RESUMEN
BACKGROUND & AIMS: Immune checkpoint inhibitors have limited efficacy in many tumors. We investigated mechanisms of tumor resistance to inhibitors of programmed cell death-1 (PDCD1, also called PD-1) in mice with gastric cancer, and the role of its ligand, PD-L1. METHODS: Gastrin-deficient mice were given N-methyl-N-nitrosourea (MNU) in drinking water along with Helicobacter felis to induce gastric tumor formation; we also performed studies with H/K-ATPase-hIL1B mice, which develop spontaneous gastric tumors at the antral-corpus junction and have parietal cells that constitutively secrete interleukin 1B. Mice were given injections of an antibody against PD-1 or an isotype control before tumors developed, or anti-PD-1 and 5-fluorouracil and oxaliplatin, or an antibody against lymphocyte antigen 6 complex locus G (also called Gr-1), which depletes myeloid-derived suppressor cells [MDSCs]), after tumors developed. We generated knock-in mice that express PD-L1 specifically in the gastric epithelium or myeloid lineage. RESULTS: When given to gastrin-deficient mice before tumors grew, anti-PD-1 significantly reduced tumor size and increased tumor infiltration by T cells. However, anti-PD-1 alone did not have significant effects on established tumors in these mice. Neither early nor late anti-PD-1 administration reduced tumor growth in the presence of MDSCs in H/K-ATPase-hIL-1ß mice. The combination of 5-fluorouracil and oxaliplatin reduced MDSCs, increased numbers of intra-tumor CD8+ T cells, and increased the response of tumors to anti-PD-1; however, this resulted in increased tumor expression of PD-L1. Expression of PD-L1 by tumor or immune cells increased gastric tumorigenesis in mice given MNU. Mice with gastric epithelial cells that expressed PD-L1 did not develop spontaneous tumors, but they developed more and larger tumors after administration of MNU and H felis, with accumulation of MDSCs. CONCLUSIONS: In mouse models of gastric cancer, 5-fluorouracil and oxaliplatin reduce numbers of MDSCs to increase the effects of anti-PD-1, which promotes tumor infiltration by CD8+ T cells. However, these chemotherapeutic agents also induce expression of PD-L1 by tumor cells. Expression of PD-L1 by gastric epithelial cells increases tumorigenesis in response to MNU and H felis, and accumulation of MDSCs, which promote tumor progression. The timing and site of PD-L1 expression is therefore important in gastric tumorigenesis and should be considered in design of therapeutic regimens.
Asunto(s)
Infecciones por Helicobacter/inmunología , Células Supresoras de Origen Mieloide/inmunología , Neoplasias Experimentales/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Neoplasias Gástricas/inmunología , Administración Oral , Animales , Carcinogénesis/inducido químicamente , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Carcinogénesis/inmunología , Mucosa Gástrica/inmunología , Mucosa Gástrica/microbiología , Mucosa Gástrica/patología , Gastrinas/genética , Infecciones por Helicobacter/inducido químicamente , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/microbiología , Helicobacter felis/inmunología , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Metilnitrosourea/administración & dosificación , Ratones , Ratones Noqueados , Neoplasias Experimentales/inducido químicamente , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/microbiología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Neoplasias Gástricas/inducido químicamente , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/microbiología , Microambiente Tumoral/inmunologíaRESUMEN
Tissue-resident memory T (TRM) cells, located in the epithelium of most peripheral tissues, constitute the first-line defense against pathogen infections. Our previous study reported that gastric subserous layer (GSL) vaccination induced a "pool" of protective tissue-resident memory CD4+T (CD4+TRM) cells in the gastric epithelium. However, the mechanistic details how CD4+TRM cells form in the gastric epithelium are unknown. Here, our results suggested that the vaccine containing CCF in combination with Silk fibroin hydrogel (SF) broadened the distribution of gastric intraepithelial CD4+TRM cells. It was revealed that the gastric intraepithelial TRM cells were even more important than circulating memory T cells against infection by Helicobacter felis. It was also shown that gastric-infiltrating neutrophils were involved as indispensable mediators which secreted CXCL10 to chemoattract CXCR3+CD4+T cells into the gastric epithelium. Blocking of CXCR3 or neutrophils significantly decreased the number of gastric intraepithelial CD4+TRM cells due to reduced recruitment of CD4+T cells. This study demonstrated the protective efficacy of gastric CD4+TRM cells against H. felis infection, and highlighted the influence of neutrophils on gastric intraepithelial CD4+TRM cells formation.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Fibroínas/administración & dosificación , Infecciones por Helicobacter/prevención & control , Helicobacter felis/inmunología , Animales , Vacunas Bacterianas , Linfocitos T CD8-positivos/metabolismo , Quimiocina CXCL10/genética , Fibroínas/química , Fibroínas/inmunología , Mucosa Gástrica/inmunología , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/inmunología , Hidrogeles , Memoria Inmunológica , Ratones , Neutrófilos/metabolismo , Receptores CXCR3/genéticaRESUMEN
