RESUMO
Therapy using anti-PD-1 immune checkpoint inhibitors (ICI) has revolutionized the treatment of many cancers including head and neck squamous cell carcinomas (HNSCC), but only a fraction of patients respond. To better understand the molecular mechanisms driving resistance, we performed extensive analysis of plasma and tumor tissues before and after a 4-week neoadjuvant trial in which HNSCC patients were treated with the anti-PD-1 inhibitor, nivolumab. Luminex cytokine analysis of patient plasma demonstrated that HPVpos nonresponders displayed high levels of the proinflammatory chemokine, interleukin-8 (IL-8), which decreased after ICI treatment, but remained higher than responders. miRNAseq analysis of tetraspanin-enriched small extracellular vesicles (sEV) purified from plasma of HPVpos nonresponders demonstrated significantly lower levels of seven miRNAs that target IL-8 including miR-146a. Levels of the pro-survival oncoprotein Dsg2, which has been to down-regulate miR-146a, are elevated with HPVpos tumors displaying higher levels than HPVneg tumors. Dsg2 levels decrease significantly following ICI in responders but not in nonresponders. In cultured HPVpos cells, restoration of miR-146a by forced expression or treatment with miR-146a-loaded sEV, reduced IL-8 level, blocked cell cycle progression, and promoted cell death. These findings identify Dsg2, miR-146a, and IL-8 as potential biomarkers for ICI response and suggest that the Dsg2/miR-146a/IL-8 signaling axis negatively impacts ICI treatment outcomes and could be targeted to improve ICI responsiveness in HPVpos HNSCC patients.
Assuntos
Vesículas Extracelulares , Neoplasias de Cabeça e Pescoço , MicroRNAs , Infecções por Papillomavirus , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Interleucina-8/genética , Nivolumabe/farmacologia , Nivolumabe/uso terapêutico , Terapia Neoadjuvante , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/genética , Vesículas Extracelulares/metabolismoRESUMO
PURPOSE: Heritable ectopic mineralization disorders comprise a group of conditions with a broad range of clinical manifestations in nonskeletal connective tissues. We report the genetic findings from a large international cohort of 478 patients afflicted with ectopic mineralization. METHODS: Sequence variations were identified using a next-generation sequencing panel consisting of 29 genes reported in association with ectopic mineralization. The pathogenicity of select splicing and missense variants was analyzed in experimental systems in vitro and in vivo. RESULTS: A total of 872 variants of unknown significance as well as likely pathogenic and pathogenic variants were disclosed in 25 genes. A total of 159 distinct variants were identified in 425 patients in ABCC6, the gene responsible for pseudoxanthoma elasticum, a heritable multisystem ectopic mineralization disorder. The interpretation of variant pathogenicity relying on bioinformatic predictions did not provide a consensus. Our in vitro and in vivo functional assessment of 14 ABCC6 variants highlighted this dilemma and provided unambiguous interpretations to their pathogenicity. CONCLUSION: The results expand the ABCC6 variant repertoire, shed new light on the genetic heterogeneity of heritable ectopic mineralization disorders, and provide evidence that functional characterization in appropriate experimental systems is necessary to determine the pathogenicity of genetic variants.
Assuntos
Heterogeneidade Genética , Pseudoxantoma Elástico , Estudos de Coortes , Tecido Conjuntivo/patologia , Humanos , Mutação de Sentido Incorreto , Pseudoxantoma Elástico/genéticaRESUMO
Characterizing self-tolerance mechanisms and their failure is critical to understand immune homeostasis, cancer immunity, and autoimmunity. However, examination of self-tolerance mechanisms has relied primarily on transgenic mice expressing TCRs targeting well-characterized, but nonphysiologic, model Ags, such as OVA and hemagglutinin. Identifying TCRs directed against bona fide self-antigens is made difficult by the extraordinary diversity of TCRs and the low prevalence of Ag-specific clones (<10-100 naive cells per organism), limiting dissection of tolerance mechanisms restricting immunity to self-proteins. In this study, we isolated and characterized TCRs recognizing the intestinal epithelial cell receptor and colorectal cancer Ag GUCY2C to establish a model to study self-antigen-specific tolerance mechanisms. GUCY2C-specific CD4+ effector T cells were isolated from immunized, nontolerant Gucy2c -/- mice. Next-generation sequencing identified GUCY2C-specific TCRs, which were engineered into CD4+ T cells in vitro to confirm TCR recognition of GUCY2C. Further, the generation of "retrogenic" mice by reconstitution with TCR-transduced hematopoietic stem cells resulted in normal CD4+ T cell development, responsiveness to immunization, and GUCY2C-induced tolerance in recipient mice, recapitulating observations in conventional models. This retrogenic model can be employed to define self-tolerance mechanisms restricting T and B cell responses to GUCY2C to optimize colorectal cancer immunotherapy without autoimmunity.
