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1.
J Allergy Clin Immunol ; 143(5): 1865-1877.e4, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30439403

RESUMO

BACKGROUND: Psychological stress and heightened mast cell (MC) activation are linked with important immunologic disorders, including allergy, anaphylaxis, asthma, and functional bowel diseases, but the mechanisms remain poorly defined. We have previously demonstrated that activation of the corticotropin-releasing factor (CRF) system potentiates MC degranulation responses during IgE-mediated anaphylaxis and psychological stress through corticotropin-releasing factor receptor subtype 1 (CRF1) expressed on MCs. OBJECTIVE: In this study we investigated the role of corticotropin-releasing factor receptor subtype 2 (CRF2) as a modulator of stress-induced MC degranulation and associated disease pathophysiology. METHODS: In vitro MC degranulation assays were performed with bone marrow-derived mast cells (BMMCs) derived from wild-type (WT) and CRF2-deficient (CRF2-/-) mice and RBL-2H3 MCs transfected with CRF2-overexpressing plasmid or CRF2 small interfering RNA. In vivo MC responses and associated pathophysiology in IgE-mediated passive systemic anaphylaxis and acute psychological restraint stress were measured in WT, CRF2-/-, and MC-deficient KitW-sh/W-sh knock-in mice. RESULTS: Compared with WT mice, CRF2-/- mice exhibited greater serum histamine levels and exacerbated IgE-mediated anaphylaxis and colonic permeability. In addition, CRF2-/- mice exhibited increased serum histamine levels and colonic permeability after acute restraint stress. Experiments with BMMCs and RBL-2H3 MCs demonstrated that CRF2 expressed on MCs suppresses store-operated Ca2+ entry signaling and MC degranulation induced by diverse MC stimuli. Experiments with MC-deficient KitW-sh/W-sh mice systemically engrafted with WT and CRF2-/- BMMCs demonstrated the functional importance of MC CRF2 in modulating stress-induced pathophysiology. CONCLUSIONS: MC CRF2 is a negative global modulator of stimuli-induced MC degranulation and limits the severity of IgE-mediated anaphylaxis and stress-related disease pathogenesis.


Assuntos
Anafilaxia/imunologia , Mucosa Intestinal/metabolismo , Mastócitos/fisiologia , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Estresse Psicológico/imunologia , Junções Íntimas/metabolismo , Animais , Degranulação Celular , Linhagem Celular , Modelos Animais de Doenças , Feminino , Liberação de Histamina/genética , Humanos , Imunoglobulina E/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos , Receptores de Hormônio Liberador da Corticotropina/genética
2.
J Leukoc Biol ; 102(6): 1299-1312, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28684600

RESUMO

Life stress is a major risk factor in the onset and exacerbation of mast cell-associated diseases, including allergy/anaphylaxis, asthma, and irritable bowel syndrome. Although it is known that mast cells are highly activated upon stressful events, the mechanisms by which stress modulates mast cell function and disease pathophysiology remains poorly understood. Here, we investigated the role of corticotropin-releasing factor receptor subtype 1 (CRF1) in mast cell degranulation and associated disease pathophysiology. In a mast cell-dependent model of IgE-mediated passive systemic anaphylaxis (PSA), prophylactic administration of the CRF1-antagonist antalarmin attenuated mast cell degranulation and hypothermia. Mast cell-deficient KitW-sh/W-sh mice engrafted with CRF1-/- bone marrow-derived mast cells (BMMCs) exhibited attenuated PSA-induced serum histamine, hypothermia, and clinical scores compared with wild-type BMMC-engrafted KitW-sh/W-sh mice. KitW-sh/W-sh mice engrafted with CRF1-/- BMMCs also exhibited suppressed in vivo mast cell degranulation and intestinal permeability in response to acute restraint stress. Genetic and pharmacologic experiments with murine BMMCs, rat RBL-2H3, and human LAD2 mast cells demonstrated that although CRF1 activation did not directly induce MC degranulation, CRF1 signaling potentiated the degranulation responses triggered by diverse mast cell stimuli and was associated with enhanced release of Ca2+ from intracellular stores. Taken together, our results revealed a prominent role for CRF1 signaling in mast cells as a positive modulator of stimuli-induced degranulation and in vivo pathophysiologic responses to immunologic and psychologic stress.


Assuntos
Degranulação Celular , Mastócitos/fisiologia , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Estresse Fisiológico , Anafilaxia/fisiopatologia , Animais , Células da Medula Óssea/citologia , Cálcio/metabolismo , Linhagem Celular , AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Imunoglobulina E/metabolismo , Mucosa Intestinal/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Permeabilidade , Ratos , Receptores de Hormônio Liberador da Corticotropina/agonistas , Receptores de Hormônio Liberador da Corticotropina/antagonistas & inibidores , Transdução de Sinais , Urocortinas/metabolismo
3.
Biol Sex Differ ; 7: 60, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27895892