BACKGROUND & AIMS: Helicobacter pylori induces strong inflammatory responses that are directed at clearing the infection, but if not controlled, these responses can be harmful to the host. We investigated the immune-regulatory effects of the innate immune molecule, nucleotide-binding oligomerization domain-like receptors (NLR) family CARD domain-containing 5 (NLRC5), in patients and mice with Helicobacter infection. METHODS: We obtained gastric biopsies from 30 patients in Australia. We performed studies with mice that lack NLRC5 in the myeloid linage (Nlrc5møKO) and mice without Nlrc5 gene disruption (controls). Some mice were gavaged with H pylori SS1 or Helicobacter felis; 3 months later, stomachs, spleens, and sera were collected, along with macrophages derived from bone marrow. Human and mouse gastric tissues and mouse macrophages were analyzed by histology, immunohistochemistry, immunoblots, and quantitative polymerase chain reaction. THP-1 cells (human macrophages, controls) and NLRC5-/- THP-1 cells (generated by CRISPR-Cas9 gene editing) were incubated with Helicobacter and gene expression and production of cytokines were analyzed. RESULTS: Levels of NLRC5 messenger RNA were significantly increased in gastric tissues from patients with H pylori infection, compared with patients without infection (P < .01), and correlated with gastritis severity (P < .05). H pylori bacteria induced significantly higher levels of chemokine and cytokine production by NLRC5-/- THP-1 macrophages than by control THP-1 cells (P < .05). After 3 months of infection with H felis, Nlrc5mø-KO mice developed gastric hyperplasia (P < .0001), splenomegaly (P < .0001), and increased serum antibody titers (P < .01), whereas control mice did not. Nlrc5mø-KO mice with chronic H felis infection had increased numbers of gastric B-cell follicles expressing CD19 (P < .0001); these follicles had features of mucosa-associated lymphoid tissue lymphoma. We identified B-cell-activating factor as a protein that promoted B-cell hyperproliferation in Nlrc5mø-KO mice. CONCLUSIONS: NLRC5 is a negative regulator of gastric inflammation and mucosal lymphoid formation in response to Helicobacter infection. Aberrant NLRC5 signaling in macrophages can promote B-cell lymphomagenesis during chronic Helicobacter infection.
Asunto(s)
Infecciones por Helicobacter/complicaciones , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Linfoma de Células B de la Zona Marginal/inmunología , Neoplasias Gástricas/inmunología , Animales , Linfocitos B/inmunología , Biopsia , Proliferación Celular , Modelos Animales de Enfermedad , Femenino , Mucosa Gástrica/inmunología , Mucosa Gástrica/microbiología , Mucosa Gástrica/patología , Regulación Neoplásica de la Expresión Génica/inmunología , Técnicas de Inactivación de Genes , Infecciones por Helicobacter/inmunología , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/patología , Helicobacter felis/inmunología , Helicobacter pylori/inmunología , Humanos , Hiperplasia/inmunología , Hiperplasia/microbiología , Inmunidad Innata , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/inmunología , Tejido Linfoide/inmunología , Tejido Linfoide/microbiología , Tejido Linfoide/patología , Linfoma de Células B de la Zona Marginal/microbiología , Linfoma de Células B de la Zona Marginal/patología , Masculino , Ratones , Ratones Noqueados , Transducción de Señal/inmunología , Neoplasias Gástricas/microbiología , Neoplasias Gástricas/patología , Células THP-1RESUMEN
Gastric adenocarcinoma is the fifth most common cancer and third most common cause of cancer-related death in the world. The majority of these cancers develop in genetically susceptible individuals who are chronically infected with the Gram-negative bacterium Helicobacter pylori. Often these individuals have also been exposed to certain environmental factors that increase susceptibility, such as dietary components. Murine models of Helicobacter-induced gastric cancer are valuable tools for investigating the mechanisms responsible for the stepwise pathological changes of chronic atrophic gastritis, intestinal metaplasia, dysplasia and gastric adenocarcinoma. Helicobacter felis colonization greatly accelerates the development of gastric neoplasia in mice, and causes pathologies similar to those observed with Helicobacter pylori-associated gastric carcinogenesis in humans. These mouse models are therefore useful for investigating genetic and environmental factors that may be involved in the pathogenesis and treatment of gastric cancer. Detailed in these protocols are procedures for inducing Helicobacter-associated carcinogenesis in mice as well as the histological analysis and interpretation of gastric pathology in these animals.
