RESUMO
Bifidobacterium bifidum strain BB1 causes a strain-specific enhancement in intestinal epithelial tight junction (TJ) barrier. Tumor necrosis factor (TNF)-α induces an increase in intestinal epithelial TJ permeability and promotes intestinal inflammation. The major purpose of this study was to delineate the protective effect of BB1 against the TNF-α-induced increase in intestinal TJ permeability and to unravel the intracellular mechanisms involved. TNF-α produces an increase in intestinal epithelial TJ permeability in Caco-2 monolayers and in mice. Herein, the addition of BB1 inhibited the TNF-α increase in Caco-2 intestinal TJ permeability and mouse intestinal permeability in a strain-specific manner. BB1 inhibited the TNF-α-induced increase in intestinal TJ permeability by interfering with TNF-α-induced enterocyte NF-κB p50/p65 and myosin light chain kinase (MLCK) gene activation. The BB1 protective effect against the TNF-α-induced increase in intestinal permeability was mediated by toll-like receptor-2/toll-like receptor-6 heterodimer complex activation of peroxisome proliferator-activated receptor γ (PPAR-γ) and PPAR-γ pathway inhibition of TNF-α-induced inhibitory kappa B kinase α (IKK-α) activation, which, in turn, resulted in a step-wise inhibition of NF-κB p50/p65, MLCK gene, MLCK kinase activity, and MLCK-induced opening of the TJ barrier. In conclusion, these studies unraveled novel intracellular mechanisms of BB1 protection against the TNF-α-induced increase in intestinal TJ permeability. The current data show that BB1 protects against the TNF-α-induced increase in intestinal epithelial TJ permeability via a PPAR-γ-dependent inhibition of NF-κB p50/p65 and MLCK gene activation.
Assuntos
Bifidobacterium bifidum , Mucosa Intestinal , Quinase de Cadeia Leve de Miosina , PPAR gama , Permeabilidade , Junções Íntimas , Receptor 2 Toll-Like , Fator de Transcrição RelA , Fator de Necrose Tumoral alfa , PPAR gama/metabolismo , Animais , Fator de Necrose Tumoral alfa/metabolismo , Junções Íntimas/metabolismo , Células CACO-2 , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Camundongos , Fator de Transcrição RelA/metabolismo , Receptor 2 Toll-Like/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Bifidobacterium bifidum/metabolismo , Bifidobacterium bifidum/fisiologia , Permeabilidade/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Probióticos/farmacologiaRESUMO
After community transmission of monkeypox virus was identified in Europe, interviews of 45 case-patients from England indicated transmission in international sexual networks of gay and bisexual men since April 2022. Interventions targeting sex-on-premises venues, geospatial dating applications, and sexual health services are likely to be critical for outbreak control.
Assuntos
Infecções por HIV , Mpox , Minorias Sexuais e de Gênero , Bissexualidade , Infecções por HIV/epidemiologia , Homossexualidade Masculina , Humanos , Masculino , Monkeypox virus , Comportamento SexualRESUMO
Defective intestinal tight junction (TJ) barrier is an important pathogenic factor of inflammatory bowel disease. To date, no effective therapies that specifically target the intestinal TJ barrier are available. The purpose of this study was to identify probiotic bacterial species or strains that induce a rapid and sustained enhancement of intestinal TJ barrier and protect against the development of intestinal inflammation by targeting the TJ barrier. After high-throughput screening of >20 Lactobacillus and other probiotic bacterial species or strains, a specific strain of Lactobacillus acidophilus, referred to as LA1, uniquely produced a marked enhancement of the intestinal TJ barrier. LA1 attached to the apical membrane surface of intestinal epithelial cells in a Toll-like receptor (TLR)-2-dependent manner and caused a rapid increase in enterocyte TLR-2 membrane expression and TLR-2/TLR-1 and TLR-2/TLR-6 hetero-complex-dependent enhancement in intestinal TJ barrier function. Oral administration of LA1 caused a rapid enhancement in mouse intestinal TJ barrier, protected against a dextran sodium sulfate (DSS) increase in intestinal permeability, and prevented the DSS-induced colitis in a TLR-2- and intestinal TJ barrier-dependent manner. In conclusion, we report for the first time that a specific strain of LA causes a strain-specific enhancement of intestinal TJ barrier through a novel mechanism that involves the TLR-2 receptor complex and protects against the DSS-induced colitis by targeting the intestinal TJ barrier.