Assuntos
Neoplasias Colorretais/imunologia , Modelos Imunológicos , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Enterotoxina/imunologia , Animais , Feminino , Tolerância Imunológica/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos TransgênicosRESUMO
Heterologous prime-boost immunization with plasmid DNA and viral vector vaccines is an emerging approach to elicit CD8+ T cell-mediated immunity targeting pathogens and tumor Ags that is superior to either monotherapy. Yet, the mechanisms underlying the synergy of prime-boost strategies remain incompletely defined. In this study, we examine a DNA and adenovirus (Ad5) combination regimen targeting guanylyl cyclase C (GUCY2C), a receptor expressed by intestinal mucosa and universally expressed by metastatic colorectal cancer. DNA immunization efficacy was optimized by i.m. delivery via electroporation, yet it remained modest compared with Ad5. Sequential immunization with DNA and Ad5 produced superior antitumor efficacy associated with increased TCR avidity, whereas targeted disruption of TCR avidity enhancement eliminated GUCY2C-specific antitumor efficacy, without affecting responding T cell number or cytokine profile. Indeed, functional TCR avidity of responding GUCY2C-specific CD8+ T cells induced by various prime or prime-boost regimens correlated with antitumor efficacy, whereas T cell number and cytokine profile were not. Importantly, although sequential immunization with DNA and Ad5 maximized antitumor efficacy through TCR avidity enhancement, it produced no autoimmunity, reflecting sequestration of GUCY2C to intestinal apical membranes and segregation of mucosal and systemic immunity. Together, TCR avidity enhancement may be leveraged by prime-boost immunization to improve GUCY2C-targeted colorectal cancer immunotherapeutic efficacy and patient outcomes without concomitant autoimmune toxicity.
Assuntos
Biomarcadores Tumorais/metabolismo , Linfócitos T CD8-Positivos/fisiologia , Neoplasias Colorretais/terapia , Imunoterapia Adotiva/métodos , Mucosa Intestinal/fisiologia , Receptores Acoplados a Guanilato Ciclase/metabolismo , Receptores de Peptídeos/metabolismo , Vacinas de DNA/imunologia , Adenoviridae/genética , Animais , Biomarcadores Tumorais/genética , Linfócitos T CD8-Positivos/transplante , Células Cultivadas , Neoplasias Colorretais/imunologia , Citotoxicidade Imunológica , Imunidade nas Mucosas , Imunização Secundária , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Enterotoxina , Receptores Acoplados a Guanilato Ciclase/genética , Receptores de Peptídeos/genética , Carga TumoralRESUMO
Recent advances in biomedical research in cancer immunotherapy have identified the use of an oxidative stress-based approach to treat cancers, which works by inducing immunogenic cell death (ICD) in cancer cells. Since the anti-cancer effects of non-thermal plasma (NTP) are largely attributed to the reactive oxygen and nitrogen species that are delivered to and generated inside the target cancer cells, it is reasonable to postulate that NTP would be an effective modality for ICD induction. NTP treatment of tumors has been shown to destroy cancer cells rapidly and, under specific treatment regimens, this leads to systemic tumor-specific immunity. The translational benefit of NTP for treatment of cancer relies on its ability to enhance the interactions between NTP-exposed tumor cells and local immune cells which initiates subsequent protective immune responses. This review discusses results from recent investigations of NTP application to induce immunogenic cell death in cancer cells. With further optimization of clinical devices and treatment protocols, NTP can become an essential part of the therapeutic armament against cancer.