Asunto(s)
Adenocarcinoma/microbiología , Modelos Animales de Enfermedad , Infecciones por Helicobacter/microbiología , Helicobacter felis/patogenicidad , Helicobacter pylori/patogenicidad , Síndromes Paraneoplásicos/microbiología , Neoplasias Gástricas/microbiología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/inmunología , Adenocarcinoma/patología , Animales , Antibacterianos/uso terapéutico , Antineoplásicos/uso terapéutico , Carcinogénesis/efectos de los fármacos , Carcinogénesis/inmunología , Carcinogénesis/metabolismo , Carcinogénesis/patología , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/inmunología , Mucosa Gástrica/microbiología , Mucosa Gástrica/patología , Infecciones por Helicobacter/tratamiento farmacológico , Infecciones por Helicobacter/inmunología , Infecciones por Helicobacter/patología , Helicobacter felis/efectos de los fármacos , Helicobacter felis/inmunología , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/inmunología , Humanos , Ratones , Síndromes Paraneoplásicos/tratamiento farmacológico , Síndromes Paraneoplásicos/inmunología , Síndromes Paraneoplásicos/patología , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/inmunología , Neoplasias Gástricas/patologíaRESUMEN
Despite the proven ability of immunization to reduce Helicobacter infection in mouse models, the precise mechanism of protection has remained elusive. In this study, we evaluated the role of inflammatory monocytes in the vaccine-induced reduction of Helicobacter felis infection. We first showed by using flow cytometric analysis that Ly6C(low) major histocompatibility complex class II-positive chemokine receptor type 2 (CCR2)-positive CD64(+) inflammatory monocytes accumulate in the stomach mucosa during the vaccine-induced reduction of H. felis infection. To determine whether inflammatory monocytes played a role in the protection, these cells were depleted with anti-CCR2 depleting antibodies. Indeed, depletion of inflammatory monocytes was associated with an impaired vaccine-induced reduction of H. felis infection on day 5 postinfection. To determine whether inflammatory monocytes had a direct or indirect role, we studied their antimicrobial activities. We observed that inflammatory monocytes produced tumor necrosis factor alpha and inducible nitric oxide synthase (iNOS), two major antimicrobial factors. Lastly, by using a Helicobacter in vitro killing assay, we showed that mouse inflammatory monocytes and activated human monocytes killed H. pylori in an iNOS-dependent manner. Collectively, these data show that inflammatory monocytes play a direct role in the immunization-induced reduction of H. felis infection from the gastric mucosa.
Asunto(s)
Vacunas Bacterianas/inmunología , Infecciones por Helicobacter/inmunología , Helicobacter felis/inmunología , Monocitos/inmunología , Animales , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/genética , Femenino , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/prevención & control , Helicobacter felis/genética , Humanos , Inmunización , Ratones , Ratones Endogámicos BALB C , Monocitos/microbiología , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
BACKGROUND: Helicobacter pylori pathogenesis results from the inflammation induced by chronic infection. CBA mice are nonresponsive to gastric Helicobacter infection, providing a useful model for examining host regulation of Helicobacter-induced gastritis. We examined whether gastric Helicobacter nonresponsiveness impacts upon vaccine efficacy and whether immune-mediated protection could occur in the absence of inflammation. METHODS: Mice were vaccinated prior to challenge with Helicobacter felis or H. pylori. Gastritis and H. felis colonization was evaluated histologically. H. pylori colonization was quantified by colony-forming assay. RESULTS: Immunizations protected CBA mice against challenge with either H. felis or H. pylori. Protection against H. felis was marked by a loss of nonresponsiveness and development of an atrophic gastritis with mucus metaplasia. However, vaccine-induced protection against H. pylori was only associated with cell infiltration into the gastric mucosa. CONCLUSIONS: Nonresponsiveness to gastric Helicobacter infection did not interfere with vaccination-induced protection. Vaccine-induced protective immunity against H. pylori was linked with the induction of cellular infiltration, but importantly not atrophic gastritis.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Vacunas Bacterianas/inmunología , Mucosa Gástrica/inmunología , Gastritis Atrófica/inmunología , Infecciones por Helicobacter/inmunología , Helicobacter/inmunología , Inmunización , Estómago/inmunología , Animales , Modelos Animales de Enfermedad , Femenino , Gastritis Atrófica/prevención & control , Infecciones por Helicobacter/prevención & control , Helicobacter felis/inmunología , Helicobacter pylori/inmunología , Humanos , Inflamación , Metaplasia/patología , Ratones , Ratones Endogámicos CBARESUMEN
BACKGROUND: Helicobacter pylori is a spiral-shaped Gram-negative microaerophilic bacterium associated with a number of gastrointestinal disorders, including gastritis, peptic ulcers, and gastric cancer. Several studies have implicated a Th17 response as a key to protective immunity against Helicobacter. MATERIALS AND METHODS: Wild type (WT) and MyD88-deficient (MyD88(-/-)) mice in the C57BL/6 background were infected with H. felis for 6 and 25 weeks and colonization density and host response evaluated. Real-time PCR was used to determine the expression of cytokines and antimicrobial peptides in the gastric tissue of mice. RESULTS: mRNA expression levels of the Th17 cytokines interleukin-17A (IL-17A) and IL-22 were markedly up-regulated in WT compared with MyD88(-/-) mice both at 6 and at 25 weeks in response to infection with H. felis, indicating that induction of Th17 responses depends on MyD88 signaling. Furthermore, reduction in the expression of Th17-dependent intestinal antimicrobial peptide lipocalin-2 was linked with increased bacterial burden in the absence of MyD88 signaling. CONCLUSION: We provide evidence showing that MyD88-dependent signaling is required for the host to induce a Th17 response for the control of Helicobacter infection.
