RESUMO
BACKGROUND & AIMS: Inflammation in the gastrointestinal tract may lead to the development of cancer. Dicarbonyl electrophiles, such as isolevuglandins (isoLGs), are generated from lipid peroxidation during the inflammatory response and form covalent adducts with amine-containing macromolecules. Thus, we sought to determine the role of dicarbonyl electrophiles in inflammation-associated carcinogenesis. METHODS: The formation of isoLG adducts was analyzed in the gastric tissues of patients infected with Helicobacter pylori from gastritis to precancerous intestinal metaplasia, in human gastric organoids, and in patients with colitis and colitis-associated carcinoma (CAC). The effect on cancer development of a potent scavenger of dicarbonyl electrophiles, 5-ethyl-2-hydroxybenzylamine (EtHOBA), was determined in transgenic FVB/N insulin-gastrin (INS-GAS) mice and Mongolian gerbils as models of H pylori-induced carcinogenesis and in C57BL/6 mice treated with azoxymethane-dextran sulfate sodium as a model of CAC. The effect of EtHOBA on mutations in gastric epithelial cells of H pylori-infected INS-GAS mice was assessed by whole-exome sequencing. RESULTS: We show increased isoLG adducts in gastric epithelial cell nuclei in patients with gastritis and intestinal metaplasia and in human gastric organoids infected with H pylori. EtHOBA inhibited gastric carcinoma in infected INS-GAS mice and gerbils and attenuated isoLG adducts, DNA damage, and somatic mutation frequency. Additionally, isoLG adducts were elevated in tissues from patients with colitis, colitis-associated dysplasia, and CAC as well as in dysplastic tumors of C57BL/6 mice treated with azoxymethane-dextran sulfate sodium. In this model, EtHOBA significantly reduced adduct formation, tumorigenesis, and dysplasia severity. CONCLUSIONS: Dicarbonyl electrophiles represent a link between inflammation and somatic genomic alterations and are thus key targets for cancer chemoprevention.
Assuntos
Transformação Celular Neoplásica/imunologia , Neoplasias Associadas a Colite/imunologia , Lipídeos/imunologia , Lesões Pré-Cancerosas/imunologia , Neoplasias Gástricas/imunologia , Animais , Benzilaminas/farmacologia , Benzilaminas/uso terapêutico , Núcleo Celular/metabolismo , Transformação Celular Neoplásica/efeitos dos fármacos , Neoplasias Associadas a Colite/microbiologia , Neoplasias Associadas a Colite/patologia , Neoplasias Associadas a Colite/prevenção & controle , Modelos Animais de Doenças , Células Epiteliais , Mucosa Gástrica/citologia , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/imunologia , Mucosa Gástrica/patologia , Gastrite/imunologia , Gastrite/microbiologia , Gastrite/patologia , Gerbillinae , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/imunologia , Helicobacter pylori/isolamento & purificação , Humanos , Lipídeos/antagonistas & inibidores , Metaplasia/imunologia , Metaplasia/microbiologia , Metaplasia/patologia , Camundongos , Camundongos Transgênicos , Organoides , Lesões Pré-Cancerosas/tratamento farmacológico , Lesões Pré-Cancerosas/microbiologia , Lesões Pré-Cancerosas/patologia , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia , Neoplasias Gástricas/prevenção & controleRESUMO
Innate responses of myeloid cells defend against pathogenic bacteria via inducible effectors. Deoxyhypusine synthase (DHPS) catalyzes the transfer of the N-moiety of spermidine to the lysine-50 residue of eukaryotic translation initiation factor 5A (EIF5A) to form the amino acid hypusine. Hypusinated EIF5A (EIF5AHyp) transports specific mRNAs to ribosomes for translation. We show that DHPS is induced in macrophages by two gastrointestinal pathogens, Helicobacter pylori and Citrobacter rodentium, resulting in enhanced hypusination of EIF5A. EIF5AHyp was also increased in gastric macrophages from patients with H. pylori gastritis. Furthermore, we identify the bacteria-induced immune effectors regulated by hypusination. This set of proteins includes essential constituents of antimicrobial response and autophagy. Mice with myeloid cell-specific deletion of Dhps exhibit reduced EIF5AHyp in macrophages and increased bacterial burden and inflammation. Thus, regulation of translation through hypusination is a critical hallmark of the defense of eukaryotic hosts against pathogenic bacteria.