RESUMO

BACKGROUND: Biological sex plays a prominent role in the prevalence and severity of a number of important stress-related gastrointestinal and immune-related diseases including IBS and allergy/anaphylaxis. Despite the establishment of sex differences in these diseases, the underlying mechanisms contributing to sex differences remain poorly understood. The objective of this study was to define the role of biological sex on mast cells (MCs), an innate immune cell central to the pathophysiology of many GI and allergic disorders. METHODS: Twelve-week-old C57BL/6 male and female mice were exposed to immunological stress (2 h of IgE-mediated passive systemic anaphylaxis (PSA)) or psychological stress (1 h of restraint stress (RS)) and temperature, clinical scores, serum histamine, and intestinal permeability (for RS) were measured. Primary bone marrow-derived MCs (BMMCs) were harvested from male and female mice and analyzed for MC degranulation, signaling pathways, mediator content, and RNA transcriptome analysis. RESULTS: Sexually dimorphic responses were observed in both models of PSA and RS and in primary MCs. Compared with male mice, female mice exhibited increased clinical scores, hypothermia, and serum histamine levels in response to PSA and had greater intestinal permeability and serum histamine responses to RS. Primary BMMCs from female mice exhibited increased release of ß-hexosaminidase, histamine, tryptase, and TNF-α upon stimulation with IgE/DNP and A23187. Increased mediator release in female BMMCs was not associated with increased upstream phospho-tyrosine signaling pathways or downstream Ca2+ mobilization. Instead, increased mediator release in female MCs was associated with markedly increased capacity for synthesis and storage of MC granule-associated immune mediators as determined by MC mediator content and RNA transcriptome analysis. CONCLUSIONS: These results provide a new understanding of sexual dimorphic responses in MCs and have direct implications for stress-related diseases associated with a female predominance and MC hyperactivity including irritable bowel syndrome, allergy, and anaphylaxis.

4.
Cell Mol Gastroenterol Hepatol ; 2(3): 340-357, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-27458604

RESUMO

BACKGROUND AND AIMS: The human intestinal peptide transporter 1, hPepT1, is expressed in the small intestine at low levels in the healthy colon and upregulated during inflammatory bowel disease. hPepT1 plays a role in mouse colitis and human studies have demonstrated that chronic intestinal inflammation leads to colorectal cancer (colitis-associated cancer; CAC). Hence, we assessed here the role of PepT1 in CAC. METHODS: Mice with hPepT1 overexpression in intestinal epithelial cells (TG) or PepT1 (PepT1-KO) deletion were used and CAC was induced by AOM/DSS. RESULTS: TG mice had larger tumor sizes, increased tumor burdens, and increased intestinal inflammation compared to WT mice. Conversely, tumor number and size and intestinal inflammation were significantly decreased in PepT1-KO mice. Proliferating crypt cells were increased in TG mice and decreased in PepT1-KO mice. Analysis of human colonic biopsies revealed an increased expression of PepT1 in patients with colorectal cancer, suggesting that PepT1 might be targeted for the treatment of CAC. The use of an anti-inflammatory tripeptide KPV (Lys-Pro-Val) transported by PepT1 was able to prevent carcinogenesis in WT mice. When administered to PepT1-KO mice, KPV did not trigger any of the inhibitory effect on tumorigenesis observed in WT mice. CONCLUSIONS: The observations that pepT1 was highly expressed in human colorectal tumor and that its overexpression and deletion in mice increased and decreased colitis associated tumorigenesis, respectively, suggest that PepT1 is a potential therapeutic target for the treatment of colitis associated tumorigenesis.

5.
Lab Invest ; 94(9): 950-65, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25068660

RESUMO

Inflammatory bowel diseases (IBD) are chronic inflammatory conditions of the gastrointestinal (GI) tract associated with an increased risk of colorectal cancer (CRC). Current treatments for both IBD and colitis-associated CRC suffer from numerous side effects. Parthenolide (PTL) is a sesquiterpene lactone with anti-inflammatory activity, and previous studies have demonstrated that PTL is a potent inhibitor of the NF-κB pathway. Micheliolide (MCL), substantially more stable than PTL in vivo, was recently developed, and this study aimed to decipher its suitability as therapeutic tool for IBD and IBD-associated diseases. Similar to PTL, MCL inhibited NF-κB activation and subsequent pro-inflammatory pathways activation in vitro. Pro-drug forms of both compounds inhibited the DSS-induced colitis when administrated intraperitoneally or encapsulated in a polysaccharide gel designed to release drugs in the colon. Interestingly, MCL was found to attenuate carcinogenesis in AOM/DSS-induced CRC, thus providing new candidate for the treatment of inflammatory bowel disease and CRC.


Assuntos
Colite/prevenção & controle , Neoplasias Colorretais/prevenção & controle , NF-kappa B/antagonistas & inibidores , Sesquiterpenos de Guaiano/farmacologia , Animais , Sequência de Bases , Western Blotting , Linhagem Celular , Colite/complicações , Neoplasias Colorretais/complicações , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Lipopolissacarídeos/farmacologia , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
6.
J Mater Chem B ; 2(11): 1499-1508, 2014 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-24729869