Assuntos
Colite/prevenção & controle , Inflamação/prevenção & controle , Lactobacillus acidophilus/fisiologia , Probióticos , Receptor 2 Toll-Like/metabolismo , Animais , Colite/induzido quimicamente , Colite/microbiologia , Colite/patologia , Sulfato de Dextrana/efeitos adversos , Células Epiteliais/patologia , Intestinos/efeitos dos fármacos , Intestinos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Permeabilidade/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/patologia , Receptor 2 Toll-Like/genéticaRESUMO
We investigated risk factors associated with COVID-19 by conducting a retrospective, frequency-matched case-control study, with three sampling periods (August-October 2020). We compared cases completing routine contact tracing to asymptomatic population controls. Multivariable analyses estimated adjusted odds ratios (aORs) for non-household community settings. Meta-analyses using random effects provided pooled odds ratios (pORs). Working in healthcare (pOR 2.87; aORs 2.72, 2.81, 3.08, for study periods 1-3 respectively), social care (pOR 4.15; aORs 2.46, 5.06, 5.41, for study periods 1-3 respectively) or hospitality (pOR 2.36; aORs 2.01, 2.54, 2.63, for study periods 1-3 respectively) were associated with increased odds of being a COVID-19 case. Additionally, working in bars, pubs and restaurants, warehouse settings, construction, educational settings were significantly associated. While definitively determining where transmission occurs is impossible, we provide evidence that in certain sectors, the impact of mitigation measures may only be partial and reinforcement of measures should be considered in these settings.
Assuntos
COVID-19 , COVID-19/epidemiologia , Estudos de Casos e Controles , Humanos , Estudos Retrospectivos , SARS-CoV-2 , Local de TrabalhoRESUMO
BACKGROUND & AIMS: Defects in the epithelial tight junction (TJ) barrier contribute to development of intestinal inflammation associated with diseases. Interleukin 1 beta (IL1B) increases intestinal permeability in mice. We investigated microRNAs that are regulated by IL1B and their effects on expression of TJ proteins and intestinal permeability. METHODS: We used Targetscan to identify microRNAs that would bind the 3' untranslated region (3'UTR) of occludin mRNA; regions that interacted with microRNAs were predicted using the V-fold server and Assemble2, and 3-dimensional models were created using UCSF Chimera linked with Assemble2. Caco-2 cells were transfected with vectors that express microRNAs, analyzed by immunoblots and real-time polymerase chain reaction (PCR), and grown as monolayers; permeability in response to IL1B was assessed with the marker inulin. Male C57BL/6 mice were given intraperitoneal injections of IL1B and intestinal recycling perfusion was measured; some mice were given dextran sodium sulfate to induce colitis and/or gavage with an antagonist to MIR200C-3p (antagomiR-200C) or the nonspecific antagomiR (control). Intestinal tissues were collected from mice and analyzed by histology and real-time PCR; enterocytes were isolated by laser capture microdissection. We also analyzed colon tissues and organoids from patients with and without ulcerative colitis. RESULTS: Incubation of Caco-2 monolayers with IL1B increased TJ permeability and reduced levels of occludin protein and mRNA without affecting the expression of other transmembrane TJ proteins. Targetscan identified MIR122, MIR200B-3p, and MIR200C-3p, as miRNAs that might bind to the occludin 3'UTR. MIR200C-3p was rapidly increased in Caco-2 cells incubated with IL1B; the antagomiR-200c prevented the IL1B-induced decrease in occludin mRNA and protein and reduced TJ permeability. Administration of IL1B to mice increased small intestinal TJ permeability, compared with mice given vehicle; enterocytes isolated from mice given IL1B had increased expression of MIR200C-3p and decreased levels of occludin messenger RNA (mRNA) and protein. Intestinal tissues from mice with colitis had increased levels of IL1B mRNA and MIR200C-3p and decreased levels of occludin mRNA; gavage of mice with antagomiR-200C reduced levels of MIR200C-3p and prevented the decrease in occludin mRNA and the increase in colonic permeability. Colon tissues and organoids from patients with ulcerative colitis had increased levels of IL1B mRNA and MIR200C-3p compared with healthy controls. Using 3-dimensional molecular modeling and mutational analyses, we identified the nucleotide bases in the occluding mRNA 3'UTR that interact with MIR200C-3p. CONCLUSIONS: Intestine tissues from patients with ulcerative colitis and mice with colitis have increased levels of IL1B mRNA and MIR200C-3p, which reduces expression of occludin by enterocytes and thereby increases TJ permeability. Three-dimensional modeling of the interaction between MIR200C-3p and the occludin mRNA 3'UTR identified sites of interaction. The antagomiR-200C prevents the decrease in occludin in enterocytes and intestine tissues of mice with colitis, maintaining the TJ barrier.