Asunto(s)
Infecciones por Helicobacter/inmunología , Helicobacter felis/inmunología , Factor 88 de Diferenciación Mieloide/fisiología , Células Th17/inmunología , Proteínas de Fase Aguda/metabolismo , Animales , Péptidos Catiónicos Antimicrobianos , Catelicidinas/metabolismo , Citocinas/metabolismo , Defensinas/metabolismo , Mucosa Gástrica/metabolismo , Infecciones por Helicobacter/genética , Lipocalina 2 , Lipocalinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Proteínas Oncogénicas/metabolismo , Transducción de Señal/inmunologíaAsunto(s)
Vacunas Bacterianas/administración & dosificación , Infecciones por Helicobacter/prevención & control , Helicobacter felis/inmunología , Helicobacter pylori/inmunología , Receptor PAR-2/metabolismo , Estómago/efectos de los fármacos , Ureasa/administración & dosificación , Animales , FemeninoRESUMEN
Human Helicobacter pylori infection leads to multiple pathological consequences, including gastritis and adenocarcinoma. Although this association has led to the classification of H. pylori as a type 1 carcinogen, it is not clear if additional nonhelicobacter gastric microbiota play a role in these diseases. In this study, we utilized either specific pathogen-free C57BL/6 mice (B6.SPF) or mice colonized with altered Schaedler flora (B6.ASF) to evaluate the role of nonhelicobacter gastric microbiota in disease development after Helicobacter felis infection. Despite similar histological changes, H. felis persisted in B6.ASF stomachs, while H. felis could no longer be detected in the majority of B6.SPF mice. The B6.SPF mice also acquired multiple Lactobacillus spp. in their stomachs after H. felis infection. Our data indicate that potential mechanisms responsible for the ineffective H. felis clearance in the B6.ASF model include the absence of new gastric microbiota to compete for the gastric niche, the lack of expression of new gastric mucins, and a reduced ratio of H. felis-specific IgG2c:IgG1 serum antibodies. These data suggest that although H. felis is sufficient to initiate gastric inflammation and atrophy, bacterial eradication and the systemic immune response to infection are significantly influenced by pre-existing and acquired gastric microbiota.
Asunto(s)
Infecciones por Helicobacter/inmunología , Infecciones por Helicobacter/patología , Helicobacter felis/inmunología , Helicobacter felis/patogenicidad , Metagenoma/fisiología , Gastropatías/microbiología , Animales , Progresión de la Enfermedad , Femenino , Helicobacter felis/aislamiento & purificación , Metagenoma/inmunología , Ratones , Ratones Endogámicos C57BL , Gastropatías/inmunología , Gastropatías/patologíaRESUMEN
BACKGROUND & AIMS: Protective immunization limits Helicobacter infection of mice by undetermined mechanisms. Protease-activated receptor 2 (PAR2) signaling is believed to regulate immune and inflammatory responses. We investigated the role of PAR2 in vaccine-induced immunity against Helicobacter infection. METHODS: Immune responses against Helicobacter infection were compared between vaccinated PAR2-/- and wild-type (WT) mice. Bacterial persistence, gastric pathology, and inflammatory and cellular responses were assessed using the rapid urease test (RUT), histologic analyses, quantitative polymerase chain reaction, and flow cytometry, respectively. RESULTS: Following vaccination, PAR2-/- mice did not have reductions in Helicobacter felis infection (RUT values were 0.01±0.01 for WT mice and 0.11±0.13 for PAR2-/- mice; P<.05). The vaccinated PAR2-/- mice had reduced inflammation-induced stomach tissue damage (tissue damage scores were 8.83±1.47 for WT mice and 4.86±1.35 for PAR2-/- mice; P<.002) and reduced T-helper (Th)17 responses, based on reduced urease-induced interleukin (IL)-17 secretion by stomach mononuclear cells (5182 ± 1265 pg/mL for WT mice and 350±436 pg/mL for PAR2-/- mice; P<.03) and reduced recruitment of CD4+ IL-17+ T cells into the gastric mucosa of PAR2-/- mice following bacterial challenge (3.7%±1.5% for WT mice and 2.6%±1.1% for PAR2-/- mice; P<.05). In vitro, H felis-stimulated dendritic cells (DCs) from WT mice induced greater secretion of IL-17 by ovalbumin-stimulated OT-II transgenic CD4+ T cells compared with DCs from PAR2-/- mice (4298±347 and 3230±779; P<.04), indicating that PAR2-/- DCs are impaired in priming of Th17 cells. Adoptive transfer of PAR2+/+ DCs into vaccinated PAR2-/- mice increased vaccine-induced protection (RUT values were 0.11±0.10 and 0.26±0.15 for injected and noninjected mice, respectively; P<.03). CONCLUSIONS: PAR2 activates DCs to mediate vaccine-induced protection against Helicobacter infection in mice.