RESUMO

Treatment strategies for inflammatory bowel disease have been constrained by limited therapeutic efficacy and serious adverse effects owing to a lack of receptor for targeted drug delivery to the inflamed colon. Upon inflammation, CD98 expression is highly elevated in colonic epithelial cells and infiltrating immune cells. To investigate whether CD98 can be used as a colitis-targeted delivery receptor, we constructed CD98 Fab'-bearing quantum dots (QDs)-loaded nanoparticles (Fab'-NPs). The resultant Fab'-NPs had desired particle size (~458 nm) with a narrow size distribution and zeta-potential (approximately +19 mV), low cytotoxicity, and excellent fluorescence properties. Electron microscopy images provided direct evidence for the well-dispersed distribution of QDs within spherical Fab'-NPs. Cellular uptake experiments demonstrated that Fab'-NPs were efficiently internalized into Colon-26 and RAW 264.7 cells through the CD98-mediated endocytosis pathway, and showed that the targeting effect of CD98 Fab' markedly increased their cellular uptake efficiency compared with control pegylated QDs-loaded NPs (PEG-NPs). Furthermore, ex vivo studies showed much more effective accumulation of Fab'-NPs in colitis tissue than that of PEG-NPs. These findings suggest that because of inflammation-dependent over-expression of CD98, active colitis-targeted delivery can be accomplished using NPs decorated with CD98 antibody.

7.
PLoS One ; 9(2): e87614, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24586284

RESUMO

PepT1 is a member of the proton-oligopeptide cotransporter family SLC15, which mediates the transport of di/tripeptides from intestinal lumen into epithelial cells. MicroRNAs (miRNAs), a small noncoding RNAs (21-23 nucleotides), post-transcriptionally regulate gene expression by binding to the 3'-untranslated regions (UTRs) of their target mRNAs. Although the role of most miRNAs remains elusive, they have been implicated in vital cellular functions such as intestinal epithelial cells differentiation, proliferation, and apoptosis. In the present study, we investigated the effect of intestinal epithelial PepT1 expression on microRNA (miRNA) expression/secretion in the colons of control mice and in mice with experimentally induced colonic inflammation (colitis). The colonic miRNA expression was deregulated in both colitis and control mice but the deregulation of miRNA expression/secretion was specific to colonic tissue and did not affect other tissues such as spleen and liver. Intestinal epithelial PepT1-dependent deregulation of colonic miRNA expression not only affects epithelial cells but also other cell types, such as intestinal macrophages. Importantly, we found the miRNA 23b which was known to be involved in inflammatory bowel disease was secreted and transported between cells to impose a gene-silencing effect on recipient intestinal macrophages. Based on our data, we may conclude that the expression of a specific protein, PepT1, in the intestine affects local miRNA expression/secretion in the colon on a tissue specific manner and may play an important role during the induction and progression of colitis. Colonic miRNA expression/secretion, regulated by intestinal epithelial PepT1, could play a crucial role in cell-to-cell communication during colitis.


Assuntos
Comunicação Celular , Colite/metabolismo , Colo/metabolismo , Mucosa Intestinal/metabolismo , MicroRNAs/genética , Simportadores/metabolismo , Regulação para Cima , Animais , Transporte Biológico , Colite/genética , Colite/patologia , Colo/patologia , Humanos , Mucosa Intestinal/patologia , Masculino , Camundongos , MicroRNAs/metabolismo , Transportador 1 de Peptídeos , Simportadores/genética
8.
Gastroenterology ; 146(5): 1289-300.e1-19, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24503126

RESUMO

BACKGROUND & AIMS: Nanoparticles have been explored as carriers of small interfering RNAs (siRNAs) and might be developed to treat patients with inflammatory bowel disease (IBD). Overexpression of CD98 on the surface of colonic epithelial cells and macrophages promotes the development and progression of IBD. We developed an orally delivered hydrogel that releases nanoparticles with single-chain CD98 antibodies on their surface (scCD98 functionalized) and loaded with CD98 siRNA (siCD98). We tested the ability of the nanoparticles to reduce levels of CD98 in the colons of mice with colitis. METHODS: scCD98-functionalized siCD98-loaded nanoparticles were fabricated using a complex coacervation technique. We investigated the cellular uptake and lysosome escape profiles of the nanoparticles in Colon-26 cells and RAW 264.7 macrophages using fluorescence microscopy. Colitis was induced by transfer of CD4(+)CD45RB(high) T cells to Rag(-/-) mice or administration of dextran sodium sulfate to C57BL/6 mice. Mice were then given hydrogel (chitosan and alginate) containing scCD98-functionalized nanoparticles loaded with siCD98 or scrambled siRNA (control) via gavage. RESULTS: The scCD98-functionalized nanoparticles were approximately 200 nm in size and had high affinity for CD98-overexpressing cells. The scCD98-functionalized siCD98-loaded nanoparticles significantly reduced levels of CD98 in Colon-26 cells and RAW 264.7 macrophages, along with production of inflammatory cytokines (tumor necrosis factor α, interleukin-6, and interleukin-12). In mice with colitis, administration of the scCD98-functionalized siCD98-loaded nanoparticles reduced colon expression of CD98. Importantly, the severity of colitis was also reduced compared with controls (based on loss of body weight, myeloperoxidase activity, inflammatory cytokine production, and histological analysis). Approximately 24.1% of colonic macrophages (CD11b(+)CD11c(-)F4/80(+)) in the mice had taken up fluorescently labeled siRNA-loaded nanoparticles within 12 hours of administration. CONCLUSIONS: Nanoparticles containing surface CD98 antibody and loaded with siCD98 reduce expression of this protein by colonic epithelial cells and macrophages, and oral administration decreases the severity of colitis in mice. This nanoparticle in hydrogel (chitosan/alginate) formulation might be developed to treat patients with IBD.