RESUMO
Obesity has emerged as a principal cause of mortality worldwide, reflecting comorbidities including cancer risk, particularly in colorectum. Although this relationship is established epidemiologically, molecular mechanisms linking colorectal cancer and obesity continue to be refined. Guanylyl cyclase C (GUCY2C), a membrane-bound guanylyl cyclase expressed in intestinal epithelial cells, binds the paracrine hormones guanylin and uroguanylin, inducing cGMP signaling in colorectum and small intestine, respectively. Guanylin is the most commonly lost gene product in sporadic colorectal cancer, and its universal loss early in transformation silences GUCY2C, a tumor suppressor, disrupting epithelial homeostasis underlying tumorigenesis. In small intestine, eating induces endocrine secretion of uroguanylin, the afferent limb of a novel gut-brain axis that activates hypothalamic GUCY2C-cGMP signaling mediating satiety opposing obesity. Recent studies revealed that diet-induced obesity suppressed guanylin and uroguanylin expression in mice and humans. Hormone loss reflects reversible calorie-induced endoplasmic reticulum stress and the associated unfolded protein response, rather than the endocrine, adipokine, or inflammatory milieu of obesity. Loss of intestinal uroguanylin secretion silences the hypothalamic GUCY2C endocrine axis, creating a feed-forward loop contributing to hyperphagia in obesity. Importantly, calorie-induced guanylin loss silences the GUCY2C-cGMP paracrine axis underlying obesity-induced epithelial dysfunction and colorectal tumorigenesis. Indeed, genetically enforced guanylin replacement eliminated diet-induced intestinal tumorigenesis in mice. Taken together, these observations suggest that GUCY2C hormone axes are at the intersection of obesity and colorectal cancer. Moreover, they suggest that hormone replacement that restores GUCY2C signaling may be a novel therapeutic paradigm to prevent both hyperphagia and intestinal tumorigenesis in obesity.
Assuntos
Neoplasias Colorretais/metabolismo , Guanilato Ciclase/metabolismo , Hormônios/metabolismo , Obesidade/metabolismo , Transdução de Sinais , Animais , Estresse do Retículo Endoplasmático , HumanosRESUMO
Enterotoxigenic Escherichia coli (ETEC) causes â¼20% of the acute infectious diarrhea (AID) episodes worldwide, often by producing heat-stable enterotoxins (STs), which are peptides structurally homologous to paracrine hormones of the intestinal guanylate cyclase C (GUCY2C) receptor. While molecular mechanisms mediating ST-induced intestinal secretion have been defined, advancements in therapeutics have been hampered for decades by the paucity of disease models that integrate molecular and functional endpoints amenable to high-throughput screening. Here, we reveal that mouse and human intestinal enteroids in three-dimensional ex vivo cultures express the components of the GUCY2C secretory signaling axis. ST and its structural analog, linaclotide, an FDA-approved oral secretagog, induced fluid accumulation quantified simultaneously in scores of enteroid lumens, recapitulating ETEC-induced intestinal secretion. Enteroid secretion depended on canonical molecular signaling events responsible for ETEC-induced diarrhea, including cyclic GMP (cGMP) produced by GUCY2C, activation of cGMP-dependent protein kinase (PKG), and opening of the cystic fibrosis transmembrane conductance regulator (CFTR). Importantly, pharmacological inhibition of CFTR abrogated enteroid fluid secretion, providing proof of concept for the utility of this model to screen antidiarrheal agents. Intestinal enteroids offer a unique model, integrating the GUCY2C signaling axis and luminal fluid secretion, to explore the pathophysiology of, and develop platforms for, high-throughput drug screening to identify novel compounds to prevent and treat ETEC diarrheal disease.