Asunto(s)
Vacunas Bacterianas/administración & dosificación , Infecciones por Helicobacter/prevención & control , Helicobacter felis/inmunología , Helicobacter pylori/inmunología , Receptor PAR-2/metabolismo , Estómago/efectos de los fármacos , Ureasa/administración & dosificación , Administración Intranasal , Traslado Adoptivo , Animales , Células Cultivadas , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Células Dendríticas/trasplante , Modelos Animales de Enfermedad , Femenino , Mucosa Gástrica/metabolismo , Infecciones por Helicobacter/inmunología , Infecciones por Helicobacter/metabolismo , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/patología , Helicobacter pylori/enzimología , Mediadores de Inflamación/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor PAR-2/deficiencia , Receptor PAR-2/genética , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/microbiología , Estómago/inmunología , Estómago/microbiología , Estómago/patología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/microbiología , Vacunas Sintéticas/administración & dosificaciónRESUMEN
B cells regulate autoimmune pathologies and chronic inflammatory conditions such as autoimmune encephalomyelitis and inflammatory bowel disease. The potential counterregulatory role of B cells in balancing pathogen-specific immune responses and the associated immunopathology is less well understood owing to the lack of appropriate persistent infection models. In this paper, we show that B cells have the ability to negatively regulate adaptive immune responses to bacterial pathogens. Using mouse models of infection with Helicobacter felis, a close relative of the human gastrointestinal pathogen H. pylori, we found that B cells activated by Helicobacter TLR-2 ligands induce IL-10-producing CD4(+)CD25(+) T regulatory-1 (Tr-1)-like cells in vitro and in vivo. Tr-1 conversion depends on TCR signaling and a direct T-/B-interaction through CD40/CD40L and CD80/CD28. B cell-induced Tr-1 cells acquire suppressive activity in vitro and suppress excessive gastric Helicobacter-associated immunopathology in vivo. Adoptive cotransfer of MyD88-proficient B cells and Tr-1 cells restores a normal gastric mucosal architecture in MyD88(-/-) and IL-10(-/-) mice in a manner that depends on T cellular, but not B cellular, IL-10 production. Our findings describe a novel mechanism of B cell-dependent Tr-1 cell generation and function in a clinically relevant disease model. In conclusion, we demonstrate that the B cell/Tr-1 cell axis is essential for balancing the control of Helicobacter infection with the prevention of excessive Th1-driven gastric immunopathology, promoting gastric mucosal homeostasis on the one hand and facilitating Helicobacter persistence on the other.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Diferenciación Celular/inmunología , Infecciones por Helicobacter/inmunología , Activación de Linfocitos/inmunología , Lesiones Precancerosas/inmunología , Neoplasias Gástricas/inmunología , Linfocitos T Reguladores/inmunología , Receptor Toll-Like 2/fisiología , Animales , Subgrupos de Linfocitos B/microbiología , Subgrupos de Linfocitos B/patología , Células Cultivadas , Infecciones por Helicobacter/patología , Infecciones por Helicobacter/prevención & control , Helicobacter felis/inmunología , Humanos , Ligandos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Factor 88 de Diferenciación Mieloide/fisiología , Lesiones Precancerosas/patología , Lesiones Precancerosas/prevención & control , Neoplasias Gástricas/patología , Neoplasias Gástricas/prevención & control , Linfocitos T Reguladores/microbiología , Linfocitos T Reguladores/patología , Receptor Toll-Like 2/deficiencia , Receptor Toll-Like 2/metabolismoRESUMEN
Helicobacter infection is the primary risk factor for gastric cancer, with the cytokine environment within the gastric mucosa the strongest predictor of disease risk. Elevated TNF-alpha, IL-1beta, and low IL-10 are associated with the highest risk. In this study, we used C57BL/6 mice to identify T-bet as a central regulator of the cytokine environment during Helicobacter felis infection. We infected male and female C57BL/6 and C57BL/6-T-bet knockout (KO) litter mates with H. felis and examined the bacterial colonization, immune response, and mucosal damage at varying time points. T-bet KO mice maintained infection for 15 mo at similar levels to wild-type mice. Infection and immune response did not differ between male and female mice. Despite sustained infection, T-bet KO mice respond with a blunted Th1 response associated with preservation of parietal and chief cells and protection from the development of gastric cancer. Unexpectedly, T-bet KO mice develop a gastric environment that would not be expected based on the phenotype of T-bet KO CD4 cells alone. T-bet KO mice respond to H. felis infection with a markedly blunted IL-1beta and TNF-alpha and elevated IL-10 levels. Activity of this one master regulator modulates the expression of the key gastric mucosal cytokines associated with gastric cancer and may be a target for therapy to restore immune balance clinically in patients at risk for gastric cancer.