Assuntos
Colite/prevenção & controle , Colo/metabolismo , Proteína-1 Reguladora de Fusão/genética , Proteína-1 Reguladora de Fusão/imunologia , Terapia Genética/métodos , Nanomedicina/métodos , Nanopartículas , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Anticorpos de Cadeia Única/administração & dosagem , Administração Oral , Alginatos/química , Animais , Linhagem Celular , Quitosana/química , Colite/induzido quimicamente , Colite/genética , Colite/imunologia , Colite/metabolismo , Colite/patologia , Colo/imunologia , Colo/patologia , Sulfato de Dextrana , Modelos Animais de Doenças , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Hidrogéis , Interleucina-12/metabolismo , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Interferente Pequeno/metabolismo , Índice de Gravidade de Doença , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismo
9.
Biomaterials ; 34(30): 7471-82, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23820013

RESUMO

The application of RNA interference (RNAi) for inflammatory bowel disease (IBD) therapy has been limited by the lack of non-cytotoxic, efficient and targetable small interfering RNA (siRNA) carriers. TNF-α is the major pro-inflammatory cytokine mainly secreted by macrophages during IBD. Here, a mannosylated bioreducible cationic polymer (PPM) was synthesized and further spontaneously assembled nanoparticles (NPs) assisted by sodium triphosphate (TPP). The TPP-PPM/siRNA NPs exhibited high uniformity (polydispersity index = 0.004), a small particle size (211-275 nm), excellent bioreducibility, and enhanced cellular uptake. Additionally, the generated NPs had negative cytotoxicity compared to control NPs fabricated by branched polyethylenimine (bPEI, 25 kDa) or Oligofectamine (OF) and siRNA. In vitro gene silencing experiments revealed that TPP-PPM/TNF-α siRNA NPs with a weight ratio of 40:1 showed the most efficient inhibition of the expression and secretion of TNF-α (approximately 69.9%, which was comparable to the 71.4% obtained using OF/siRNA NPs), and its RNAi efficiency was highly inhibited in the presence of mannose (20 mm). Finally, TPP-PPM/siRNA NPs showed potential therapeutic effects on colitis tissues, remarkably reducing TNF-α level. Collectively, these results suggest that non-toxic TPP-PPM/siRNA NPs can be exploited as efficient, macrophage-targeted carriers for IBD therapy.


Assuntos
Doenças Inflamatórias Intestinais/terapia , Macrófagos/metabolismo , Manose/metabolismo , Nanopartículas/química , Interferência de RNA , Fator de Necrose Tumoral alfa/metabolismo , Animais , Células CACO-2 , Morte Celular/efeitos dos fármacos , Eletroforese em Gel de Ágar , Endocitose/efeitos dos fármacos , Humanos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Nanopartículas/ultraestrutura , Tamanho da Partícula , Polímeros/síntese química , Polímeros/química , Polímeros/farmacologia , Polifosfatos/química , Polifosfatos/farmacologia , Interferência de RNA/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Titulometria
10.
Lab Invest ; 93(8): 888-99, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23797361

RESUMO

We and others have shown that the dipeptide cotransporter PepT1 is expressed in immune cells, including macrophages that are in close contact with the lamina propria of the small and large intestines. In the present study, we used PepT1-knockout (KO) mice to explore the role played by PepT1 in immune cells during dextran sodium sulfate (DSS)-induced colitis. DSS treatment caused less severe body weight loss, diminished rectal bleeding, and less diarrhea in PepT1-KO mice than in wild-type (WT) animals. A histological examination of colonic sections revealed that the colonic architecture was less disrupted and the extent of immune cell infiltration into the mucosa and submucosa following DSS treatment was reduced in PepT1-KO mice compared with WT animals. Consistent with these results, the DSS-induced colitis increase in colonic myeloperoxidase activity was significantly less in PepT1-KO mice than in WT littermates. The colonic levels of mRNAs encoding the inflammatory cytokines CXCL1, interleukin (IL)-6, monocyte chemotactic protein-1, IL-12, and interferon-γ were significantly lower in DSS-treated PepT1-KO mice than in DSS-treated WT animals. Colonic immune cells from WT had significantly higher level of proinflammatory cytokines then PepT1 KO. In addition, we observed that knocking down the PepT1 expression decreases chemotaxis of immune cells recruited during intestinal inflammation. Antibiotic treatment before DSS-induced colitis eliminated the differential expression of inflammatory cytokines between WT and PepT1-KO mice. In conclusion, PepT1 in immune cells regulates the secretion of proinflammatory cytokines triggered by bacteria and/or bacterial products, and thus has an important role in the induction of colitis. PepT1 may transport small bacterial products, such as muramyl dipeptide and the tripeptide L-Ala-gamma-D-Glu-meso-DAP, into macrophages. These materials may be sensed by members of the nucleotide-binding site-leucine-rich repeat family of intracellular receptors, ultimately resulting in altered homeostasis of the intestinal microbiota.


