objectives of the DPCC for reducing diarrhoea mortality was to increase the use of oral rehydration salts (ORS) and zinc. This study aimed to describe and evaluate efforts by the DPCC to increase ORS and zinc coverage. Methods We conducted a retrospective mixed-methods evaluation to describe the activities conducted under the DPCC and evaluate the extent to which the committee's goal of increasing ORS and zinc use was achieved. We conducted secondary analysis using Uganda's Demographic and Health Survey from 2011 and 2016, analyzed cross-sectional private medicine outlet surveys from 2014 and 2016, analyzed ORS and zinc distribution data from the Uganda National Medical Stores, and reviewed program documents from DPCC partners. Results Nationally, the proportion of children under five with diarrhoea treated with ORS and zinc increased from 1% (95% confidence interval (CI) = 1%, 2%) in 2011 to 30% (95% CI = 27%, 32%) in 2016. Among private medicine outlets, the adjusted odds of having any zinc in-stock was 1.5 (95% CI = 1.14, 1.97) times higher in 2016 than in 2014, and the retail price for a complete treatment (2 ORS sachets and 10 zinc tablets) declined by $0.19 (95% CI = -0.31, -0.06), or 14%. Conclusions Use of combined ORS and zinc for treatment of diarrhoea in children under five significantly increased in Uganda during the program period. The range of activities conducted by the various members of the DPCC likely contributed to the increase in the use of combined ORS and zinc.

Background In Kenya, diarrheal disease is the second leading cause of death among children under five. The Government of Kenya launched a national plan to increase coverage of oral rehydration solution (ORS) and zinc by addressing demand and supply-side barriers. This study evaluates progress of ORS and zinc uptake in Kenya according to the national plan from 2011 to 2016. Methods In 2016, we conducted a nationally representative population-based household survey to estimate coverage of ORS and zinc for treatment of diarrhea cases among children under five. We also used ORS and zinc coverage data from the two most recent Kenya Demographic and Health Surveys in 2008/09 and 2014 to estimate annual changes in coverage rates during the program period. Based on these inputs, we used the Lives Saved Tool to estimate the number of diarrhea deaths averted between 2011 and 2016 due to increased use of ORS and zinc. Results The 2016 survey results showed that ORS coverage was 42% (95% confidence interval (CI) = 38%, 47%) and zinc coverage was 18% (95% CI = 15%, 23%). The estimated coverage for the combined use of ORS and zinc was 15% in 2016 (95% CI = 12%, 19%). Compared to 2011, an additional 3340 (sensitivity bounds = 2 670, 3 920) diarrhea deaths among children under five were averted due to increases in ORS and zinc coverage. Conclusions Kenya was successful in catalyzing uptake of combined treatment with ORS and zinc, which rose from 0.8% in 2011 to 15% in 2016. Ongoing efforts are necessary to further build on these gains.

Uropathogenic Escherichia coli (UPEC) is the major cause of urinary tract infections (UTIs). The multidrug-resistant E. coli sequence type 131 (ST131) clone is a serious threat to human health, yet its effects on immune responses are not well understood. Here we screened a panel of ST131 isolates, finding that only strains expressing the toxin hemolysin A (HlyA) killed primary human macrophages and triggered maturation of the inflammasome-dependent cytokine IL-1β. Using a representative strain, the requirement for the hlyA gene in these responses was confirmed. We also observed considerable heterogeneity in levels of cell death initiated by different HlyA+ve ST131 isolates, and this correlated with secreted HlyA levels. Investigation into the biological significance of this variation revealed that an ST131 strain producing low levels of HlyA initiated cell death that was partly dependent on the nod-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, with this response being associated with a host-protective role in a mouse UTI model. When the same ST131 strain was engineered to overexpress high HlyA levels, macrophage cell death occurred even when NLRP3 function was abrogated, and bladder colonization was significantly increased. Thus, variation in HlyA expression in UPEC affects mechanisms by which macrophages die, as well as host susceptibility vs. resistance to colonization.-Murthy, A. M. V., Sullivan, M. J., Nhu, N. T. K., Lo, A. W., Phan, M.-D., Peters, K. M., Boucher, D., Schroder, K., Beatson, S. A., Ulett, G. C., Schembri, M. A., Sweet, M. J. Variation in hemolysin A expression between uropathogenic Escherichia coli isolates determines NLRP3-dependent vs. -independent macrophage cell death and host colonization. Refereed/Peer-reviewed