Assuntos
Colite Ulcerativa/patologia , Interleucina-1beta/metabolismo , MicroRNAs/metabolismo , Ocludina/metabolismo , Junções Íntimas/metabolismo , Animais , Células CACO-2 , Técnicas de Cultura de Células , Colite Ulcerativa/etiologia , Colite Ulcerativa/metabolismo , Enterócitos , Humanos , Absorção Intestinal/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ocludina/genética , Permeabilidade , RNA Mensageiro/metabolismo , Regulação para CimaRESUMO
INTRODUCTION: Previous investigations have identified high rates of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection among residents and staff in care homes reporting an outbreak of coronavirus disease 2019 (COVID-19). We investigated care homes reporting a single suspected or confirmed case to assess whether early mass testing might reduce risk of transmission during the peak of the pandemic in London. METHODS: Between 18 and 27 April 2020, residents and staff in care homes reporting a single case of COVID-19 to Public Health England had a nasal swab to test for SARS-CoV-2 infection by reverse transcription polymerase chain reaction and subsequent whole-genome sequencing. Residents and staff in two care homes were re-tested 8 days later. RESULTS: Four care homes were investigated. SARS-CoV-2 positivity was 20% (65/333) overall, ranging between 3 and 59%. Among residents, positivity ranged between 3 and 76% compared with 3 and 40% in staff. Half of the SARS-CoV-2-positive residents (23/46, 50%) and 63% of staff (12/19) reported symptoms within 14 days before or after testing. Repeat testing 8 days later in two care homes with the highest infection rates identified only two new cases. Genomic analysis demonstrated a small number of introduction of the virus into care homes, and distinct clusters within three of the care homes. CONCLUSIONS: We found extensive but variable rates of SARS-CoV-2 infection among residents and staff in care homes reporting a single case of COVID-19. Although routine whole-home testing has now been adopted into practice, care homes must remain vigilant and should be encouraged to report a single suspected case, which should trigger appropriate outbreak control measures.
Assuntos
COVID-19/diagnóstico , Programas de Rastreamento/estatística & dados numéricos , SARS-CoV-2/isolamento & purificação , Idoso , Idoso de 80 Anos ou mais , COVID-19/epidemiologia , COVID-19/transmissão , Teste de Ácido Nucleico para COVID-19 , Teste para COVID-19 , Inglaterra , Feminino , Humanos , Controle de Infecções , Londres/epidemiologia , Assistência de Longa Duração , Masculino , Pandemias , Políticas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2/genética , Sequenciamento Completo do GenomaRESUMO
Defective intestinal tight junction (TJ) barrier is a hallmark in the pathogenesis of inflammatory bowel disease (IBD). To date, there are no effective therapies that specifically target the intestinal TJ barrier. Among the various probiotic bacteria, Bifidobacterium, is one of the most widely studied to have beneficial effects on the intestinal TJ barrier. The main purpose of this study was to identify Bifidobacterium species that cause a sustained enhancement in the intestinal epithelial TJ barrier and can be used therapeutically to target the intestinal TJ barrier and to protect against or treat intestinal inflammation. Our results showed that Bifidobacterium bifidum caused a marked, sustained enhancement in the intestinal TJ barrier in Caco-2 monolayers. The Bifidobacterium bifidum effect on TJ barrier was strain-specific, and only the strain designated as BB1 caused a maximal enhancement in TJ barrier function. The mechanism of BB1 enhancement of intestinal TJ barrier required live bacterial cell/enterocyte interaction and was mediated by the BB1 attachment to Toll-like receptor-2 (TLR-2) at the apical membrane surface. The BB1 enhancement of the intestinal epithelial TJ barrier function was mediated by the activation of the p38 kinase pathway, but not the NF-κB signaling pathway. Moreover, the BB1 caused a marked enhancement in mouse intestinal TJ barrier in a TLR-2-dependent manner and protected against dextran sodium sulfate (DSS)-induced increase in mouse colonic permeability, and treated the DSS-induced colitis in a TJ barrier-dependent manner. These studies show that probiotic bacteria BB1 causes a strain-specific enhancement of the intestinal TJ barrier through a novel mechanism involving BB1 attachment to the enterocyte TLR-2 receptor complex and activation of p38 kinase pathway.