Assuntos
Anti-Infecciosos/uso terapêutico , Lisina/análogos & derivados , Macrófagos/imunologia , Animais , Anti-Infecciosos/farmacologia , Modelos Animais de Doenças , Humanos , Lisina/uso terapêutico , CamundongosRESUMO
The reverse transsulfuration pathway is the major route for the metabolism of sulfur-containing amino acids. The role of this metabolic pathway in macrophage response and function is unknown. We show that the enzyme cystathionine γ-lyase (CTH) is induced in macrophages infected with pathogenic bacteria through signaling involving phosphatidylinositol 3-kinase (PI3K)/MTOR and the transcription factor SP1. This results in the synthesis of cystathionine, which facilitates the survival of pathogens within myeloid cells. Our data demonstrate that the expression of CTH leads to defective macrophage activation by (i) dysregulation of polyamine metabolism by depletion of S-adenosylmethionine, resulting in immunosuppressive putrescine accumulation and inhibition of spermidine and spermine synthesis, and (ii) increased histone H3K9, H3K27, and H3K36 di/trimethylation, which is associated with gene expression silencing. Thus, CTH is a pivotal enzyme of the innate immune response that disrupts host defense. The induction of the reverse transsulfuration pathway by bacterial pathogens can be considered an unrecognized mechanism for immune escape.IMPORTANCE Macrophages are professional immune cells that ingest and kill microbes. In this study, we show that different pathogenic bacteria induce the expression of cystathionine γ-lyase (CTH) in macrophages. This enzyme is involved in a metabolic pathway called the reverse transsulfuration pathway, which leads to the production of numerous metabolites, including cystathionine. Phagocytized bacteria use cystathionine to better survive in macrophages. In addition, the induction of CTH results in dysregulation of the metabolism of polyamines, which in turn dampens the proinflammatory response of macrophages. In conclusion, pathogenic bacteria can evade the host immune response by inducing CTH in macrophages.
Assuntos
Bactérias/imunologia , Bactérias/metabolismo , Imunidade Inata , Macrófagos/metabolismo , Redes e Vias Metabólicas/fisiologia , Enxofre/metabolismo , Animais , Bactérias/patogenicidade , Inativação Gênica , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Histonas/metabolismo , Humanos , Evasão da Resposta Imune , Imunoglobulinas , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Poliaminas/metabolismo , Células RAW 264.7 , Espermidina/metabolismo , Espermina/metabolismo , Fatores de TranscriçãoRESUMO
Polyamines have been implicated in numerous biological processes, including inflammation and carcinogenesis. Homeostatic regulation leads to interconversion of the polyamines putrescine and the downstream metabolites spermidine and spermine. The enzyme spermine oxidase (SMOX), which back-converts spermine to spermidine, contributes to regulation of polyamine levels, but can also have other effects. We have implicated SMOX in gastric inflammation and carcinogenesis due to infection by the pathogen Helicobacter pylori. In addition, we reported that SMOX can be upregulated in humans with inflammatory bowel disease. Herein, we utilized Smox-deficient mice to examine the role of SMOX in two murine colitis models, Citrobacter rodentium infection and dextran sulfate sodium (DSS)-induced epithelial injury. In C. rodentium-infected wild-type (WT) mice, there were marked increases in colon weight/length and histologic injury, with mucosal hyperplasia and inflammatory cell infiltration; these changes were ameliorated in Smox-/- mice. In contrast, with DSS, Smox-/- mice exhibited substantial mortality, and increased body weight loss, colon weight/length, and histologic damage. In C. rodentium-infected WT mice, there were increased colonic levels of the chemokines CCL2, CCL3, CCL4, CXCL1, CXCL2, and CXCL10, and the cytokines IL-6, TNF-α, CSF3, IFN-γ, and IL-17; each were downregulated in Smox-/- mice. In DSS colitis, increased levels of IL-6, CSF3, and IL-17 were further increased in Smox-/- mice. In both models, putrescine and spermidine were increased in WT mice; in Smox-/- mice, the main effect was decreased spermidine and spermidine/spermine ratio. With C. rodentium, polyamine levels correlated with histologic injury, while with DSS, spermidine was inversely correlated with injury. Our studies indicate that SMOX has immunomodulatory effects in experimental colitis via polyamine flux. Thus, SMOX contributes to the immunopathogenesis of C. rodentium infection, but is protective in DSS colitis, indicating the divergent effects of spermidine.