Assuntos
Colite/imunologia , Expressão Gênica , Imunidade Celular/imunologia , Macrófagos/metabolismo , Simportadores/genética , Animais , Antibacterianos/farmacologia , Colite/induzido quimicamente , Colite/patologia , Colo/efeitos dos fármacos , Colo/enzimologia , Colo/microbiologia , Citocinas/genética , Citocinas/metabolismo , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Homeostase , Imunidade Celular/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transportador 1 de Peptídeos , Peroxidase/metabolismo , RNA Mensageiro/metabolismo , Simportadores/metabolismo
11.
Infect Immun ; 81(3): 923-34, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23297381

RESUMO

CD98 is a type II transmembrane glycoprotein whose expression increases in intestinal epithelial cells (IECs) during intestinal inflammation. Enteropathogenic Escherichia coli (EPEC) is a food-borne human pathogen that attaches to IECs and injects effector proteins directly into the host cells, thus provoking an inflammatory response. In the present study, we investigated CD98 and EPEC interactions in vitro and ex vivo and examined FVB wild-type (WT) and villin-CD98 transgenic mice overexpressing human CD98 in IECs (hCD98 Tg mice) and infected with Citrobacter rodentium as an in vivo model. In vivo studies indicated that CD98 overexpression, localized to the apical domain of colonic cells, increased the attachment of C. rodentium in mouse colons and resulted in increased expression of proinflammatory markers and decreased expression of anti-inflammatory markers. The proliferative markers Ki-67 and cyclin D1 were significantly increased in the colonic tissue of C. rodentium-infected hCD98 Tg mice compared to that of WT mice. Ex vivo studies correlate with the in vivo data. Small interfering RNA (siRNA) studies with Caco2-BBE cells showed a decrease in adherence of EPEC to Caco2 cells in which CD98 expression was knocked down. In vitro surface plasmon resonance (SPR) experiments showed direct binding between recombinant hCD98 and EPEC/C. rodentium proteins. We also demonstrated that the partial extracellular loop of hCD98 was sufficient for direct binding to EPEC/C. rodentium. These findings demonstrate the importance of the extracellular loop of CD98 in the innate host defense response to intestinal infection by attaching and effacing (A/E) pathogens.


Assuntos
Infecções por Enterobacteriaceae/imunologia , Proteína-1 Reguladora de Fusão/metabolismo , Imunidade Inata , Mucosa Intestinal/metabolismo , Animais , Células CACO-2 , Citrobacter rodentium , Colo , Infecções por Enterobacteriaceae/microbiologia , Escherichia coli Enteropatogênica , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Feminino , Proteína-1 Reguladora de Fusão/genética , Regulação da Expressão Gênica/imunologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Peroxidase , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
12.
Lab Invest ; 92(8): 1203-12, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22641098

RESUMO

The transmembrane glycoprotein CD98 regulates integrin signaling that in turn controls cell proliferation and survival. CD98 expression is upregulated in various carcinomas, including colorectal cancer. Recently, by generating gain- and loss-of-function mouse models featuring genetic manipulation of CD98 expression specifically in intestinal epithelial cells (IECs), we have explored the crucial role of CD98 in the regulation of intestinal homeostasis and inflammation-associated tumorigenesis. In the present study, we investigated the contribution of CD98 to intestinal tumorigenesis in Apc(Min/+) mice and the underlying mechanism of action. Mice featuring IEC-specific CD98 overexpression (Tg animals) were crossed with Apc(Min/+) mice, and the characteristics of intestinal adenoma formation were assessed. Compared with Apc(Min/+) mice, Tg/Apc(Min/+) animals exhibited increases in both intestinal tumor incidence and tumor size; these parameters correlated with enhanced proliferation and decreased apoptosis of IECs. IEC-specific CD98 overexpression resulted in increased synthesis of the oncogenic proteins c-myc and cyclin-D1 in Apc(Min/+) mice, independently of the Wnt-APC-ß-catenin pathway, suggesting the implication of CD98 overexpression-mediated Erk activation. IEC-specific CD98 overexpression enhanced the production of proinflammatory cytokines and chemokines that are crucial for tumorigenesis. We validated our results in mice exhibiting IEC-specific CD98 downregulation (CD98(flox/+)VillinCre animals). IEC-specific CD98 downregulation efficiently attenuated tumor incidence and growth in Apc(Min/+) mice. The reduction of intestinal tumorigenesis upon IEC-specific CD98 downregulation was caused by the attenuation of IEC proliferation and cytokine/chemokine production. In conclusion, we show that CD98 exerts an oncogenic activity in terms of intestinal tumorigenesis, via an ability to regulate tumor growth and survival.


Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Transformação Celular Neoplásica/metabolismo , Células Epiteliais/metabolismo , Proteína-1 Reguladora de Fusão/biossíntese , Mucosa Intestinal/metabolismo , Neoplasias Intestinais/metabolismo , Adenoma/genética , Adenoma/metabolismo , Adenoma/patologia , Animais , Apoptose/fisiologia , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Citocinas/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Proteína-1 Reguladora de Fusão/genética , Proteína-1 Reguladora de Fusão/metabolismo , Histocitoquímica , Mucosa Intestinal/patologia , Neoplasias Intestinais/genética , Neoplasias Intestinais/patologia , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Transgênicos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo
13.
Am J Physiol Gastrointest Liver Physiol ; 302(11): G1282-91, 2012 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-22499850

