RESUMO
The NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome is a multiprotein complex and component of the innate immune system that is activated by exogenous and endogenous danger signals to promote activation of caspase-1 and the maturation and release of the proinflammatory cytokines interleukin (IL)-1ß and IL-18. Inappropriate activation of NLRP3 has been implicated in the pathophysiology of multiple inflammatory and autoimmune diseases, including cardiovascular disease, neurodegenerative diseases, and nonalcoholic steatohepatitis (NASH), thus increasing the clinical interest of this target. We describe in this study the preclinical pharmacologic, pharmacokinetic, and pharmacodynamic properties of a novel and highly specific NLRP3 inhibitor, JT001 (6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-sulfonylurea). In cell-based assays, JT001 potently and selectively inhibited NLRP3 inflammasome assembly, resulting in the inhibition of cytokine release and the prevention of pyroptosis, a form of inflammatory cell death triggered by active caspase-1. Oral administration of JT001 to mice inhibited IL-1ß production in peritoneal lavage fluid at plasma concentrations that correlated with mouse in vitro whole blood potency. Orally administered JT001 was effective in reducing hepatic inflammation in three different murine models, including the Nlrp3A350V /+CreT model of Muckle-Wells syndrome (MWS), a diet-induced obesity NASH model, and a choline-deficient diet-induced NASH model. Significant reductions in hepatic fibrosis and cell damage were also observed in the MWS and choline-deficient models. Our findings demonstrate that blockade of NLRP3 attenuates hepatic inflammation and fibrosis and support the use of JT001 to investigate the role of NLRP3 in other inflammatory disease models. SIGNIFICANCE STATEMENT: Persistent inflammasome activation is the consequence of inherited mutations of NLRP3 and results in the development of cryopyrin-associated periodic syndromes associated with severe systemic inflammation. NLRP3 is also upregulated in nonalcoholic steatohepatitis, a metabolic chronic liver disease currently missing a cure. Selective and potent inhibitors of NLRP3 hold great promise and have the potential to overcome an urgent unmet need.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Camundongos , Animais , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/metabolismo , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas NLR , Domínio Pirina , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/prevenção & controle , Caspase 1/metabolismo , Inflamação , Colina/efeitos adversos , Interleucina-1beta/metabolismoRESUMO
Sensing of pathogens by Toll-like receptor 4 (TLR4) induces an inflammatory response; controlled responses confer immunity but uncontrolled responses cause harm. Here we define how a multimodular scaffold, GIV (a.k.a. Girdin), titrates such inflammatory response in macrophages. Upon challenge with either live microbes or microbe-derived lipopolysaccharides (a ligand for TLR4), macrophages with GIV mount a more tolerant (hypo-reactive) transcriptional response and suppress proinflammatory cytokines and signaling pathways (i.e., NFkB and CREB) downstream of TLR4 compared to their GIV-depleted counterparts. Myeloid-specific gene-depletion studies confirmed that the presence of GIV ameliorates dextran sodium sulfate-induced colitis and sepsis-induced death. The antiinflammatory actions of GIV are mediated via its C-terminally located TIR-like BB-loop (TILL) motif which binds the cytoplasmic TIR modules of TLR4 in a manner that precludes receptor dimerization; such dimerization is a prerequisite for proinflammatory signaling. Binding of GIV's TILL motif to TIR modules inhibits proinflammatory signaling via other TLRs, suggesting a convergent paradigm for fine-tuning macrophage inflammatory responses.
Assuntos
Proteínas dos Microfilamentos/metabolismo , Receptor 4 Toll-Like/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Colite/metabolismo , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Células RAW 264.7 , Sepse/metabolismo , Transdução de SinaisRESUMO
Macrophages are specialized phagocytic cells involved in clearing invading pathogens. Previously we reported that engulfment and cell motility protein 1 (ELMO1) in macrophages mediates bacterial internalization and intestinal inflammation. Here we studied the role of ELMO1 in the fate of internalized targets. ELMO1 is present in the intracellular vesicles and enhances accumulation of the protein LC3B following engulfment of Salmonella or treatment with autophagy-inducing rapamycin. The protein ATG5 and the kinase ULK1 are involved in classical autophagy, while LC3-associated phagocytosis is ULK1 independent. ATG5 but not ULK1 cooperated with ELMO1 in LC3 accumulation after infection, suggesting the ELMO1 preferentially regulated LC3-associated phagocytosis. Because LC3-associated phagocytosis delivers cargo for degradation, the contribution of ELMO1 to the lysosome degradation pathways was evaluated by studying pH and cathepsin B activity. ELMO1-depleted macrophages showed a time-dependent increase in pH and a decrease in cathepsin B activity associated with bacterial survival. Together, ELMO1 regulates LC3B accumulation and antimicrobial responses involved in the clearance of enteric pathogens. This paper investigated how innate immune pathways involving ELMO1 work in a coordinated fashion to eliminate bacterial threats. ELMO1 is present in the phagosome and enhances bacterial clearance by differential regulation of lysosomal acidification and enzymatic activity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia , Macrófagos/imunologia , Macrófagos/microbiologia , Infecções por Salmonella/patologia , Salmonella/crescimento & desenvolvimento , Salmonella/imunologia , Animais , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Catepsina B/análise , Linhagem Celular , Modelos Animais de Doenças , Concentração de Íons de Hidrogênio , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