Assuntos
Bifidobacterium bifidum/fisiologia , Colite/microbiologia , Mucosa Intestinal/microbiologia , Transdução de Sinais , Junções Íntimas , Receptor 2 Toll-Like/metabolismo , Animais , Células CACO-2 , Colite/prevenção & controle , Humanos , Mucosa Intestinal/metabolismo , Camundongos , NF-kappa B , Permeabilidade , ProbióticosRESUMO
Lipopolysaccharides (LPSs) are a major component of Gram-negative bacterial cell wall and play an important role in promoting intestinal inflammatory responses. Recent studies have shown that physiologically relevant concentrations of LPS (0 to 2000 pg/mL) cause an increase in intestinal epithelial tight junction (TJ) permeability without causing cell death. However, the intracellular pathways and the mechanisms that mediate LPS-induced increase in intestinal TJ permeability remain unclear. The aim was to delineate the intracellular pathways that mediate the LPS-induced increase in intestinal permeability using in vitro and in vivo intestinal epithelial models. LPS-induced increase in intestinal epithelial TJ permeability was preceded by an activation of transforming growth factor-ß-activating kinase-1 (TAK-1) and canonical NF-κB (p50/p65) pathways. The siRNA silencing of TAK-1 inhibited the activation of NF-κB p50/p65. The siRNA silencing of TAK-1 and p65/p50 subunit inhibited the LPS-induced increase in intestinal TJ permeability and the increase in myosin light chain kinase (MLCK) expression, confirming the regulatory role of TAK-1 and NF-κB p65/p50 in up-regulating MLCK expression and the subsequent increase in TJ permeability. The data also showed that toll-like receptor (TLR)-4/myeloid differentiation primary response (MyD)88 pathway was crucial upstream regulator of TAK-1 and NF-κB p50/p65 activation. In conclusion, activation of TAK-1 by the TLR-4/MyD88 signal transduction pathway and MLCK by NF-κB p65/p50 regulates the LPS-induced increase in intestinal epithelial TJ permeability.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Quinase I-kappa B/metabolismo , Mucosa Intestinal/fisiologia , Lipopolissacarídeos/farmacologia , MAP Quinase Quinase Quinases/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Animais , Células CACO-2 , Proteínas de Ligação ao Cálcio/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Quinase I-kappa B/genética , Mucosa Intestinal/efeitos dos fármacos , MAP Quinase Quinase Quinases/genética , Masculino , Camundongos Endogâmicos C57BL , Quinase de Cadeia Leve de Miosina/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Transdução de Sinais , Junções Íntimas/efeitos dos fármacos , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
A defective tight junction (TJ) barrier is a key pathogenic factor for inflammatory bowel disease. Previously, we have shown that autophagy, a cell survival mechanism, enhances intestinal epithelial TJ barrier function. Autophagy-related protein-6 (ATG6/beclin 1), a key protein in the autophagy pathway, also plays a role in the endocytic pathway. The constitutive role of beclin 1 in the intestinal TJ barrier is not known. In Caco-2 cells, beclin 1 was found to be coimmunoprecipitated with the TJ protein occludin and colocalized with occludin on the membrane. Treatment of Caco-2 cells with beclin 1 peptide [transactivating regulatory protein (Tat)-beclin 1] reduced TJ barrier function. Activation of beclin 1 increased occludin endocytosis and reduced total occludin protein level. In contrast, beclin 1 siRNA transfection enhanced Caco-2 TJ barrier function. In pharmacologic and genetic autophagy inhibition studies, the constitutive function of beclin 1 in the TJ barrier was found to be autophagy independent. However, de novo induction of autophagy with starvation or rapamycin prevented Tat-beclin 1-induced increase in TJ permeability and reduction in occludin level. Induction of autophagy also resulted in reduced beclin 1-occludin association. In mouse colon, beclin 1 colocalized with occludin on the epithelial membrane. Perfusion of mouse colon with beclin 1 peptide caused an increase in colonic TJ permeability that was prevented by in vivo induction of autophagy. These findings show that beclin 1 plays a constitutive, autophagy-independent role in the regulation of intestinal TJ barrier function via endocytosis of occludin. Autophagy terminates constitutive beclin 1 function in the TJ barrier and enhances the TJ barrier.