We aimed to characterize antimicrobial zinc trafficking within macrophages and to determine whether the professional intramacrophage pathogen Salmonella enterica serovar Typhimurium (S Typhimurium) subverts this pathway. Using both Escherichia coli and S Typhimurium, we show that TLR signaling promotes the accumulation of vesicular zinc within primary human macrophages. Vesicular zinc is delivered to E. coli to promote microbial clearance, whereas S. Typhimurium evades this response via Salmonella pathogenicity island (SPI)-1. Even in the absence of SPI-1 and the zinc exporter ZntA, S Typhimurium resists the innate immune zinc stress response, implying the existence of additional host subversion mechanisms. We also demonstrate the combinatorial antimicrobial effects of zinc and copper, a pathway that S. Typhimurium again evades. Our use of complementary tools and approaches, including confocal microscopy, direct assessment of intramacrophage bacterial zinc stress responses, specific E. coli and S Typhimurium mutants, and inductively coupled plasma mass spectroscopy, has enabled carefully controlled characterization of this novel innate immune antimicrobial pathway. In summary, our study provides new insights at the cellular level into the well-documented effects of zinc in promoting host defense against infectious disease, as well as the complex host subversion strategies employed by S Typhimurium to combat this pathway.-Kapetanovic, R., Bokil, N. J., Achard, M. E. S., Ong, C.-L. Y., Peters, K. M., Stocks, C. J., Phan, M.-D., Monteleone, M., Schroder, K., Irvine, K. M., Saunders, B. M., Walker, M. J., Stacey, K. J., McEwan, A. G., Schembri, M. A., Sweet, M. J. Salmonella employs multiple mechanisms to subvert the TLR-inducible zinc-mediated antimicrobial response of human macrophages.

Differences in human and mouse immune responses may partly reflect species-specific adaptations and can provide important insights into human immunity. In this study, we show that RNF144B, which encodes an E3 ubiquitin ligase, was lipopolysaccharide-inducible in primary human macrophages and in human macrophage–like THP-1 cells. In contrast, Rnf144b was not lipopolysaccharide-inducible in several mouse cell populations, including primary macrophages from C57BL/6 and BALB/c mice and RAW264.7 macrophages. Similarly, Rnf144b was not up-regulated by infection of C57BL/6 mice with Escherichia coli. Although the human and mouse RNF144B genes have conserved transcription start sites, cap analysis of gene expression data confirmed that the RNF144B promoter directs transcription in human but not mouse macrophages. The human and mouse RNF144B genes are controlled by highly conserved TATA-containing promoters, but subtle differences in transcription factor binding sites may account for differential regulation. Using gene silencing, we showed that RNF144B is necessary for priming of inflammasome responses in primary human macrophages. Specifically, RNF144B promotes lipopolysaccharide-inducible IL-1b mRNA expression but does not regulate expression of several other lipopolysaccharide-inducible cytokines (e.g., interleukin-10, interferon-γ) or affect expression of inflammasome components or substrates (e.g., procaspase-1, pro-interleukin-18). Our findings thus revealed a species-specific regulatory mechanism for selective inflammasome priming in human macrophages.

Many programmed cell death pathways are essential for organogenesis, development, immunity and the maintenance of homeostasis in multicellular organisms. Pyroptosis, a highly proinflammatory form of cell death, is a critical innate immune response to prevent intracellular infection. Pyroptosis is induced upon the activation of proinflammatory caspases within macromolecular signalling platforms called inflammasomes. This article reviews our understanding of pyroptosis induction, the function of inflammatory caspases in pyroptosis execution, and the importance of pyroptosis for pathogen clearance. It also highlights the situations in which extensive pyroptosis may in fact be detrimental to the host, leading to immune cell depletion or cytokine storm. Current efforts to understand the beneficial and pathological roles of pyroptosis bring the promise of new approaches to fight infectious diseases.