RESUMO

The transmembrane glycoprotein CD98 is known to be involved in intestinal inflammation. In the present study, we found that CD98 overexpression in intestinal epithelial cells does not normally affect the expression of colonic (epithelial and immune cell) microRNAs (miRNAs), small noncoding RNAs that posttranscriptionally regulate a wide variety of biological processes. However, upon dextran sulfate sodium (DSS) treatment, the expression of several colonic miRNAs, but not miRNAs from other tissues such as liver and spleen, were differentially regulated in mice overexpressing CD98 in epithelial cells compared with wild-type (WT) animals. For example, the level of colonic miRNA 132 was not affected by DSS treatment in WT animals but was upregulated in mice overexpressing CD98 in intestinal epithelial cells. Other colonic miRNAs, including colonic miRNA 23a and 23b, were downregulated in WT animals after DSS treatment but not in colonic epithelial cell CD98-overexpressing mice. Interestingly, the expression of potential miRNA target genes affected intestinal epithelial cells that overexpress CD98 and cell types that did not overexpress CD98 but were in close proximity to CD98-overexpressing intestinal epithelial cells. Taken together, these observations show that the combination of an inflammatory context and intestinal epithelial cell expression of CD98 affects the regulation of miRNA expression in colonic epithelial and immune cells. This is new evidence that protein expression modulates miRNA expression and suggests the existence of regulatory crosstalk between proteins and miRNAs in diseases such as colitis.


Assuntos
Colite/metabolismo , Colo/metabolismo , Proteína-1 Reguladora de Fusão/biossíntese , Mucosa Intestinal/metabolismo , MicroRNAs/metabolismo , Animais , Colite/genética , Células Epiteliais/metabolismo , Inflamação , Camundongos , MicroRNAs/genética
14.
PLoS One ; 7(3): e32084, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22427817

RESUMO

Inflammatory bowel diseases (IBDs), primarily ulcerative colitis and Crohn's disease, are inflammatory disorders caused by multiple factors. Research on IBD has often used the dextran sodium sulfate (DSS)-induced colitis mouse model. DSS induces in vivo but not in vitro intestinal inflammation. In addition, no DSS-associated molecule (free glucose, sodium sulfate solution, free dextran) induces in vitro or in vivo intestinal inflammation. We find that DSS but not dextran associated molecules established linkages with medium-chain-length fatty acids (MCFAs), such as dodecanoate, that are present in the colonic lumen. DSS complexed to MCFAs forms nanometer-sized vesicles ~200 nm in diameter that can fuse with colonocyte membranes. The arrival of nanometer-sized DSS/MCFA vesicles in the cytoplasm may activate intestinal inflammatory signaling pathways. We also show that the inflammatory activity of DSS is mediated by the dextran moieties. The deleterious effect of DSS is localized principally in the distal colon, therefore it will be important to chemically modify DSS to develop materials beneficial to the colon without affecting colon-targeting specificity.


Assuntos
Colite/induzido quimicamente , Sulfato de Dextrana/efeitos adversos , Sulfato de Dextrana/metabolismo , Ácidos Graxos/metabolismo , Substâncias Macromoleculares/metabolismo , Nanoestruturas/química , Análise de Variância , Animais , Colite/metabolismo , Colite/patologia , Citocinas/sangue , Primers do DNA/genética , Dieta Hiperlipídica , Impedância Elétrica , Endoscopia Gastrointestinal , Feminino , Técnicas Histológicas , Camundongos , Camundongos Endogâmicos C57BL , Tamanho da Partícula , Peroxidase/metabolismo , Vesículas Transportadoras/metabolismo
15.
Am J Physiol Gastrointest Liver Physiol ; 302(5): G484-92, 2012 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-22194420

RESUMO

Intestinal inflammation is characterized by epithelial disruption, leading to loss of barrier function and the recruitment of immune cells, including neutrophils. Although the mechanisms are not yet completely understood, interactions between environmental and immunological factors are thought to be critical in the initiation and progression of intestinal inflammation. In recent years, it has become apparent that the di/tripeptide transporter PepT1 may play an important role in the pathogenesis of such inflammation. In healthy individuals, PepT1 is primarily expressed in the small intestine and transports di/tripeptides for metabolic purposes. However, during chronic inflammation such as that associated with inflammatory bowel disease, PepT1 expression is upregulated in the colon, wherein the protein is normally expressed either minimally or not at all. Several recent studies have shown that PepT1 binds to and transports various bacterial di/tripeptides into colon cells, leading to activation of downstream proinflammatory responses via peptide interactions with innate immune receptors. In the present review, we examine the relationship between colonic PepT1-mediated peptide transport in the colon and activation of innate immune responses during disease. It is important to understand the mechanisms of PepT1 action during chronic intestinal inflammation to develop future therapies addressing inappropriate immune activation in the colon.