Assuntos
Autofagia/fisiologia , Proteína Beclina-1/fisiologia , Mucosa Intestinal/fisiologia , Junções Íntimas/fisiologia , Animais , Células CACO-2 , Feminino , Humanos , Mucosa Intestinal/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Matrix metalloproteinase-9 (MMP-9) has been implicated as being an important pathogenic factor in inflammatory bowel disease (IBD). MMP-9 is markedly elevated in intestinal tissue of patients with IBD, and IBD patients have a defective intestinal tight-junction (TJ) barrier manifested by an increase in intestinal permeability. The loss of intestinal epithelial barrier function is an important contributing factor in the development and prolongation of intestinal inflammation; however, the role of MMP-9 in intestinal barrier function remains unclear. The purpose of this study was to investigate the effect of MMP-9 on the intestinal epithelial TJ barrier and to delineate the intracellular mechanisms involved by using in vitro (filter-grown Caco-2 monolayers) and in vivo (mouse small intestine recycling perfusion) systems. MMP-9 caused a time- and dose-dependent increase in Caco-2 TJ permeability. MMP-9 also caused an increase in myosin light-chain kinase (MLCK) gene activity, protein expression, and enzymatic activity. The pharmacological MLCK inhibition and siRNA-induced knockdown of MLCK inhibited the MMP-9-induced increase in Caco-2 TJ permeability. MMP-9 caused a rapid activation of the p38 kinase signaling pathway and inhibition of p38 kinase activity prevented the MMP-9-induced increase in MLCK gene activity and the increase in Caco-2 TJ permeability. MMP-9 also caused an increase in mouse intestinal permeability in vivo, which was accompanied by an increase in MLCK expression. The MMP-9-induced increase in mouse intestinal permeability was inhibited in MLCK-deficient mice. These data show for the first time that the MMP-9-induced increase in intestinal TJ permeability in vitro and in vivo was mediated by the p38 kinase signal transduction pathway upregulation of MLCK gene activity and that therapeutic targeting of these pathways can prevent the MMP-9-induced increase in intestinal TJ permeability. NEW & NOTEWORTHY MMP-9 is highly elevated in patients with IBD. IBD patients have compromised intestinal TJ barrier function manifested by an increase in intestinal permeability and intestinal inflammation. This study shows that MMP-9, at clinically achievable concentrations, causes an increase in intestinal TJ permeability in vitro and in vivo. In addition, a MMP-9-induced increase in intestinal TJ permeability was mediated by an increase in MLCK gene and protein expression via the p38 kinase pathway.