Assuntos
Toxinas Bacterianas/metabolismo , Escherichia coli Enterotoxigênica/fisiologia , Enterotoxinas/fisiologia , Infecções por Escherichia coli/microbiologia , Mucosa Intestinal/metabolismo , Receptores Acoplados a Guanilato Ciclase/metabolismo , Receptores de Peptídeos/metabolismo , Análise de Variância , Animais , GMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Diarreia/metabolismo , Modelos Animais de Doenças , Escherichia coli Enterotoxigênica/metabolismo , Enterotoxinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Infecções por Escherichia coli/fisiopatologia , Proteínas de Escherichia coli/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Enterotoxina , Transdução de Sinais/fisiologiaRESUMO
Self-tolerance, presumably through lineage-unbiased elimination of self-antigen-specific lymphocytes (CD4(+) T, CD8(+) T, and B cells), creates a formidable barrier to cancer immunotherapy. In contrast to this prevailing paradigm, we demonstrate that for some antigens, self-tolerance reflects selective elimination of antigen-specific CD4(+) T cells, but preservation of CD8(+) T- and B-cell populations. In mice, antigen-specific CD4(+) T-cell tolerance restricted CD8(+) T- and B-cell responses targeting the endogenous self-antigen guanylyl cyclase c (GUCY2C) in colorectal cancer. Although selective CD4(+) T-cell tolerance blocked GUCY2C-specific antitumor immunity and memory responses, it offered a unique solution to the inefficacy of GUCY2C vaccines through recruitment of self-antigen-independent CD4(+) T-cell help. Incorporating CD4(+) T-cell epitopes from foreign antigens into vaccines against GUCY2C reconstituted CD4(+) T-cell help, revealing the latent functional capacity of GUCY2C-specific CD8(+) T- and B-cell pools, producing durable antitumor immunity without autoimmunity. Incorporating CD4(+) T-cell epitopes from foreign antigens into vaccines targeting self-antigens in melanoma (Trp2) and breast cancer (Her2) produced similar results, suggesting selective CD4(+) T-cell tolerance underlies ineffective vaccination against many cancer antigens. Thus, identification of self-antigens characterized by selective CD4(+) T-cell tolerance and abrogation of such tolerance through self-antigen-independent T-cell help is essential for future immunotherapeutics.
Assuntos
Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Tolerância Imunológica , Neoplasias/terapia , Animais , Autoantígenos/imunologia , Epitopos de Linfócito T/imunologia , Memória Imunológica , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Receptores de Enterotoxina , Receptores Acoplados a Guanilato Ciclase/imunologia , Receptores de Peptídeos/imunologiaRESUMO
We assessed several routes of immunization with vaccinia virus (VACV) in protecting mice against ectromelia virus (ECTV). By a wide margin, skin scarification provided the greatest protection. Humoral immunity and resident-memory T cells notwithstanding, several approaches revealed that circulating, memory CD8(+) T cells primed via scarification were functionally superior and conferred enhanced virus control. Immunization via the epithelial route warrants further investigation, as it may also provide enhanced defense against other infectious agents.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Vírus da Ectromelia/imunologia , Ectromelia Infecciosa/imunologia , Epitélio/imunologia , Vaccinia virus/imunologia , Animais , Imunidade Humoral/imunologia , Imunização/métodos , Memória Imunológica/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Vacinação/métodosRESUMO
Veriflow® Listeria monocytogenes (LM) is a molecular based assay for the presumptive detection of Listeria monocytogenes from environmental surfaces, dairy, and ready-to-eat (RTE) food matrixes (hot dogs and deli meat). The assay utilizes a PCR detection method coupled with a rapid, visual, flow-based assay that develops in 3 min post PCR amplification and requires only 24 h of enrichment for maximum sensitivity. The Veriflow LM system eliminates the need for sample purification, gel electrophoresis, or fluorophore-based detection of target amplification, and does not require complex data analysis. This Performance Tested Method(SM) validation study demonstrated the ability of the Veriflow LM method to detect low levels of artificially inoculated L. monocytogenes in seven distinct environmental and food matrixes. In each unpaired reference comparison study, probability of detection analysis indicated no significant difference between the Veriflow LM method and the U.S. Department of Agriculture, Food Safety and Inspection Service Microbiology Laboratory Guidebook 8.08 or AOAC 993.12 reference method. Fifty strains of L. monocytogenes were detected in the inclusivity study, while 39 nonspecific organisms were undetected in the exclusivity study. The study results show that Veriflow LM is a sensitive, selective, and robust assay for the presumptive detection of L. monocytogenes sampled from environmental, dairy, or RTE (hot dogs and deli meat) food matrixes.
Assuntos
Laticínios/microbiologia , Análise de Alimentos/normas , Listeria monocytogenes/genética , Carne/análise , Reação em Cadeia da Polimerase/normas , Animais , Automação Laboratorial , Bovinos , Análise de Alimentos/métodos , Contaminação de Alimentos/análise , Humanos , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade , Estados Unidos , United States Department of AgricultureRESUMO
Neurological disorders are the leading cause of cognitive and physical disability worldwide, affecting 15% of the global population. Due to the demographics of aging, the prevalence of neurological disorders, including neurodegenerative diseases, will double over the next two decades. Unfortunately, while available therapies provide symptomatic relief for cognitive and motor impairment, there is an urgent unmet need to develop disease-modifying therapies that slow the rate of pathological progression. In that context, biomarkers could identify at-risk and prodromal patients, monitor disease progression, track responses to therapy, and parse the causality of molecular events to identify novel targets for further clinical investigation. Thus, identifying biomarkers that discriminate between diseases and reflect specific stages of pathology would catalyze the discovery and development of therapeutic targets. This review will describe the prevalence, known mechanisms, ongoing or recently concluded therapeutic clinical trials, and biomarkers of three of the most prevalent neurodegenerative diseases, including Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and Parkinson's disease (PD).