Assuntos
Colite/etiologia , Colite/metabolismo , Imunomodulação , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Animais , Citrobacter rodentium/fisiologia , Colite/patologia , Citocinas/metabolismo , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Deleção de Genes , Imunidade nas Mucosas/genética , Imunomodulação/efeitos dos fármacos , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Knockout , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Espermidina/metabolismo , Espermidina/farmacologia , Espermina/metabolismo , Espermina/farmacologia , Poliamina OxidaseRESUMO
Blood vessel epicardial substance (BVES), or POPDC1, is a tight junction-associated transmembrane protein that modulates epithelial-to-mesenchymal transition (EMT) via junctional signaling pathways. There have been no in vivo studies investigating the role of BVES in colitis. We hypothesized that BVES is critical for maintaining colonic epithelial integrity. At baseline, Bves-/- mouse colons demonstrate increased crypt height, elevated proliferation, decreased apoptosis, altered intestinal lineage allocation, and dysregulation of tight junctions with functional deficits in permeability and altered intestinal immunity. Bves-/- mice inoculated with Citrobacter rodentium had greater colonic injury, increased colonic and mesenteric lymph node bacterial colonization, and altered immune responses after infection. We propose that increased bacterial colonization and translocation result in amplified immune responses and worsened injury. Similarly, dextran sodium sulfate (DSS) treatment resulted in greater histologic injury in Bves-/- mice. Two different human cell lines (Caco2 and HEK293Ts) co-cultured with enteropathogenic E. coli showed increased attaching/effacing lesions in the absence of BVES. Finally, BVES mRNA levels were reduced in human ulcerative colitis (UC) biopsy specimens. Collectively, these studies suggest that BVES plays a protective role both in ulcerative and infectious colitis and identify BVES as a critical protector of colonic mucosal integrity.
Assuntos
Colite Ulcerativa/metabolismo , Colo/metabolismo , Células Epiteliais/metabolismo , Absorção Intestinal/fisiologia , Proteínas de Membrana/metabolismo , Adulto , Animais , Células CACO-2 , Moléculas de Adesão Celular , Linhagem Celular , Linhagem Celular Tumoral , Citrobacter rodentium/patogenicidade , Técnicas de Cocultura , Colo/efeitos dos fármacos , Sulfato de Dextrana/farmacologia , Células Epiteliais/efeitos dos fármacos , Escherichia coli/metabolismo , Feminino , Células HEK293 , Humanos , Absorção Intestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Proteínas Musculares , Permeabilidade/efeitos dos fármacos , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismoRESUMO
OBJECTIVE: Gastric cancer is the third leading cause of cancer death worldwide and infection by Helicobacter pylori is the strongest risk factor. We have reported increased epidermal growth factor receptor (EGFR) phosphorylation in the H. pylori-induced human carcinogenesis cascade, and association with DNA damage. Our goal was to determine the role of EGFR activation in gastric carcinogenesis. DESIGN: We evaluated gefitinib, a specific EGFR inhibitor, in chemoprevention of H. pylori-induced gastric inflammation and cancer development. Mice with genetically targeted epithelial cell-specific deletion of Egfr (EfgrΔepi mice) were also used. RESULTS: In C57BL/6 mice, gefitinib decreased Cxcl1 and Cxcl2 expression by gastric epithelial cells, myeloperoxidase-positive inflammatory cells in the mucosa and epithelial DNA damage induced by H. pylori infection. Similar reductions in chemokines, inflammatory cells and DNA damage occurred in infected EgfrΔepi versus Egfrfl/fl control mice. In H. pylori-infected transgenic insulin-gastrin (INS-GAS) mice and gerbils, gefitinib treatment markedly reduced dysplasia and carcinoma. Gefitinib blocked H. pylori-induced activation of mitogen-activated protein kinase 1/3 (MAPK1/3) and activator protein 1 in gastric epithelial cells, resulting in inhibition of chemokine synthesis. MAPK1/3 phosphorylation and JUN activation was reduced in gastric tissues from infected wild-type and INS-GAS mice treated with gefitinib and in primary epithelial cells from EfgrΔepi versus Egfrfl/fl mice. Epithelial EGFR activation persisted in humans and mice after H. pylori eradication, and gefitinib reduced gastric carcinoma in INS-GAS mice treated with antibiotics. CONCLUSIONS: These findings suggest that epithelial EGFR inhibition represents a potential strategy to prevent development of gastric carcinoma in H. pylori-infected individuals.