Assuntos
Permeabilidade da Membrana Celular/genética , Sistema de Sinalização das MAP Quinases/genética , Metaloproteinase 9 da Matriz/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Células CACO-2 , Células Epiteliais , Humanos , Intestinos/fisiologia , Metaloproteinase 9 da Matriz/genética , Permeabilidade , Junções Íntimas/genética , Junções Íntimas/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Lipopolysaccharides (LPSs) are a major component of the Gram-negative bacterial cell wall and play an important role in mediating intestinal inflammatory responses in inflammatory bowel disease. Although recent studies suggested that physiologically relevant concentrations of LPS (0 to 1 ng/mL) cause an increase in intestinal epithelial tight junction (TJ) permeability, the mechanisms that mediate an LPS-induced increase in intestinal TJ permeability remain unclear. Herein, we show that myosin light chain kinase (MLCK) plays a central role in the LPS-induced increase in TJ permeability. Filter-grown Caco-2 intestinal epithelial monolayers and C57BL/6 mice were used as an in vitro and in vivo intestinal epithelial model system, respectively. LPS caused a dose- and time-dependent increase in MLCK expression and kinase activity in Caco-2 monolayers. The pharmacologic MLCK inhibition and siRNA-induced knock-down of MLCK inhibited the LPS-induced increase in Caco-2 TJ permeability. The LPS increase in TJ permeability was mediated by toll-like receptor 4 (TLR-4)/MyD88 signal-transduction pathway up-regulation of MLCK expression. The LPS-induced increase in mouse intestinal permeability also required an increase in MLCK expression. The LPS-induced increase in intestinal permeability was inhibited in MLCK-/- and TLR-4-/- mice. These data show, for the first time, that the LPS-induced increase in intestinal permeability was mediated by TLR-4/MyD88 signal-transduction pathway up-regulation of MLCK. Therapeutic targeting of these pathways can prevent an LPS-induced increase in intestinal permeability.
Assuntos
Mucosa Intestinal/metabolismo , Lipopolissacarídeos/toxicidade , Fator 88 de Diferenciação Mieloide/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Junções Íntimas/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Células CACO-2 , Humanos , Inflamação/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Permeabilidade/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Junções Íntimas/efeitos dos fármacosRESUMO
Previous studies have demonstrated that the chloride channel ClC-2 plays a critical role in intestinal epithelial tight junction (TJ) barrier function via intracellular trafficking of TJ protein occludin. To study the mechanism of ClC-2-mediated TJ barrier function and intracellular trafficking of occludin, we established ClC-2 over-expressing Caco-2 cell line (Caco-2CLCN2) by full length ClC-2 ORF transfection. ClC-2 over-expression (Caco-2CLCN2) significantly enhanced TJ barrier (increased TER by ≥2 times and reduced inulin flux by 50%) compared to control Caco-2pEZ cells. ClC-2 over-expression (Caco-2CLCN2) increased occludin protein level compared to control Caco-2pEZ cells. Surface biotinylation assay revealed reduced steady state endocytosis of occludin in Caco-2CLCN2 cells. Furthermore, ClC-2 over-expression led to reduction in caveolin-1 protein level and diminishment of caveolae assembly. Caveolae disruption increased TJ permeability in control but not ClC-2 over-expressing Caco-2CLCN2 cells. Selective ClC-2 channel blocker GaTx2 caused an increase in caveolin-1 protein level and reduced occludin level. Delivery of cell permeable caveolin-1 scaffolding domain reduced the occludin protein level. Over all, these results suggest that ClC- 2 enhances TJ barrier function in intestinal epithelial cells via regulation of caveolin-1 and caveolae-mediated trafficking of occludin.