Assuntos
Doença de Alzheimer , Biomarcadores , Doenças Neurodegenerativas , Humanos , Biomarcadores/metabolismo , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/diagnóstico , Doenças Neurodegenerativas/terapia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/terapia , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/terapia , Esclerose Lateral Amiotrófica/diagnóstico , Doença de Parkinson/metabolismo , Doença de Parkinson/diagnóstico , Doença de Parkinson/terapia , AnimaisRESUMO
This chapter introduces four commonly used in vitro chimeric antigen receptor (CAR)-T cell cytotoxicity assays (lactate dehydrogenase release assay, 51Cr release assay, IncuCyte live cell killing assay, and xCELLigence real-time analysis) and provides a detailed protocol for xCELLigence real-time analysis. Focusing on in vitro assays, this chapter starts with explaining the mechanisms and discussing the utilization of each assay to quantify T-cell-induced cytotoxicity. Due to the high-throughput quantification and straightforward workflow of xCELLigence real-time analysis, a protocol entailing reagents and equipment, a 3-day step-by-step procedure, and instructions for data analysis are provided.
Assuntos
Apoptose , Linfócitos T , Linhagem Celular TumoralRESUMO
Mitochondrial dysfunction and reactive oxygen species (ROS) accumulation within the substantia nigra pars compacta (SNpc) are central drivers of dopaminergic (DA) neuron death in Parkinson's disease (PD). Guanylyl cyclases and their second messenger cyclic (c)GMP support mitochondrial function, protecting against ROS and promoting cell survival in several tissues. However, the role of the guanylyl cyclase-cGMP axis in defining the vulnerability of DA neurons in the SNpc in PD remains unclear, in part due to the challenge of manipulating cGMP levels selectively in midbrain DA neurons. In that context, guanylyl cyclase C (GUCY2C), a receptor primarily expressed by intestinal epithelial cells, was discovered recently in midbrain DA neurons. Here, we demonstrate that GUCY2C promotes mitochondrial function, reducing oxidative stress and protecting DA neurons from degeneration in the 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP) mouse model. GUCY2C is overexpressed in the SNpc in PD patients and in mice treated with MPTP, possibly reflecting a protective response to oxidative stress. Moreover, cGMP signaling protects against oxidative stress, mitochondrial impairment, and cell death in cultured DA neurons. These observations reveal a previously unexpected role for the GUCY2C-cGMP signaling axis in controlling mitochondrial dysfunction and toxicity in SNpc DA neurons, highlighting the therapeutic potential of targeting DA neuron GUCY2C to prevent neurodegeneration in PD.
RESUMO
Most patients treated with US Food and Drug Administration (FDA)-approved chimeric antigen receptor (CAR) T cells eventually experience disease progression. Furthermore, CAR T cells have not been curative against solid cancers and several hematological malignancies such as T cell lymphomas, which have very poor prognoses. One of the main barriers to the clinical success of adoptive T cell immunotherapies is CAR T cell dysfunction and lack of expansion and/or persistence after infusion. In this study, we found that CD5 inhibits CAR T cell activation and that knockout (KO) of CD5 using CRISPR-Cas9 enhances the antitumor effect of CAR T cells in multiple hematological and solid cancer models. Mechanistically, CD5 KO drives increased T cell effector function with enhanced cytotoxicity, in vivo expansion, and persistence, without apparent toxicity in preclinical models. These findings indicate that CD5 is a critical inhibitor of T cell function and a potential clinical target for enhancing T cell therapies.