Asunto(s)
Adenocarcinoma/microbiología , Adenocarcinoma/prevención & control , Infecciones por Helicobacter/prevención & control , Helicobacter felis/inmunología , Neoplasias Gástricas/microbiología , Neoplasias Gástricas/prevención & control , Proteínas de Dominio T Box/deficiencia , Proteínas de Dominio T Box/genética , Adenocarcinoma/inmunología , Animales , Femenino , Mucosa Gástrica/inmunología , Mucosa Gástrica/microbiología , Mucosa Gástrica/patología , Predisposición Genética a la Enfermedad , Infecciones por Helicobacter/inmunología , Infecciones por Helicobacter/patología , Interleucina-1beta/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias Gástricas/inmunología , Proteínas de Dominio T Box/fisiología , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
Nuclear factor kappaB (NF-kappaB) plays a key regulatory role in host cell responses to Helicobacter pylori infection in humans. Although mice are routinely used as a model to study H. pylori pathogenesis, the role of NF-kappaB in murine cell responses to helicobacters has not been studied in detail. We thus investigated the abilities of different Helicobacter isolates to induce NF-kappaB-dependent responses in murine gastric epithelial cells (GECs) and in transgenic mice harboring an NF-kappaB-responsive lacZ reporter gene. H. pylori and Helicobacter felis strains up-regulated the synthesis in mouse GECs of the NF-kappaB-dependent chemokines KC (CXCL1) and MIP-2 (CXCL2). These responses were cag pathogenicity island (cagPAI) independent and could be abolished by pretreatment with a pharmacological inhibitor of NF-kappaB. Consistent with the in vitro data, experimental Helicobacter infection of transgenic mice resulted in increased numbers of GECs with nuclear beta-galactosidase activity, which is indicative of specific NF-kappaB activation. The numbers of beta-galactosidase-positive cells in mice were significantly increased at day 1 postinoculation with wild-type H. pylori strains harboring or not harboring a functional cagPAI, compared to naive animals (P = 0.007 and P = 0.04, respectively). Strikingly, however, no differences were observed in the levels of gastric NF-kappaB activation at day 1 postinoculation with H. felis or at day 30 or 135 postinoculation with H. pylori. This work demonstrates for the first time the induction of NF-kappaB activation within gastric mucosal cells during acute H. pylori infection. Furthermore, the data suggest that helicobacters may be able to regulate NF-kappaB signaling during chronic infection.
Asunto(s)
Células Epiteliales/microbiología , Infecciones por Helicobacter/inmunología , Helicobacter pylori/inmunología , FN-kappa B/metabolismo , Animales , Quimiocina CXCL1/biosíntesis , Quimiocina CXCL2/biosíntesis , Femenino , Mucosa Gástrica/inmunología , Mucosa Gástrica/microbiología , Genes Reporteros , Helicobacter felis/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , FN-kappa B/inmunología , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genéticaRESUMEN
BACKGROUND: The mucin Muc1 is constitutively expressed by the gastric mucosa and is likely the first point of direct contact between the host stomach and the adherent pathogens. The expression of Muc1 has been shown to limit colonization of mice by Helicobacter pylori, known to adhere to the gastric epithelium, as well as associated pathology. However, the potential role of this mucin against nonadherent Helicobacter has not been previously studied. We therefore examined the importance of Muc1 on the pathogenesis of Helicobacter felis, believed not to adhere to the murine mucosa. METHODS AND RESULTS: Using primary cell cultures, we found that H. felis can bind gastric epithelial cells in vitro, and adherence to epithelial cells deficient in Muc1 was increased compared to controls that expressed the mucin. However, following infection of deficient mice, we found that Muc1 did not impact on H. felis colonization or pathogenesis in vivo, in contrast to previous observations with H. pylori. CONCLUSIONS: This demonstrates a variable effect of Muc1 on protection against closely related adherent and nonadherent Helicobacter species, and supports a key role for Muc1 in limiting attachment of adherent bacteria to the gastric mucosal surface.
Asunto(s)
Adhesión Bacteriana/inmunología , Células Epiteliales/microbiología , Infecciones por Helicobacter/inmunología , Infecciones por Helicobacter/patología , Helicobacter felis/inmunología , Mucina-1/inmunología , Animales , Células Cultivadas , Infecciones por Helicobacter/microbiología , Ratones , Ratones NoqueadosRESUMEN
BACKGROUND: Although previous studies have reported important roles of CD4(+) type 1-helper T cells and regulatory T cells in Helicobacter-associated gastritis, the significance of CD8(+) cytotoxic T cells remains unknown. To study the roles of CD8(+) T cells, we examined the immune response in the gastric mucosa of Helicobacter felis-infected major histocompatibility complex (MHC) class II-deficient (II(-/-)) mice, which lack CD4(+) T cells. MATERIALS AND METHODS: Stomachs from H. felis-infected wild-type and infected MHC II(-/-) mice were examined histologically and immunohistochemically. Gastric acidity and serum levels of anti-H. felis antibodies were measured. The expression of pro-inflammatory and anti-inflammatory cytokine, Fas-ligand, perforin, and Foxp3 genes in the gastric mucosa was investigated. RESULTS: H. felis-infected MHC II(-/-) mice developed severe gastritis, accompanied by marked infiltration of CD8(+) cells. At 1 and 2 months after inoculation, mucosal inflammation and atrophy were more severe in MHC II(-/-) mice, although gastritis had reached similar advanced stages at 3 months after inoculation. There was little infiltration of CD4(+) cells, and no Foxp3-positive cells were detected in the gastric mucosa of the infected MHC II(-/-) mice. The expression of the interleukin-1beta and Fas-ligand genes was up regulated, but that of Foxp3 was down regulated in the infected MHC II(-/-) mice. Serum levels of anti-H. felis antibodies were lower in the infected MHC II(-/-) mice, despite severe gastritis. CONCLUSIONS: The present study suggests that cross-primed CD8(+) cytotoxic T cells can induce severe H.-associated gastritis in the absence of CD4(+) helper T cells and that Foxp3-positive cells may have an important role in the control of gastric inflammation.