Background: Genetic factors and microbial imbalances play crucial roles in colorectal cancers (CRCs), yet the impact of infections on cancer initiation remains poorly understood. While bioinformatic approaches offer valuable insights, the rising incidence of CRCs creates a pressing need to precisely identify early CRC events. We constructed a network model to identify continuum states during CRC initiation spanning normal colonic tissue to pre-cancer lesions (adenomatous polyps) and examined the influence of microbes and host genetics. Methods: A Boolean network was built using a publicly available transcriptomic dataset from healthy and adenoma affected patients to identify an invariant Microbe-Associated Colorectal Cancer Signature (MACS). We focused on Fusobacterium nucleatum ( Fn ), a CRC-associated microbe, as a model bacterium. MACS-associated genes and proteins were validated by RT-qPCR, RNA seq, ELISA, IF and IHCs in tissues and colon-derived organoids from genetically predisposed mice ( CPC-APC Min+/- ) and patients (FAP, Lynch Syndrome, PJS, and JPS). Results: The MACS that is upregulated in adenomas consists of four core genes/proteins: CLDN2/Claudin-2 (leakiness), LGR5/leucine-rich repeat-containing receptor (stemness), CEMIP/cell migration-inducing and hyaluronan-binding protein (epithelial-mesenchymal transition) and IL8/Interleukin-8 (inflammation). MACS was induced upon Fn infection, but not in response to infection with other enteric bacteria or probiotics. MACS induction upon Fn infection was higher in CPC-APC Min+/- organoids compared to WT controls. The degree of MACS expression in the patient-derived organoids (PDOs) generally corresponded with the known lifetime risk of CRCs. Conclusions: Computational prediction followed by validation in the organoid-based disease model identified the early events in CRC initiation. MACS reveals that the CRC-associated microbes induce a greater risk in the genetically predisposed hosts, suggesting its potential use for risk prediction and targeted cancer prevention.
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Crohn's disease (CD) is a complex, clinically heterogeneous disease of multifactorial origin; there is no perfect pre-clinical model, little insight into the basis for such heterogeneity, and still no cure. To address these unmet needs, we sought to explore the translational potential of adult stem cell-derived organoids that not only retain their tissue identity, but also their genetic and epigenetic disease-driving traits. We prospectively created a biobank of CD patient-derived organoid cultures (PDOs) using biopsied tissues from colons of 34 consecutive subjects representing all clinical subtypes (Montreal Classification B1-B3 and perianal disease). PDOs were generated also from healthy subjects. Comparative gene expression analyses enabled benchmarking of PDOs as tools for modeling the colonic epithelium in active disease and revealed that despite the clinical heterogeneity there are two major molecular subtypes: immune-deficient infectious-CD [IDICD] and stress and senescence-induced fibrostenotic-CD [S2FCD]. The transcriptome, genome and phenome show a surprising degree of internal consistency within each molecular subtype. The spectrum of morphometric, phenotypic, and functional changes within the "living biobank" reveals distinct differences between the molecular subtypes. These insights enabled drug screens that reversed subtype-specific phenotypes, e.g., impaired microbial clearance in IDICD was reversed using agonists for nuclear receptors, and senescence in S2FCD was rectified using senotherapeutics, but not vice versa . Phenotyped-genotyped CD-PDOs may fill the gap between basic biology and patient trials by enabling pre-clinical Phase '0' human trials for personalized therapeutics. In Brief: This work creates a prospectively biobanked phenotyped-genotyped Crohn's disease patient-derived organoids (CD-PDOs) as platforms for molecular subtyping of disease and for ushering personalized therapeutics. HIGHLIGHTS: Prospectively biobanked CD-organoids recapitulate the disease epithelium in patientsThe phenome-transcriptome-genome of CD-organoids converge on two molecular subtypesOne subtype shows impaired microbial clearance, another increased cellular senescencePhenotyped-genotyped PDOs are then used for integrative and personalized therapeutics.
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E-cigarette usage continues to rise, yet the safety of e-cigarette aerosols is questioned. Using murine models of acute and chronic e-cigarette aerosol inhalation, murine colon transcriptomics, and murine and human gut-derived organoids in co-culture models, we assessed the effects of e-cigarette use on the gut barrier. Histologic and transcriptome analyses revealed that chronic, but not acute, nicotine-free e-cigarette use increased inflammation and reduced expression of tight junction (TJ) markers. Exposure of murine and human enteroid-derived monolayers (EDMs) to nicotine-free e-cigarette aerosols alone or in co-culture with bacteria also causes barrier disruption, downregulation of TJ protein, and enhanced inflammation in response to infection. These data highlight the harmful effects of "non-nicotine" component of e-cigarettes on the gut barrier. Considering the importance of an intact gut barrier for host fitness and the impact of gut mucosal inflammation on a multitude of chronic diseases, these findings are broadly relevant to both medicine and public health.
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Modeling human diseases as networks simplify complex multi-cellular processes, helps understand patterns in noisy data that humans cannot find, and thereby improves precision in prediction. Using Inflammatory Bowel Disease (IBD) as an example, here we outline an unbiased AI-assisted approach for target identification and validation. A network was built in which clusters of genes are connected by directed edges that highlight asymmetric Boolean relationships. Using machine-learning, a path of continuum states was pinpointed, which most effectively predicted disease outcome. This path was enriched in gene-clusters that maintain the integrity of the gut epithelial barrier. We exploit this insight to prioritize one target, choose appropriate pre-clinical murine models for target validation and design patient-derived organoid models. Potential for treatment efficacy is confirmed in patient-derived organoids using multivariate analyses. This AI-assisted approach identifies a first-in-class gut barrier-protective agent in IBD and predicted Phase-III success of candidate agents.