Assuntos
Antineoplásicos/uso terapêutico , Receptores ErbB/antagonistas & inibidores , Gastrite/patologia , Infecções por Helicobacter/patologia , Quinazolinas/uso terapêutico , Neoplasias Gástricas/prevenção & controle , Animais , Técnicas de Cultura de Células , Células Epiteliais , Gastrite/microbiologia , Gefitinibe , Gerbillinae , Helicobacter pylori , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologiaRESUMO
The intestinal microbiota of patients with constipated-predominant irritable bowel syndrome (C-IBS) displays chronic dysbiosis. Our aim was to determine whether this microbial imbalance instigates perturbation of the host intestinal mucosal immune response, using a model of human microbiota-associated rats (HMAR) and dextran sulfate sodium (DSS)-induced experimental colitis. The analysis of the microbiota composition revealed a decrease of the relative abundance of Bacteroides, Roseburia-Eubacterium rectale and Bifidobacterium and an increase of Enterobacteriaceae, Desulfovibrio sp., and mainly Akkermansia muciniphila in C-IBS patients compared to healthy individuals. The bacterial diversity of the gut microbiota of healthy individuals or C-IBS patients was maintained in corresponding HMAR. Animals harboring a C-IBS microbiota had reduced DSS colitis with a decreased expression of pro-inflammatory cytokines from innate, Th1, and Th17 responses. The pre-treatment of conventional C57BL/6 mice or HMAR with A. muciniphila, but not with Escherichia coli, prior exposure to DSS also resulted in a reduction of colitis severity, highlighting that the anti-inflammatory effect of the gut microbiota of C-IBS patients is mediated, in part, by A. muciniphila. This work highlights a novel aspect of the crosstalk between the gut microbiota of C-IBS patients and host intestinal homeostasis.
Assuntos
Anti-Inflamatórios/imunologia , Microbioma Gastrointestinal/imunologia , Intestinos/microbiologia , Síndrome do Intestino Irritável/microbiologia , Animais , Colite/imunologia , Colite/microbiologia , Citocinas/imunologia , Sulfato de Dextrana/imunologia , Disbiose/imunologia , Humanos , Inflamação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Microbiota/imunologia , RatosRESUMO
Solute carrier family 7 member 2 (SLC7A2) is an inducible transporter of the semi-essential amino acid L-arginine (L-Arg), which has been implicated in immune responses to pathogens. We assessed the role of SLC7A2 in murine infection with Citrobacter rodentium, an attaching and effacing enteric pathogen that causes colitis. Induction of SLC7A2 was upregulated in colitis tissues, and localized predominantly to colonic epithelial cells. Compared to wild-type mice, Slc7a2-/-mice infected with C. rodentium had improved survival and decreased weight loss, colon weight, and histologic injury; this was associated with decreased colonic macrophages, dendritic cells, granulocytes, and Th1 and Th17 cells. In infected Slc7a2-/-mice, there were decreased levels of the proinflammatory cytokines G-CSF, TNF-α, IL-1α, IL-1ß, and the chemokines CXCL1, CCL2, CCL3, CCL4, CXCL2, and CCL5. In bone marrow chimeras, the recipient genotype drove the colitis phenotype, indicative of the importance of epithelial, rather than myeloid SLC7A2. Mice lacking Slc7a2 exhibited reduced adherence of C. rodentium to the colonic epithelium and decreased expression of Talin-1, a focal adhesion protein involved in the attachment of the bacterium. The importance of SLC7A2 and Talin-1 in the intimate attachment of C. rodentium and induction of inflammatory response was confirmed in vitro, using conditionally-immortalized young adult mouse colon (YAMC) cells with shRNA knockdown of Slc7a2 or Tln1. Inhibition of L-Arg uptake with the competitive inhibitor, L-lysine (L-Lys), also prevented attachment of C. rodentium and chemokine expression. L-Lys and siRNA knockdown confirmed the role of L-Arg and SLC7A2 in human Caco-2 cells co-cultured with enteropathogenic Escherichia coli. Overexpression of SLC7A2 in human embryonic kidney cells increased bacterial adherence and chemokine expression. Taken together, our data indicate that C. rodentium enhances its own pathogenicity by inducing the expression of SLC7A2 to favor its attachment to the epithelium and thus create its ecological niche.