Asunto(s)
Reactividad Cruzada/inmunología , Gastritis/fisiopatología , Infecciones por Helicobacter/fisiopatología , Helicobacter felis/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Factores de Transcripción Forkhead/metabolismo , Mucosa Gástrica/química , Mucosa Gástrica/metabolismo , Mucosa Gástrica/fisiología , Gastritis/inmunología , Gastritis/microbiología , Infecciones por Helicobacter/inmunología , Infecciones por Helicobacter/microbiología , Concentración de Iones de Hidrógeno , Ratones , Ratones Endogámicos C57BL , Linfocitos T Reguladores/metabolismoRESUMEN
Immune responses to microorganisms in the gastrointestinal tract must be carefully controlled to avoid disease. Helicobacter are Gram-negative bacteria which cause persistent infection and, in a minority of hosts, peptic ulceration or gastric cancer. Lymphocyte responses are important determinants of the outcome of infection. Therefore, it is important to identify the genetic determinants of lymphocyte responses to this mucosal pathogen. Using a (C57BL/6xBALB/c) F2 mouse model of Helicobacter infection, we mapped a region of linkage for lymphoproliferation to chromosome 9. Analysis of candidate genes in this region revealed variation of DNA sequence and gene expression in the TLR9 gene between C57BL/6 and BALB/c mouse strains. Reporter assays demonstrated higher levels of TLR9 transcriptional activity and increased NF-kappaB activation associated with the C57BL/6 TLR9 promoter and coding sequences. The importance of TLR9 in the control of lymphocyte responses was confirmed by demonstrating that lymphoproliferation and IFN-gamma secretion was diminished in the TLR9-/- mouse. Furthermore, neutrophil infiltration of the gastric epithelium is reduced in the absence of TLR9. Regulation of TLR9 expression and signalling therefore appears to play an important role in the control of lymphocyte responses to Helicobacter and potentially other luminal microorganisms.
Asunto(s)
Infecciones por Helicobacter/inmunología , Helicobacter felis/inmunología , Linfocitos/inmunología , Polimorfismo Genético , Receptor Toll-Like 9/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proliferación Celular/efectos de los fármacos , Cruzamientos Genéticos , Femenino , Mucosa Gástrica/metabolismo , Gastritis/inmunología , Gastritis/patología , Infecciones por Helicobacter/patología , Interferón gamma/metabolismo , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Neutrófilos/patología , Oligodesoxirribonucleótidos/farmacología , Regiones Promotoras Genéticas , Bazo/inmunología , Bazo/metabolismo , Estómago/inmunología , Estómago/patología , Receptor Toll-Like 9/metabolismoRESUMEN
BACKGROUND: Surfactant protein D (SP-D), a component of innate immunity, is expressed in the gastric mucosa and is up-regulated in the presence of Helicobacter infection. SP-D binds to Helicobacter in vitro, suggesting the involvement of SP-D in Helicobacter-induced immune responses. The aim of this study was to determine the role of SP-D in gastric epithelial defense in vivo. METHODS: Specific pathogen-free SP-D-deficient mice (SP-D(-/-)) and C57BL/6 wild-type controls were challenged by gavage with different doses of Helicobacter felis, a mouse-adapted Helicobacter strain. Mice were assessed for colonization rates and density of infection. Inflammatory responses were measured by neutrophil counting and T-cell responses by proliferation assays on spleen cells stimulated with H. felis sonicate. The in vitro effect of SP-D on Helicobacter uptake by monocyte-derived dendritic cells was assessed by confocal microscopy and FACS analyses. RESULTS: SP-D(-/-) mice were more susceptible to low-dose infectious challenge than C57BL/6 controls (p = .02). The density of colonization was higher in the SP-D(-/-) infected mice. Neutrophil infiltrates were lower in the SP-D(-/-) mice, particularly in the acid-secreting regions of the stomach. T-cell proliferative responses to Helicobacter antigen were reduced in SP-D(-/-) mice (p = .001) after 12 weeks infection. In vitro uptake of Helicobacter by dendritic cells was significantly enhanced in the presence of SP-D (p = .001). CONCLUSION: In the absence of SP-D, Helicobacter uptake by dendritic cells is impaired. This provides an explanation for the diminished inflammation and immune responses in the SP-D(-/-) mice.
