ABSTRACT
Recently, we showed that double-transgenic rats overexpressing guanylin (Gn), a bioactive peptide, and its receptor, guanylyl cyclase-C (GC-C), specifically in macrophages demonstrate an antiobesity phenotype and low-expression levels of proinflammatory cytokines in the mesenteric fat even when fed a high-fat diet. Here, we examined the levels and mechanism of Gn and GC-C transcription following saturated fatty acid and lipopolysaccharide (LPS), an activator of Toll-like receptor 4 (TLR4), exposure by using the NR8383 macrophage cell line. In addition, the levels of guanylin and cGMP were increased by addition of either palmitic acid or LPS. Next, we investigated the interaction of the gene transcription and nuclear factor-κB (NF-κB) by using an NF-κB inhibitor and chromatin immunoprecipitation assay. We showed that palmitic acid induced Gn gene expression via TLR4 and NF-κB. Moreover, we demonstrated that NF-κB binding to the Gn promoter was responsible for the induction of gene transcription by palmitic acid or LPS. Our results indicate that saturated fatty acids such as palmitic acid activate Gn gene expression via the NF-κB pathway, raising the possibility that the activated Gn-GC-C system may contribute to the inhibition of high-fat diet-induced proinflammatory cytokines in macrophages.
Subject(s)
Gastrointestinal Hormones/genetics , Lipopolysaccharides/pharmacology , Macrophages, Alveolar/drug effects , NF-kappa B/genetics , Natriuretic Peptides/genetics , Palmitic Acid/pharmacology , Toll-Like Receptor 4/genetics , Animals , Cell Line , Cyclic GMP/immunology , Cyclic GMP/metabolism , Gastrointestinal Hormones/agonists , Gastrointestinal Hormones/immunology , Gene Expression Regulation , Genes, Reporter , Humans , Luciferases/genetics , Luciferases/metabolism , Macrophages, Alveolar/cytology , Macrophages, Alveolar/immunology , Mice , NF-kappa B/immunology , Natriuretic Peptides/agonists , Natriuretic Peptides/immunology , RAW 264.7 Cells , Rats , Receptors, Guanylate Cyclase-Coupled/genetics , Receptors, Guanylate Cyclase-Coupled/immunology , Signal Transduction , THP-1 Cells , Toll-Like Receptor 4/immunologyABSTRACT
Nontyphoidal Salmonella disease contributes toward significant morbidity and mortality across the world. Host factors, including gamma interferon, tumor necrosis factor alpha, and gut microbiota, significantly influence the outcome of Salmonella pathogenesis. However, the entire repertoire of host protective mechanisms contributing to Salmonella pathogenicity is not completely appreciated. Here, we investigated the roles of receptor guanylyl cyclase C (GC-C), which is predominantly expressed in the intestine and regulates intestinal cell proliferation and fluid-ion homeostasis. Mice deficient in GC-C (Gucy2c-/-) displayed accelerated mortality compared with that for wild-type mice following infection via the oral route, even though both groups possessed comparable systemic Salmonella infection burdens. Survival following intraperitoneal infection remained similar in both groups, indicating that GC-C offered protection via a gut-mediated response. The serum cortisol level was higher in Gucy2c-/- mice than wild-type (Gucy2c+/+) mice, and an increase in infection-induced thymic atrophy with a loss of immature CD4+ CD8+ double-positive thymocytes was observed. Accelerated and enhanced damage in the ileum, including submucosal edema, epithelial cell damage, focal tufting, and distortion of the villus architecture, was seen in Gucy2c-/- mice concomitantly with a larger number of ileal tissue-associated bacteria. Transcription of key mediators of Salmonella-induced inflammation (interleukin-22/Reg3ß) was altered in Gucy2c-/- mice in comparison to that in Gucy2c+/+ mice. A reduction in fecal lactobacilli, which are protective against Salmonella infection, was observed in Gucy2c-/- mice. Gucy2c-/- mice cohoused with wild-type mice continued to show reduced amounts of lactobacilli and increased susceptibility to infection. Our study, therefore, suggests that the receptor GC-C confers a survival advantage during gut-mediated Salmonella enterica serovar Typhimurium pathogenesis, presumably by regulating Salmonella effector mechanisms and maintaining a beneficial microbiome.