Assuntos
Cavéolas/metabolismo , Caveolina 1/metabolismo , Canais de Cloreto/metabolismo , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Ocludina/metabolismo , Junções Íntimas/metabolismo , Western Blotting , Canais de Cloro CLC-2 , Permeabilidade da Membrana Celular , Proliferação de Células , Células Cultivadas , Endocitose/fisiologia , Células Epiteliais/citologia , Imunofluorescência , Humanos , Intestinos/citologia , Transporte ProteicoRESUMO
Tumor necrosis factor (TNF)-α, a key mediator of intestinal inflammation, causes an increase in intestinal epithelial tight junction (TJ) permeability by activating myosin light chain kinase (MLCK; official name MYLK3) gene. However, the precise signaling cascades that mediate the TNF-α-induced activation of MLCK gene and increase in TJ permeability remain unclear. Our aims were to delineate the upstream signaling mechanisms that regulate the TNF-α modulation of intestinal TJ barrier function with the use of in vitro and in vivo intestinal epithelial model systems. TNF-α caused a rapid activation of both canonical and noncanonical NF-κB pathway. NF-κB-inducing kinase (NIK) and mitogen-activated protein kinase kinase-1 (MEKK-1) were activated in response to TNF-α. NIK mediated the TNF-α activation of inhibitory κB kinase (IKK)-α, and MEKK1 mediated the activation of IKK complex, including IKK-ß. NIK/IKK-α axis regulated the activation of both NF-κB p50/p65 and RelB/p52 pathways. Surprisingly, the siRNA induced knockdown of NIK, but not MEKK-1, prevented the TNF-α activation of both NF-κB p50/p65 and RelB/p52 and the increase in intestinal TJ permeability. Moreover, NIK/IKK-α/NF-κB p50/p65 axis mediated the TNF-α-induced MLCK gene activation and the subsequent MLCK increase in intestinal TJ permeability. In conclusion, our data show that NIK/IKK-α/regulates the activation of NF-κB p50/p65 and plays an integral role in the TNF-α-induced activation of MLCK gene and increase in intestinal TJ permeability.
Assuntos
Quinase I-kappa B/metabolismo , MAP Quinase Quinase Quinase 1/metabolismo , NF-kappa B/metabolismo , Junções Íntimas/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Células CACO-2 , Células Cultivadas , Humanos , Intestino Delgado/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/fisiologia , NF-kappa B/antagonistas & inibidores , Subunidade p50 de NF-kappa B/metabolismo , Subunidade p52 de NF-kappa B/metabolismo , Permeabilidade , Regiões Promotoras Genéticas/fisiologia , RNA Interferente Pequeno/metabolismo , Fator de Transcrição RelA/metabolismo , Transfecção , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Gut-derived bacterial LPS plays an essential role in inducing intestinal and systemic inflammatory responses and have been implicated as a pathogenic factor in necrotizing enterocolitis and inflammatory bowel disease. The defective intestinal tight junction barrier was shown to be an important factor contributing to the development of intestinal inflammation. LPS, at physiological concentrations, causes an increase in intestinal tight junction permeability (TJP) via a TLR4-dependent process; however, the intracellular mechanisms that mediate LPS regulation of intestinal TJP remain unclear. The aim of this study was to investigate the adaptor proteins and the signaling interactions that mediate LPS modulation of intestinal tight junction barrier using in vitro and in vivo model systems. LPS caused a TLR4-dependent activation of membrane-associated adaptor protein focal adhesion kinase (FAK) in Caco-2 monolayers. LPS caused an activation of both MyD88-dependent and -independent pathways. Small interfering RNA silencing of MyD88 prevented an LPS-induced increase in TJP. LPS caused MyD88-dependent activation of IL-1R-associated kinase 4. TLR4, FAK, and MyD88 were colocalized. Small interfering silencing of TLR4 inhibited TLR4-associated FAK activation, and FAK knockdown prevented MyD88 activation. In vivo studies also confirmed that the LPS-induced increase in mouse intestinal permeability was associated with FAK and MyD88 activation; knockdown of intestinal epithelial FAK prevented an LPS-induced increase in intestinal permeability. Additionally, high-dose LPS-induced intestinal inflammation was dependent on the TLR4/FAK/MyD88 signal transduction axis. To our knowledge, our data show for the first time that the LPS-induced increases in intestinal TJP and intestinal inflammation were regulated by TLR4-dependent activation of the FAK/MyD88/IL-1R-associated kinase 4 signaling pathway.