Asunto(s)
Infecciones por Helicobacter/metabolismo , Helicobacter felis/patogenicidad , Proteína D Asociada a Surfactante Pulmonar/fisiología , Aglutinación , Animales , Células Cultivadas , Células Dendríticas/microbiología , Femenino , Gastritis/microbiología , Gastritis/patología , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/patología , Helicobacter felis/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Proteína D Asociada a Surfactante Pulmonar/genética , Linfocitos T/patologíaRESUMEN
Trefoil family factor 2 (TFF2), also known as spasmolytic peptide, is a low-molecular-weight protein that is upregulated in gastric tissues infected with Helicobacter or having other inflammatory conditions, but a precise function is yet to be elucidated. The role of TFF2 in the development of gastritis, colitis, and inflammatory cytokine responses was examined both in vivo and in vitro using wild-type and TFF2 knockout mice. TFF2 knockout and wild-type mice were infected with Helicobacter felis (H. felis) to induce gastritis. Colitis was induced in TFF2 knockout and wild-type mice by administering dextran sodium sulfate (DSS) in drinking water. Histopathology, clinical disease (colitis), and antibody levels (H. felis) were examined. TFF2 expression in tissues was determined by reverse transcriptase PCR, and the inflammatory and proliferative responses of TFF2-expressing macrophages and spleen cells were examined by cytokine enzyme-linked immunosorbent assay, thymidine incorporation, and gene array studies. TFF2 knockout mice have increased susceptibility to H. felis-induced gastritis, with enhanced gastric inflammation. They were also more susceptible to DSS-induced colitis, with prolonged colonic hemorrhage and persistent weight loss. Remarkably, TFF2 expression was not limited to the gastrointestinal tract, as suggested in previous studies, but was also present in macrophages and lymphocytes. The inflammatory and proliferative responses of these immune cell types were dysregulated in TFF2 knockout mice. TFF2-/- cells were hyperresponsive to interleukin 1 beta stimulation but showed normal responses to lipopolysaccharide, suggesting a specific role for TFF2 in interleukin 1 receptor but not Toll-like receptor 4 signaling via their Toll-interleukin 1 resistance domains. TFF2-/- lymphocytes also produced higher levels of interleukin 2 than wild-type cells. Thus, TFF2 was expressed in the gastrointestinal cells and in immune cells and was a negative regulator of gastrointestinal inflammation and immune cell cytokine responses. Our studies suggest that TFF2 not only controls gastrointestinal repair but also regulates mononuclear cell inflammatory responses.
Asunto(s)
Mucosa Gástrica/inmunología , Enfermedades Gastrointestinales/inmunología , Inflamación/inmunología , Linfocitos/inmunología , Macrófagos/inmunología , Mucinas/inmunología , Proteínas Musculares/inmunología , Péptidos/inmunología , Animales , Citocinas/inmunología , Ensayo de Inmunoadsorción Enzimática , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiología , Enfermedades Gastrointestinales/microbiología , Enfermedades Gastrointestinales/prevención & control , Expresión Génica , Perfilación de la Expresión Génica , Infecciones por Helicobacter/inmunología , Infecciones por Helicobacter/metabolismo , Infecciones por Helicobacter/patología , Helicobacter felis/inmunología , Inflamación/microbiología , Inflamación/patología , Mucosa Intestinal/metabolismo , Intestinos/inmunología , Intestinos/patología , Linfocitos/microbiología , Macrófagos/microbiología , Ratones , Ratones Noqueados , Mucinas/genética , Mucinas/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Péptidos/genética , Péptidos/metabolismo , Receptores de Interleucina-1/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estómago/inmunología , Estómago/patología , Factor Trefoil-2RESUMEN
Helicobacter infections are present in approximately 50% of humans, causing severe illnesses such as gastritis and malignancies. Dendritic cells (DC) are critical antigen-presenting cells which link innate and adaptive immune responses. The mechanism of dendritic cell regulation in Helicobacter-induced gastritis is poorly understood. These studies characterized DC isolated from the lamina propria of Helicobacter-infected mice and analyzed innate and adaptive immune responses elicited by Helicobacter antigen (Ag)-pulsed DC. The presence of DC was elevated in the gastric lamina propria infiltrate of infected mice in comparison with controls. After treatment with Helicobacter felis Ag, DC were polarized to secrete interleukin-6 as the dominant cytokine. In the presence of DC and Helicobacter Ag, responder allogeneic T cells in culture exhibited limited cell division. We suggest that the response of DC and T cells to Helicobacter Ag is critical to the chronic persistence of Helicobacter-induced gastritis.
Asunto(s)
Antígenos Bacterianos/inmunología , Células Dendríticas/inmunología , Helicobacter felis/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Gatos , Células Cultivadas , Técnicas de Cocultivo , Mucosa Gástrica/citología , Mucosa Gástrica/inmunología , Gastritis/inmunología , Gastritis/microbiología , Infecciones por Helicobacter , Interleucina-6/biosíntesis , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Linfocitos T/inmunologíaRESUMEN
The initiating molecular events in Helicobacter-induced gastric carcinogenesis are not known. Early in infection, Fas antigen-mediated apoptosis depletes parietal and chief cell populations, leading to architectural distortion. As infection progresses, metaplastic and dysplastic glands appear, which are resistant to Fas-mediated apoptosis. These abnormal lineages precede, and are thought to be the precursor lesions of, gastric cancer. Acquisition of an antiapoptotic phenotype before transformation of cells suggests that loss of Fas sensitivity may be an early required trait for gastric cancer. We reasoned that forced Fas-apoptosis resistance would result in earlier and more aggressive gastric cancer in our mouse model. Fas antigen-deficient (lpr) mice or C57BL/6 wild-type mice were irradiated and reconstituted with C57BL/6 marrow forming partial lpr/wt chimera or wt/wt control mice, extending the life span of the lpr and ensuring a competent immune response to Helicobacter felis infection. Infected lpr/wt mice developed gastric cancer as early as 7 months after infection (compared with 15 months in wt/wt mice). At 10 months (90%) and 15 months (100%), mice developed aggressive invasive lesions. This earlier onset and more aggressive histology strongly argues that Fas-apoptosis resistance is an early and important feature of gastric cancer formation.