Assuntos
Antígenos CD5 , Imunoterapia Adotiva , Linfócitos T , Animais , Imunoterapia Adotiva/métodos , Antígenos CD5/imunologia , Camundongos , Humanos , Linfócitos T/imunologia , Linfócitos T/transplante , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/genética , Linhagem Celular Tumoral , Sistemas CRISPR-Cas , FemininoRESUMO
Mitochondrial dysfunction and reactive oxygen species (ROS) accumulation within the substantia nigra pars compacta (SNpc) are central drivers of dopaminergic (DA) neuron death in Parkinson's disease (PD). Guanylyl cyclases, and their second messengers cyclic (c)GMP, support mitochondrial function, protecting against ROS and promoting cell survival in a number of tissues. However, the role of the guanylyl cyclase-cGMP axis in defining the vulnerability of DA neurons in the SNpc in PD remains unclear, in part due to the challenge of manipulating cGMP levels selectively in midbrain DA neurons. In that context, guanylyl cyclase C (GUCY2C), a receptor primarily expressed by intestinal epithelial cells, was discovered recently in midbrain DA neurons. Here, we demonstrate that GUCY2C promotes mitochondrial function, reducing oxidative stress and protecting DA neurons from degeneration in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of neurodegeneration. GUCY2C is overexpressed in the SNpc in PD patients and in mice treated with MPTP, possibly reflecting a protective response to oxidative stress. Moreover, cGMP signaling protects against oxidative stress, mitochondrial impairment, and cell death in cultured DA neurons. These observations reveal a previously unexpected role for the GUCY2C-cGMP signaling axis in controlling mitochondrial dysfunction and toxicity in nigral DA neurons, highlighting the therapeutic potential of targeting DA neuron GUCY2C to prevent neurodegeneration in PD.
RESUMO
Visceral pain (VP) is a global problem with complex etiologies and limited therapeutic options. Guanylyl cyclase C (GUCY2C), an intestinal receptor producing cyclic GMP(cGMP), which regulates luminal fluid secretion, has emerged as a therapeutic target for VP. Indeed, FDA-approved GUCY2C agonists ameliorate VP in patients with chronic constipation syndromes, although analgesic mechanisms remain obscure. Here, we revealed that intestinal GUCY2C was selectively enriched in neuropod cells, a type of enteroendocrine cell that synapses with submucosal neurons in mice and humans. GUCY2Chi neuropod cells associated with cocultured dorsal root ganglia neurons and induced hyperexcitability, reducing the rheobase and increasing the resulting number of evoked action potentials. Conversely, the GUCY2C agonist linaclotide eliminated neuronal hyperexcitability produced by GUCY2C-sufficient - but not GUCY2C-deficient - neuropod cells, an effect independent of bulk epithelial cells or extracellular cGMP. Genetic elimination of intestinal GUCY2C amplified nociceptive signaling in VP that was comparable with chemically induced VP but refractory to linaclotide. Importantly, eliminating GUCY2C selectively in neuropod cells also increased nociceptive signaling and VP that was refractory to linaclotide. In the context of loss of GUCY2C hormones in patients with VP, these observations suggest a specific role for neuropod GUCY2C signaling in the pathophysiology and treatment of these pain syndromes.
Assuntos
Células Enteroendócrinas , Receptores de Enterotoxina , Dor Visceral , Animais , Humanos , Camundongos , GMP Cíclico/metabolismo , Células Enteroendócrinas/metabolismo , Células Enteroendócrinas/fisiologia , Intestinos/metabolismo , Intestinos/fisiologia , Receptores de Enterotoxina/metabolismo , Receptores Acoplados a Guanilato Ciclase/metabolismo , Transdução de Sinais/fisiologia , Dor Visceral/genética , Dor Visceral/metabolismoRESUMO
Environmental factors are the major contributor to the onset of immunological disorders such as ulcerative colitis. However, their identities remain unclear. Here, we discover that the amount of consumed L-Tryptophan (L-Trp), a ubiquitous dietary component, determines the transcription level of the colonic T cell homing receptor, GPR15, hence affecting the number of colonic FOXP3+ regulatory T (Treg) cells and local immune homeostasis. Ingested L-Trp is converted by host IDO1/2 enzymes, but not by gut microbiota, to compounds that induce GPR15 transcription preferentially in Treg cells via the aryl hydrocarbon receptor. Consequently, two weeks of dietary L-Trp supplementation nearly double the colonic GPR15+ Treg cells via GPR15-mediated homing and substantially reduce the future risk of colitis. In addition, humans consume 3-4 times less L-Trp per kilogram of body weight and have fewer colonic GPR15+ Treg cells than mice. Thus, we uncover a microbiota-independent mechanism linking dietary L-Trp and colonic Treg cells, that may have therapeutic potential.