NOD-like receptors (NLR) are a family of cytosolic pattern recognition receptors that include many key drivers of innate immune responses. NLRP12 is an emerging member of the NLR family that is closely related to the well-known inflammasome scaffold, NLRP3. Since its discovery, various functions have been proposed for NLRP12, including the positive regulation of dendritic cell (DC) and neutrophil migration and the inhibition of NF-κB and ERK signalling in DC and macrophages. We show here that NLRP12 is poorly expressed in murine macrophages and DC, but is strongly expressed in neutrophils. Using myeloid cells from WT and Nlrp12(-/)(-) mice, we show that, contrary to previous reports, NLRP12 does not suppress LPS- or infection-induced NF-κB or ERK activation in myeloid cells, and is not required for DC migration in vitro. Surprisingly, we found that Nlrp12 deficiency caused increased rather than decreased neutrophil migration towards the chemokine CXCL1 and the neutrophil parasite Leishmania major, revealing NLRP12 as a negative regulator of directed neutrophil migration under these conditions.

This study provides a detailed molecular mechanism for caspase-11 activation within the non-canonical inflammasome, giving new insight into host defence against cytosolic bacterial infection., Caspase-11 is a cytosolic sensor and protease that drives innate immune responses to the bacterial cell wall component, LPS. Caspase-11 provides defence against cytosolic Gram-negative bacteria; however, excessive caspase-11 responses contribute to murine endotoxic shock. Upon sensing LPS, caspase-11 assembles a higher order structure called the non-canonical inflammasome that enables the activation of caspase-11 protease function, leading to gasdermin D cleavage and cell death. The mechanism by which caspase-11 acquires protease function is, however, poorly defined. Here, we show that caspase-11 dimerization is necessary and sufficient for eliciting basal caspase-11 protease function, such as the ability to auto-cleave. We further show that during non-canonical inflammasome signalling, caspase-11 self-cleaves at site (D285) within the linker connecting the large and small enzymatic subunits. Self-cleavage at the D285 site is required to generate the fully active caspase-11 protease (proposed here to be p32/p10) that mediates gasdermin D cleavage, macrophage death, and NLRP3-dependent IL-1β production. This study provides a detailed molecular mechanism by which LPS induces caspase-11–driven inflammation and cell death to provide host defence against cytosolic bacterial infection.

Parkinson’s disease (PD) is characterized by a profound loss of dopaminergic neurons in the substantia nigra, accompanied by chronic neuroinflammation, mitochondrial dysfunction, and widespread accumulation of α-synuclein-rich protein aggregates in the form of Lewy bodies. However, the mechanisms linking α-synuclein pathology and dopaminergic neuronal death to chronic microglial neuroinflammation have not been completely elucidated. We show that activation of the microglial NLR family pyrin domain containing 3 (NLRP3) inflammasome is a common pathway triggered by both fibrillar α-synuclein and dopaminergic degeneration in the absence of α-synuclein aggregates. Cleaved caspase-1 and the inflammasome adaptor protein apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC) were elevated in the substantia nigra of the brains of patients with PD and in multiple preclinical PD models. NLRP3 activation by fibrillar α-synuclein in mouse microglia resulted in a delayed but robust activation of the NLRP3 inflammasome leading to extracellular interleukin-1β and ASC release in the absence of pyroptosis. Nanomolar doses of a small-molecule NLRP3 inhibitor, MCC950, abolished fibrillar α-synuclein-mediated inflammasome activation in mouse microglial cells and extracellular ASC release. Furthermore, oral administration of MCC950 in multiple rodent PD models inhibited inflammasome activation and effectively mitigated motor deficits, nigrostriatal dopaminergic degeneration, and accumulation of α-synuclein aggregates. These findings suggest that microglial NLRP3 may be a sustained source of neuroinflammation that could drive progressive dopaminergic neuropathology and highlight NLRP3 as a potential target for disease-modifying treatments for PD.