Subject(s)
Cytokines/immunology , Guanylate Cyclase/immunology , Receptors, Guanylate Cyclase-Coupled/immunology , Salmonella Infections, Animal/immunology , Salmonella enterica/genetics , Salmonella enterica/immunology , Salmonella typhimurium/immunology , Salmonella typhimurium/pathogenicity , Animals , Cytokines/metabolism , Guanylate Cyclase/metabolism , Ileum/immunology , Ileum/microbiology , Mice , Models, Animal , Receptors, Guanylate Cyclase-Coupled/metabolism , Salmonella Infections, Animal/microbiology , Serogroup , Signal Transduction/physiologyABSTRACT
Cancer immunotherapy is becoming a routine treatment modality in the oncology clinic, in spite of the fact that it is a relatively nascent field. The challenge in developing effective immunotherapeutics is the identification of target molecules that promote anti-tumor efficacy across the patient population while sparing healthy tissue from damaging autoimmunity. The intestinally restricted receptor guanylyl cyclase C (GUCY2C) is a target that has been investigated for the treatment of colorectal cancer and numerous animal, and clinical studies have demonstrated both efficacy and safety. Here, we describe the current state of GUCY2C-directed cancer immunotherapy and the future directions of this work.
Subject(s)
Colorectal Neoplasms/therapy , Immunotherapy , Receptors, Guanylate Cyclase-Coupled/immunology , Animals , Colorectal Neoplasms/immunology , Humans , Molecular Targeted TherapyABSTRACT
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.
Subject(s)
B-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cancer Vaccines/immunology , Immune Tolerance , Neoplasms/therapy , Animals , Autoantigens/immunology , Epitopes, T-Lymphocyte/immunology , Immunologic Memory , Melanoma/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Receptors, Enterotoxin , Receptors, Guanylate Cyclase-Coupled/immunology , Receptors, Peptide/immunologyABSTRACT
BACKGROUND: Guanylate Cyclase C (GC-C; Gucy2c) is a transmembrane receptor expressed in intestinal epithelial cells. Activation of GC-C by its secreted ligand guanylin stimulates intestinal fluid secretion. Familial mutations in GC-C cause chronic diarrheal disease or constipation and are associated with intestinal inflammation and infection. Here, we investigated the impact of GC-C activity on mucosal immune responses. METHODS: We utilized intraperitoneal injection of lipopolysaccharide to elicit a systemic cytokine challenge and then measured pro-inflammatory gene expression in colonic mucosa. GC-C(+/+) and GC-C(-/-) mice were bred with interleukin (IL)-10 deficient animals and colonic inflammation were assessed. Immune cell influx and cytokine/chemokine expression was measured in the colon of wildtype, IL-10(-/-), GC-C(+/+)IL-10(-/-) and GC-C(-/-)IL-10(-/-) mice. GC-C and guanylin production were examined in the colon of these animals and in a cytokine-treated colon epithelial cell line. RESULTS: Relative to GC-C(+/+) animals, intraperitoneal lipopolysaccharide injection into GC-C(-/-) mice increased proinflammatory gene expression in both whole colon tissue and in partially purified colonocyte isolations. Spontaneous colitis in GC-C(-/-)IL-10(-/-) animals was significantly more severe relative to GC-C(+/+)IL-10(-/-) mice. Unlike GC-C(+/+)IL-10(-/-) controls, colon pathology in GC-C(-/-)IL-10(-/-) animals was apparent at an early age and was characterized by severely altered mucosal architecture, crypt abscesses, and hyperplastic subepithelial lesions. F4/80 and myeloperoxidase positive cells as well as proinflammatory gene expression were elevated in GC-C(-/-)IL-10(-/-) mucosa relative to control animals. Guanylin was diminished early in colitis in vivo and tumor necrosis factor α suppressed guanylin mRNA and protein in intestinal goblet cell-like HT29-18-N2 cells. CONCLUSIONS: The GC-C signaling pathway blunts colonic mucosal inflammation that is initiated by systemic cytokine burst or loss of mucosal immune cell immunosuppression. These data as well as the apparent intestinal inflammation in human GC-C mutant kindred underscore the importance of GC-C in regulating the response to injury and inflammation within the gut.