Assuntos
Colite Ulcerativa , Colite , Humanos , Camundongos , Animais , Linfócitos T Reguladores , Triptofano , Colite/induzido quimicamente , Colo , Receptores de Peptídeos , Receptores Acoplados a Proteínas G/genéticaRESUMO
Guanylyl cyclase C (GUCY2C) is the index cancer mucosa antigen, an emerging class of immunotherapeutic targets for the prevention of recurrent metastases originating in visceral epithelia. GUCY2C is an autoantigen principally expressed by intestinal epithelium, and universally by primary and metastatic colorectal tumors. Immunization with adenovirus expressing the structurally unique GUCY2C extracellular domain (GUCY2C(ECD); Ad5-GUCY2C) produces prophylactic and therapeutic protection against GUCY2C-expressing colon cancer metastases in mice, without collateral autoimmunity. GUCY2C antitumor efficacy is mediated by a unique immunological mechanism involving lineage-specific induction of antigen-targeted CD8(+) T cells, without CD4(+) T cells or B cells. Here, the unusual lineage specificity of this response was explored by integrating high-throughput peptide screening and bioinformatics, revealing the role for GUCY2C-directed CD8(+) T cells targeting specific epitopes in antitumor efficacy. In BALB/c mice vaccinated with Ad5-GUCY2C, CD8(+) T cells recognize the dominant GUCY2C(254-262) epitope in the context of H-2K(d), driving critical effector functions including interferon gamma secretion, cytolysis ex vivo and in vivo, and antitumor efficacy. The ability of GUCY2C to induce lineage-specific responses targeted to cytotoxic CD8(+) T cells recognizing a single epitope mediating antitumor efficacy without autoimmunity highlights the immediate translational potential of cancer mucosa antigen-based vaccines for preventing metastases of mucosa-derived cancers.
Assuntos
Antígenos de Neoplasias/imunologia , Epitopos de Linfócito T/imunologia , Receptores Acoplados a Guanilato Ciclase/imunologia , Receptores de Peptídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Adenoviridae/imunologia , Animais , Autoantígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/farmacologia , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/terapia , Imunoterapia/métodos , Interferon gama/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mucosa/imunologia , Receptores de EnterotoxinaRESUMO
Major histocompatibility complex (MHC) class II-presented peptides can be derived from both exogenous (extracellular) and endogenous (biosynthesized) sources of antigen. Although several endogenous antigen-processing pathways have been reported, little is known about their relative contributions to global CD4(+) T cell responses against complex antigens. Using influenza virus for this purpose, we assessed the role of macroautophagy, a process in which cytosolic proteins are delivered to the lysosome by de novo vesicle formation and membrane fusion. Influenza infection triggered productive macroautophagy, and autophagy-dependent presentation was readily observed with model antigens that naturally traffic to the autophagosome. Furthermore, treatments that enhance or inhibit macroautophagy modulated the level of presentation from these model antigens. However, validated enzyme-linked immunospot (ELISpot) assays of influenza-specific CD4(+) T cells from infected mice using a variety of antigen-presenting cells, including primary dendritic cells, revealed no detectable macroautophagy-dependent component. In contrast, the contribution of proteasome-dependent endogenous antigen processing to the global influenza CD4(+) response was readily appreciated. The contribution of macroautophagy to the MHC class II-restricted response may vary depending upon the pathogen.
Assuntos
Apresentação de Antígeno/imunologia , Autofagia/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A Subtipo H2N2/patogenicidade , Animais , Autofagia/fisiologia , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , ELISPOT , Feminino , Fibroblastos/fisiologia , Fibroblastos/virologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H2N2/imunologia , Células L , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Pancreatic cancer is one of the most lethal cancers, due to its uniquely aggressive behavior and resistance to therapy. The tumor microenvironment of pancreatic cancer is immunosuppressive, and attempts at utilizing immunotherapies have been unsuccessful. Radiation therapy (RT) results in immune activation and antigen presentation in other cancers, but in pancreatic cancer has had limited success in stimulating immune responses. RT activates common pathways of fibrosis and chronic inflammation seen in pancreatic cancer, resulting in immune suppression. Here we describe the pancreatic tumor microenvironment with regard to fibrosis, myeloid and lymphoid cells, and the impact of RT. We also describe strategies of targeting these pathways that have promise to improve outcomes by harnessing the cytotoxic and immune-activating aspects of RT.