Mitochondria-derived signals are associated with NLRP3 inflammasome activation. A recent study in Nature by Zhong et al. shows that inflammasome priming drives new mitochondrial DNA synthesis, and proposes this to be a prerequisite for NLRP3 inflammasome signaling.

Inflammation is the host response to microbial infection or sterile injury that aims to eliminate the insult, repair the tissue and restore homeostasis. Macrophages and the NLRP3 inflammasome are key sentinels for both types of insult. Although it is well established that the NLRP3 inflammasome is activated by microbial products and molecules released during sterile injury, it is unclear whether the responses elicited by these different types of signals are distinct. In this study, we used lipopolysaccharide and tumor necrosis factor as prototypical microbial and sterile signal 1 stimuli, respectively, to prime the NLRP3 inflammasome. We then used the bacterial toxin nigericin and a common product released from necrotic cells, ATP, as prototypical microbial and sterile signal 2 stimuli, respectively, to trigger the assembly of the NLRP3 inflammasome complex in mouse and human macrophages. We found that NLRP3 inflammasome responses were weakest when both signal 1 and signal 2 were sterile, but responses were faster and stronger when at least one of the two signals was microbial. Ultimately, the most rapid and potent responses were elicited when both signals were microbial. Together, these data suggest that microbial versus sterile signals are distinct, both kinetically and in magnitude, in their ability to generate inflammasome-dependent responses. This hierarchy of NLRP3 responses to sterile versus microbial stimuli likely reflects the urgent need for the immune system to respond rapidly to the presence of infection to halt pathogen dissemination.

MCC950 a potent, highly specific small molecule inhibitor of canonical and noncanonical activation of NLRP3 inflammasome has been evaluated in a multitude of NLRP3 driven inflammatory diseases. However, the effect of MCC950 on colonic inflammation has not yet been reported. In the present study we investigated the effect of MCC950 in a spontaneous chronic colitis mouse model Winnie, which mimics human ulcerative colitis. Oral administration of 40 mg/kg MCC950 commencing at Winnie week seven for three weeks significantly improved body weight gain, colon length, colon weight to body weight ratio, disease activity index and histopathological scores. MCC950 significantly suppressed release of proinflammatory cytokines IL-1β, IL-18, IL1-α, IFNγ, TNF-α, IL6, IL17, chemokine MIP1a and Nitric Oxide in colonic explants. Moreover, MCC950 resulted in a significant decrease of IL-1β release and activation of caspase-1 in colonic explants and macrophage cells isolated from Winnie. Complete inhibition with MCC950 in Winnie colonic explants shows, for the first time, the contribution of inflammatory effects resulting exclusively from canonical and noncanonical NLRP3 inflammasome activation in colitis. Taken together, our results illustrate the efficacy of MCC950 in the treatment of murine ulcerative colitis and provides avenue for a potential novel therapeutic agent for human inflammatory bowel diseases.

Protracted bacterial bronchitis (PBB) in young children is characterised by prolonged wet cough, prominent airway interleukin (IL)-1β expression and infection, often with nontypeable Haemophilus influenzae (NTHi). The mechanisms responsible for IL-1-driven inflammation in PBB are poorly understood. We hypothesised that the inflammation in PBB involves the NLRP3 and/or AIM2 inflammasome/IL-1β axis. Lung macrophages obtained from bronchoalveolar lavage (BAL), peripheral blood mononuclear cells (PBMCs), blood monocytes and monocyte-derived macrophages from patients with PBB and age-matched healthy controls were cultured in control medium or exposed to live NTHi. In healthy adult PBMCs, CD14+ monocytes contributed to 95% of total IL-1β-producing cells upon NTHi stimulation. Stimulation of PBB PBMCs with NTHi significantly increased IL-1β expression (p, Airway IL-1β activation in protracted bacterial bronchitis http://ow.ly/ut9r30iqim2