Assuntos
Gastroenterite/etiologia , Doenças Inflamatórias Intestinais/etiologia , Simportadores/fisiologia , Animais , Neoplasias Colorretais/fisiopatologia , Gastroenterite/tratamento farmacológico , Humanos , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/fisiopatologia , Proteínas Adaptadoras de Sinalização NOD/fisiologia , Oligopeptídeos/metabolismo , Transportador 1 de Peptídeos , Simportadores/genética
16.
J Biol Chem ; 286(35): 31003-31013, 2011 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-21757725

RESUMO

The oligopeptide transporter PepT1 expressed in inflamed colonic epithelial cells transports small bacterial peptides, such as muramyl dipeptide (MDP) and l-Ala-γ-D-Glu-meso-diaminopimelic acid (Tri-DAP) into cells. The innate immune system uses various proteins to sense pathogen-associated molecular patterns. Nucleotide-binding oligomerization domain (NOD)-like receptors of which there are more than 20 related family members are present in the cytosol and recognize intracellular ligands. NOD proteins mediate NF-κB activation via receptor-interacting serine/threonine-protein kinase 2 (RICK or RIPK). The specific ligands for some NOD-like receptors have been identified. NOD type 1 (NOD1) is activated by peptides that contain a diaminophilic acid, such as the PepT1 substrate Tri-DAP. In other words, PepT1 transport activity plays an important role in controlling intracellular loading of ligands for NOD1 in turn determining the activation level of downstream inflammatory pathways. However, no direct interaction between Tri-DAP and NOD1 has been identified. In the present work, surface plasmon resonance and atomic force microscopy experiments showed direct binding between NOD1 and Tri-DAP with a K(d) value of 34.5 µM. In contrast, no significant binding was evident between muramyl dipeptide and NOD1. Furthermore, leucine-rich region (LRR)-truncated NOD1 did not interact with Tri-DAP, indicating that Tri-DAP interacts with the LRR domain of NOD1. Next, we examined binding between RICK and NOD1 proteins and found that such binding was significant with a K(d) value of 4.13 µM. However, NOD1/RICK binding was of higher affinity (K(d) of 3.26 µM) when NOD1 was prebound to Tri-DAP. Furthermore, RICK phosphorylation activity was increased when NOD was prebound to Tri-DAP. In conclusion, we have shown that Tri-DAP interacts directly with the LRR domain of NOD1 and consequently increases RICK/NOD1 association and RICK phosphorylation activity.


Assuntos
Ácido Diaminopimélico/análogos & derivados , Leucina/química , Proteína Adaptadora de Sinalização NOD1/química , Oligopeptídeos/química , Biofísica/métodos , Células CACO-2 , Ácido Diaminopimélico/química , Ácido Diaminopimélico/metabolismo , Humanos , Imunidade Inata , Inflamação , Microscopia de Força Atômica/métodos , Nucleotídeos/química , Oligopeptídeos/metabolismo , Peptídeos/química , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
17.
Gastroenterology ; 141(4): 1334-45, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21762661

RESUMO

BACKGROUND & AIMS: The human di/tripeptide transporter human intestinal H-coupled oligonucleotide transporter (hPepT1) is abnormally expressed in colons of patients with inflammatory bowel disease, although its exact role in pathogenesis is unclear. We investigated the contribution of PepT1 to intestinal inflammation in mouse models of colitis and the involvement of the nucleotide-binding oligomerization domain 2 (NOD2) signaling pathway in the pathogenic activity of colonic epithelial hPepT1. METHODS: Transgenic mice were generated in which hPepT1 expression was regulated by the ß-actin or villin promoters; colitis was induced using 2,4,6-trinitrobenzene sulfonic acid (TNBS) or dextran sodium sulfate (DSS) and the inflammatory responses were assessed. The effects of NOD2 deletion in the hPepT1 transgenic mice also was studied to determine the involvement of the PepT1-NOD2 signaling pathway. RESULTS: TNBS and DSS induced more severe levels of inflammation in ß-actin-hPepT1 transgenic mice than wild-type littermates. Intestinal epithelial cell-specific hPepT1 overexpression in villin-hPepT1 transgenic mice increased the severity of inflammation induced by DSS, but not TNBS. Bone marrow transplantation studies showed that hPepT1 expression in intestinal epithelial cells and immune cells has an important role in the proinflammatory response. Antibiotics abolished the effect of hPepT1 overexpression on the inflammatory response in DSS-induced colitis in ß-actin-hPepT1 and villin-hPepT1 transgenic mice, indicating that commensal bacteria are required to aggravate intestinal inflammation. Nod2-/-, ß-actin-hPepT1 transgenic/Nod2-/-, and villin-hPepT1 transgenic/Nod2-/- littermates had similar levels of susceptibility to DSS-induced colitis, indicating that hPepT1 overexpression increased intestinal inflammation in a NOD2-dependent manner. CONCLUSIONS: The PepT1-NOD2 signaling pathway is involved in aggravation of DSS-induced colitis in mice.


Assuntos
Colite/metabolismo , Colo/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Transdução de Sinais , Simportadores/metabolismo , Actinas/genética , Animais , Antibacterianos/farmacologia , Transplante de Medula Óssea , Colite/induzido quimicamente , Colite/genética , Colite/imunologia , Colite/microbiologia , Colite/patologia , Colite/prevenção & controle , Colo/efeitos dos fármacos , Colo/imunologia , Colo/microbiologia , Colo/patologia , Sulfato de Dextrana , Modelos Animais de Doenças , Humanos , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/genética , Proteína Adaptadora de Sinalização NOD2/deficiência , Proteína Adaptadora de Sinalização NOD2/genética , Transportador 1 de Peptídeos , Regiões Promotoras Genéticas , Índice de Gravidade de Doença , Transdução de Sinais/efeitos dos fármacos , Simportadores/genética , Fatores de Tempo , Ácido Trinitrobenzenossulfônico
18.
J Immunol ; 187(3): 1496-505, 2011 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-21705622