Subject(s)
Colitis/immunology , Immune Tolerance , Intestinal Mucosa/immunology , Mutation , Receptors, Guanylate Cyclase-Coupled/immunology , Receptors, Peptide/immunology , Signal Transduction/immunology , Animals , Colitis/genetics , Colitis/pathology , Gastrointestinal Hormones/genetics , Gastrointestinal Hormones/immunology , Humans , Interleukin-10/genetics , Interleukin-10/immunology , Intestinal Mucosa/pathology , Mice , Mice, Knockout , Natriuretic Peptides/genetics , Natriuretic Peptides/immunology , Receptors, Enterotoxin , Receptors, Guanylate Cyclase-Coupled/genetics , Receptors, Peptide/genetics , Signal Transduction/genetics , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunologyABSTRACT
BACKGROUND: Guanylate Cyclase C (GC-C) is an apically-oriented transmembrane receptor that is expressed on epithelial cells of the intestine. Activation of GC-C by the endogenous ligands guanylin or uroguanylin elevates intracellular cGMP and is implicated in intestinal ion secretion, cell proliferation, apoptosis, intestinal barrier function, as well as the susceptibility of the intestine to inflammation. Our aim was to determine if GC-C is required for host defense during infection by the murine enteric pathogen Citrobacter rodentium of the family Enterobacteriacea. METHODS: GC-C+/+ control mice or those having GC-C genetically ablated (GC-C-/-) were administered C. rodentium by orogastric gavage and analyzed at multiple time points up to post-infection day 20. Commensal bacteria were characterized in uninfected GC-C+/+ and GC-C-/- mice using 16S rRNA PCR analysis. RESULTS: GC-C-/- mice had an increase in C. rodentium bacterial load in stool relative to GC-C+/+. C. rodentium infection strongly decreased guanylin expression in GC-C+/+ mice and, to an even greater degree, in GC-C-/- animals. Fluorescent tracer studies indicated that mice lacking GC-C, unlike GC-C+/+ animals, had a substantial loss of intestinal barrier function early in the course of infection. Epithelial cell apoptosis was significantly increased in GC-C-/- mice following 10 days of infection and this was associated with increased frequency and numbers of C. rodentium translocation out of the intestine. Infection led to significant liver histopathology in GC-C-/- mice as well as lymphocyte infiltration and elevated cytokine and chemokine expression. Relative to naïve GC-C+/+ mice, the commensal microflora load in uninfected GC-C-/- mice was decreased and bacterial composition was imbalanced and included outgrowth of the Enterobacteriacea family. CONCLUSIONS: This work demonstrates the novel finding that GC-C signaling is an essential component of host defense during murine enteric infection by reducing bacterial load and preventing systemic dissemination of attaching/effacing-lesion forming bacterial pathogens such as C. rodentium.
Subject(s)
Colon/immunology , Enterobacteriaceae Infections/immunology , Intestinal Mucosa/immunology , Receptors, Guanylate Cyclase-Coupled/immunology , Receptors, Peptide/immunology , Animals , Apoptosis/immunology , Bacterial Load , Bacterial Translocation/physiology , Citrobacter rodentium/physiology , Colon/pathology , Enterobacteriaceae Infections/genetics , Intestinal Mucosa/pathology , Liver/pathology , Mice , Mice, Knockout , Permeability , Receptors, Enterotoxin , Receptors, Guanylate Cyclase-Coupled/genetics , Receptors, Peptide/genetics , Signal Transduction/immunologyABSTRACT
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.