Background In Nigeria, diarrhea is the second leading killer of children under five. Between 2012-2017, the Clinton Health Access Initiative, Inc. (CHAI) and the Government of Nigeria implemented a comprehensive program in eight states aimed at increasing the percentage of children under five with diarrhea who were treated with zinc and oral rehydration solution (ORS). The program addressed demand, supply, and policy barriers to ORS and zinc uptake through interventions in both public and private sectors. The interventions included: (1) policy revision and partner coordination; (2) market shaping to improve availability of affordable, high-quality ORS and zinc; (3) provider training and mentoring; and (4) caregiver demand generation. Methods We conducted cross-sectional household surveys in program states at baseline, midline, and endline and constructed logistic regression models with generalized estimating equations to assess changes in ORS and zinc treatment during the program period. Results In descriptive analysis, we found 38% (95% CI = 34%-42%) received ORS at baseline and 4% (95% CI = 3%-5%) received both ORS and zinc. At endline, we found 55% (95% CI = 51%-58%) received ORS and 30% (95% CI = 27%-33%) received both ORS and zinc. Adjusting for other covariates, the odds of diarrhea being treated with ORS were 1.88 (95% CI = 1.46, 2.43) times greater at endline. The odds of diarrhea being treated with ORS and zinc combined were 15.14 (95% CI = 9.82, 23.34) times greater at endline. When we include the interaction term to investigate whether the odds ratios between the endline and baseline survey were modified by source of care, we found statistically significant results among diarrhea episodes that sought care in the public and private sector. Among cases that sought care in the public sector, the predictive probability of treatment with ORS increased from 57% (95% CI = 50%-65%) to 83% (95% CI = 79%-87%). Among cases that sought care in the private sector, the predictive probability increased from 41% (95% CI = 34%-48%) to 58% (95% CI = 54%-63%). Conclusions Use of ORS and combined ORS and zinc for treatment of diarrhea significantly increased in program states during the program period.

Chronic liver disease (CLD) is associated with tissue-destructive fibrosis. Considering that common mechanisms drive fibrosis across etiologies, and that steatosis is an important cofactor for pathology, we performed RNA sequencing on liver biopsies of patients with different fibrosis stages, resulting from infection with hepatitis C virus (HCV) (with or without steatosis) or fatty liver disease. In combination with enhanced liver fibrosis score correlation analysis, we reveal a common set of genes associated with advanced fibrosis, as exemplified by those encoding the transcription factor ETS-homologous factor (EHF) and the extracellular matrix protein versican (VCAN). We identified 17 fibrosis-associated genes as candidate EHF targets and demonstrated that EHF regulates multiple fibrosis-associated genes, including VCAN, in hepatic stellate cells. Serum VCAN levels were also elevated in advanced fibrosis patients. Comparing biopsies from patients with HCV with or without steatosis, we identified a steatosis-enriched gene set associated with advanced fibrosis, validating follistatin-like protein 1 (FSTL1) as an exemplar of this profile. In patients with advanced fibrosis, serum FSTL1 levels were elevated in those with steatosis (versus those without). Liver Fstl1 mRNA levels were also elevated in murine CLD models. We thus reveal a common gene signature for CLD-associated liver fibrosis and potential biomarkers and/or targets for steatosis-associated liver fibrosis.

The caspase-1 protease is a core component of multiprotein inflammasome complexes, which play a critical role in regulating the secretion of mature, bioactive pro-inflammatory cytokines interleukin (IL)-1β and IL-18. The activity of caspase-1 is often measured indirectly, by monitoring cleavage of cellular caspase-1 substrates, processing of caspase-1 itself, or by quantifying cell death. Here we describe methods for eliciting caspase-1 activity in murine macrophages, via activation of the NLRP3, NAIP/NLRC4 or AIM2 inflammasomes. We then describe a simple fluorogenic assay for directly quantifying cellular caspase-1 activity.