RESUMO

Inflammatory bowel disease, mainly Crohn's disease and ulcerative colitis, are characterized by epithelial barrier disruption and altered immune regulation. Colonic Ste20-like proline/alanine-rich kinase (SPAK) plays a role in intestinal inflammation, but its underlying mechanisms need to be defined. Both SPAK-transfected Caco2-BBE cells and villin-SPAK transgenic (TG) FVB/6 mice exhibited loss of intestinal barrier function. Further studies demonstrated that SPAK significantly increased paracellular intestinal permeability to FITC-dextran. In vivo studies using the mouse models of colitis induced by dextran sulfate sodium (DSS) and trinitrobenzene sulfonic acid showed that TG FVB/6 mice were more susceptible to DSS and trinitrobenzene sulfonic acid treatment than wild-type FVB/6 mice, as demonstrated by clinical and histological characteristics and enzymatic activities. Consistent with this notion, we found that SPAK increased intestinal epithelial permeability, which likely facilitated the production of inflammatory cytokines in vitro and in vivo, aggravated bacterial translocation in TG mice under DSS treatment, and consequently established a context favorable for the triggering of intestinal inflammation cascades. In conclusion, overexpression of SPAK inhibits maintenance of intestinal mucosal innate immune homeostasis, which makes regulation of SPAK important to attenuate pathological responses in inflammatory bowel disease.


Assuntos
Predisposição Genética para Doença , Doenças Inflamatórias Intestinais/enzimologia , Doenças Inflamatórias Intestinais/genética , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Imunidade Adaptativa/genética , Animais , Células CACO-2 , Células Cultivadas , Modelos Animais de Doenças , Humanos , Imunidade Inata/genética , Doenças Inflamatórias Intestinais/imunologia , Mucosa Intestinal/enzimologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Camundongos , Camundongos Transgênicos , Permeabilidade , Proteínas Serina-Treonina Quinases/fisiologia
19.
PLoS One ; 6(4): e19293, 2011 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-21559394

RESUMO

Microbiota are known to modulate host gene expression, yet the underlying molecular mechanisms remain elusive. MicroRNAs (miRNAs) are importantly implicated in many cellular functions by post-transcriptionally regulating gene expression via binding to the 3'-untranslated regions (3'-UTRs) of the target mRNAs. However, a role for miRNAs in microbiota-host interactions remains unknown. Here we investigated if miRNAs are involved in microbiota-mediated regulation of host gene expression. Germ-free mice were colonized with the microbiota from pathogen-free mice. Comparative profiling of miRNA expression using miRNA arrays revealed one and eight miRNAs that were differently expressed in the ileum and the colon, respectively, of colonized mice relative to germ-free mice. A computational approach was then employed to predict genes that were potentially targeted by the dysregulated miRNAs during colonization. Overlapping the miRNA potential targets with the microbiota-induced dysregulated genes detected by a DNA microarray performed in parallel revealed several host genes that were regulated by miRNAs in response to colonization. Among them, Abcc3 was identified as a highly potential miRNA target during colonization. Using the murine macrophage RAW 264.7 cell line, we demonstrated that mmu-miR-665, which was dysregulated during colonization, down-regulated Abcc3 expression by directly targeting the Abcc3 3'-UTR. In conclusion, our study demonstrates that microbiota modulate host microRNA expression, which could in turn regulate host gene expression.


Assuntos
Metagenoma/genética , MicroRNAs/genética , Regiões 3' não Traduzidas , Animais , Linhagem Celular , Biologia Computacional/métodos , DNA Complementar/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos
20.
BMC Microbiol ; 10: 285, 2010 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-21073689

RESUMO

BACKGROUND: Accurate identification is necessary to discriminate harmless environmental Yersinia species from the food-borne pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis and from the group A bioterrorism plague agent Yersinia pestis. In order to circumvent the limitations of current phenotypic and PCR-based identification methods, we aimed to assess the usefulness of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) protein profiling for accurate and rapid identification of Yersinia species. As a first step, we built a database of 39 different Yersinia strains representing 12 different Yersinia species, including 13 Y. pestis isolates representative of the Antiqua, Medievalis and Orientalis biotypes. The organisms were deposited on the MALDI-TOF plate after appropriate ethanol-based inactivation, and a protein profile was obtained within 6 minutes for each of the Yersinia species. RESULTS: When compared with a 3,025-profile database, every Yersinia species yielded a unique protein profile and was unambiguously identified. In the second step of analysis, environmental and clinical isolates of Y. pestis (n = 2) and Y. enterocolitica (n = 11) were compared to the database and correctly identified. In particular, Y. pestis was unambiguously identified at the species level, and MALDI-TOF was able to successfully differentiate the three biotypes. CONCLUSION: These data indicate that MALDI-TOF can be used as a rapid and accurate first-line method for the identification of Yersinia isolates.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Yersinia/química , Yersinia/isolamento & purificação , Microbiologia Ambiental , Humanos , Yersinia/classificação , Yersinia/genética , Yersiniose/microbiologia , Yersinia pestis/química , Yersinia pestis/classificação , Yersinia pestis/genética , Yersinia pestis/isolamento & purificação
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