Assuntos
Transportador 2 de Aminoácidos Catiônicos/metabolismo , Infecções por Enterobacteriaceae/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Animais , Western Blotting , Transportador 2 de Aminoácidos Catiônicos/imunologia , Linhagem Celular , Citrobacter rodentium , Modelos Animais de Doenças , Infecções por Enterobacteriaceae/imunologia , Humanos , Imunofenotipagem , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , TransfecçãoRESUMO
EGFR signaling regulates macrophage function, but its role in bacterial infection has not been investigated. Here, we assessed the role of macrophage EGFR signaling during infection with Helicobacter pylori, a bacterial pathogen that causes persistent inflammation and gastric cancer. EGFR was phosphorylated in murine and human macrophages during H. pylori infection. In human gastric tissues, elevated levels of phosphorylated EGFR were observed throughout the histologic cascade from gastritis to carcinoma. Deleting Egfr in myeloid cells attenuated gastritis and increased H. pylori burden in infected mice. EGFR deficiency also led to a global defect in macrophage activation that was associated with decreased cytokine, chemokine, and NO production. We observed similar alterations in macrophage activation and disease phenotype in the Citrobacter rodentium model of murine infectious colitis. Mechanistically, EGFR signaling activated NF-κB and MAPK1/3 pathways to induce cytokine production and macrophage activation. Although deletion of Egfr had no effect on DC function, EGFR-deficient macrophages displayed impaired Th1 and Th17 adaptive immune responses to H. pylori, which contributed to decreased chronic inflammation in infected mice. Together, these results indicate that EGFR signaling is central to macrophage function in response to enteric bacterial pathogens and is a potential therapeutic target for infection-induced inflammation and associated carcinogenesis.
Assuntos
Infecções Bacterianas/metabolismo , Receptores ErbB/metabolismo , Ativação de Macrófagos , Macrófagos/metabolismo , Animais , Quimiocinas/metabolismo , Citrobacter rodentium , Citocinas/metabolismo , Progressão da Doença , Feminino , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori , Humanos , Inflamação , Masculino , Camundongos , Camundongos Transgênicos , NF-kappa B/metabolismo , Fosforilação , Transdução de SinaisRESUMO
Nonalcoholic fatty liver disease occurs frequently in the setting of metabolic syndrome, but the factors leading to nonalcoholic steatohepatitis (NASH) are not fully understood. This study investigated Toll-like receptor 4 (TLR4) signaling in human liver with the goal of delineating whether activation of this pathway segregates those with nonalcoholic fatty liver from those with NASH. Experiments were performed using liver biopsy tissue obtained from class III obese subjects undergoing bariatric surgery, and extended to an immortalized human hepatocyte HepaRG cell line and primary human hepatocytes. The bacterial endotoxin lipopolysaccharide (LPS) and total free fatty acid levels were significantly increased in plasma of NASH patients. TLR4 mRNA levels were significantly increased in subjects with NASH compared with NAFL as was interferon regulatory factor (IRF) 3 in the myeloid differentiation factor 88-independent signaling pathway. In HepaRG cells, nuclear factor-κB (NF-κB) nuclear translocation and functional activity increased following treatment with the fatty acid, palmitate, and following exposure to LPS compared with hepatocytes stimulated with a lipogenic treatment that induced de novo lipogenesis. Palmitate and LPS induction of NF-κB activity was partially attenuated by chemical- or small-interfering RNA-mediated inhibition of TLR4. Expression of TLR4 and its downstream mediators was upregulated with palmitate and LPS. Similar results were observed using primary human hepatocytes from a lean donor. Interestingly, NF-κB activity assays showed obese donor hepatocytes were resistant to chemical TLR4 inhibition. In conclusion, TLR4 expression is upregulated in a large cohort of NASH patients, compared with those with NAFL, and this occurs within the setting of increased LPS and fatty acids.