Subject(s)
Antigens, Neoplasm/immunology , Epitopes, T-Lymphocyte/immunology , Receptors, Guanylate Cyclase-Coupled/immunology , Receptors, Peptide/immunology , T-Lymphocytes, Cytotoxic/immunology , Adenoviridae/immunology , Animals , Autoantigens/immunology , CD8-Positive T-Lymphocytes/immunology , Cancer Vaccines/immunology , Cancer Vaccines/pharmacology , Colorectal Neoplasms/immunology , Colorectal Neoplasms/therapy , Immunotherapy/methods , Interferon-gamma/immunology , Mice , Mice, Inbred BALB C , Mucous Membrane/immunology , Receptors, EnterotoxinABSTRACT
For the last decade, we have focused on guanylyl cyclase C (GUCY2C) as a potentially ideal target antigen for colorectal cancer immunotherapy. GUCY2C is expressed only in intestinal epithelial cells and by nearly 100% of colorectal cancers. We have developed and tested a recombinant adenoviral vector possessing GUCY2C (Ad5-GUCY2C) as a candidate vaccine for colorectal cancer patients. Murine studies have revealed that this vaccine is safe and effective against GUCY2C-expressing targets, and Ad5-GUCY2C is poised for phase I clinical testing in colorectal cancer patients with minimal residual disease. Moreover, we are developing second-generation GUCY2C-targeted therapeutics, including the use of chimeric antigen receptor (CAR)-expressing T cells, for treatment of patients with advanced colorectal cancer for whom Ad5-GUCY2C immunization is not appropriate. Thus, a family of GUCY2C-targeted immunotherapeutics may bridge the gap in effective treatments for the 500,000 patients worldwide who die annually from colorectal cancer.
Subject(s)
Adenocarcinoma/therapy , Colorectal Neoplasms/therapy , Immunotherapy/methods , Molecular Targeted Therapy/methods , Receptors, Guanylate Cyclase-Coupled/metabolism , Receptors, Peptide/metabolism , Adenocarcinoma/drug therapy , Adenocarcinoma/immunology , Adenoviridae/genetics , Animals , Clinical Trials as Topic , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/immunology , Genetic Vectors , Humans , Mice , Receptors, Enterotoxin , Receptors, Guanylate Cyclase-Coupled/genetics , Receptors, Guanylate Cyclase-Coupled/immunology , Receptors, Peptide/genetics , Receptors, Peptide/immunologyABSTRACT
Crustacean Y-organs synthesize ecdysteroid molting hormones. Synthesis of ecdysteroids by Y-organs is negatively regulated by a polypeptide neurohormone, molt-inhibiting hormone (MIH). Our laboratory has recently cloned from Y-organs of the blue crab (Callinectes sapidus) a cDNA (CsGC-YO1) encoding a putative receptor guanylyl cyclase (CsGC-YO1). We hypothesize that CsGC-YO1 is an MIH receptor. In studies reported here, antipeptide antibodies (anti-CsGC-YO1) were raised against a fragment of the extracellular domain of CsGC-YO1. Western blots showed affinity purified anti-CsGC-YO1 bound to the heterologously expressed extracellular domain, and to a protein in Y-organs that corresponded in size to the theoretical molecular mass of CsGC-YO1. Immunocytochemical studies with anti-CsGC-YO1 as primary antibody, showed CsGC-YO1 immunoreactivity was restricted to the peripheral margins of cells, and was not present in cytoplasm or nuclei. The results strongly suggest that CsGC-YO1 is a membrane-associated protein. Preincubation of Y-organs with anti-CsCG-YO1 blunted MIH-induced suppression of ecdysteroidogenesis. This finding represents the first demonstration of a link between CsGC-YO1 and MIH action. A real-time PCR assay for quantifying CsCG-YO1 was developed and validated. The assay was used to determine the abundance of the CsCG-YO1 transcript in Y-organs during a molt cycle: the level of CsGC-YO1 in Y-organs was elevated during intermolt (C(4)) and lower during premolt stages D(1)-D(3). The data suggest that the biological action of CsGC-YO1 in Y-organs is likely to be most pronounced during intermolt. The combined results are consistent with the hypothesis that CsGC-YO1 is an MIH receptor.