Assuntos
Quinase 1 de Adesão Focal/imunologia , Lipopolissacarídeos/farmacologia , Fator 88 de Diferenciação Mieloide/imunologia , Transdução de Sinais/efeitos dos fármacos , Junções Íntimas/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Células CACO-2 , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Quinase 1 de Adesão Focal/genética , Humanos , Intestinos/imunologia , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Permeabilidade/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Junções Íntimas/genética , Receptor 4 Toll-Like/genéticaRESUMO
Autophagy is an intracellular degradation pathway and is considered to be an essential cell survival mechanism. Defects in autophagy are implicated in many pathological processes, including inflammatory bowel disease. Among the innate defense mechanisms of intestinal mucosa, a defective tight junction (TJ) barrier has been postulated as a key pathogenic factor in the causation and progression of inflammatory bowel disease by allowing increased antigenic permeation. The cross-talk between autophagy and the TJ barrier has not yet been described. In this study, we present the novel finding that autophagy enhances TJ barrier function in Caco-2 intestinal epithelial cells. Nutrient starvation-induced autophagy significantly increased transepithelial electrical resistance and reduced the ratio of sodium/chloride paracellular permeability. Nutrient starvation reduced the paracellular permeability of small-sized urea but not larger molecules. The role of autophagy in the modulation of paracellular permeability was confirmed by pharmacological induction as well as pharmacological and genetic inhibition of autophagy. Consistent with the autophagy-induced reduction in paracellular permeability, a marked decrease in the level of the cation-selective, pore-forming TJ protein claudin-2 was observed after cell starvation. Starvation reduced the membrane presence of claudin-2 and increased its cytoplasmic, lysosomal localization. Therefore, our data show that autophagy selectively reduces epithelial TJ permeability of ions and small molecules by lysosomal degradation of the TJ protein claudin-2.
Assuntos
Autofagia , Claudina-2/metabolismo , Células Epiteliais/citologia , Mucosa Intestinal/citologia , Proteólise , Junções Íntimas/metabolismo , Células CACO-2 , Células Epiteliais/metabolismo , Humanos , Mucosa Intestinal/metabolismo , PermeabilidadeRESUMO
Recent studies have implicated a pathogenic role for matrix metalloproteinases 9 (MMP-9) in inflammatory bowel disease. Although loss of epithelial barrier function has been shown to be a key pathogenic factor for the development of intestinal inflammation, the role of MMP-9 in intestinal barrier function remains unclear. The aim of this study was to investigate the role of MMP-9 in intestinal barrier function and intestinal inflammation. Wild-type (WT) and MMP-9(-/-) mice were subjected to experimental dextran sodium sulfate (DSS) colitis by administration of 3% DSS in drinking water for 7 days. The mouse colonic permeability was measured in vivo by recycling perfusion of the entire colon using fluorescently labeled dextran. The DSS-induced increase in the colonic permeability was accompanied by an increase in intestinal epithelial cell MMP-9 expression in WT mice. The DSS-induced increase in intestinal permeability and the severity of DSS colitis was found to be attenuated in MMP-9(-/-) mice. The colonic protein expression of myosin light chain kinase (MLCK) and phospho-MLC was found to be significantly increased after DSS administration in WT mice but not in MMP-9(-/-) mice. The DSS-induced increase in colonic permeability and colonic inflammation was attenuated in MLCK(-/-) mice and MLCK inhibitor ML-7-treated WT mice. The DSS-induced increase in colonic surface epithelial cell MLCK mRNA was abolished in MMP-9(-/-) mice. Lastly, increased MMP-9 protein expression was detected within the colonic surface epithelial cells in ulcerative colitis cases. These data suggest a role of MMP-9 in modulation of colonic epithelial permeability and inflammation via MLCK.
Assuntos
Colite/enzimologia , Colo/enzimologia , Sulfato de Dextrana , Mucosa Intestinal/enzimologia , Metaloproteinase 9 da Matriz/metabolismo , Junções Íntimas/enzimologia , Animais , Colite/induzido quimicamente , Colite/genética , Colite/patologia , Colite/prevenção & controle , Colo/efeitos dos fármacos , Colo/patologia , Modelos Animais de Doenças , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Metaloproteinase 9 da Matriz/análise , Metaloproteinase 9 da Matriz/deficiência , Metaloproteinase 9 da Matriz/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/metabolismo , Permeabilidade , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Índice de Gravidade de Doença , Transdução de Sinais , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/patologia , Fatores de TempoAssuntos
Síndrome da Imunodeficiência Adquirida , Linfo-Histiocitose Hemofagocítica , Transfusão de Plaquetas/efeitos adversos , Reação Transfusional , Síndrome da Imunodeficiência Adquirida/sangue , Síndrome da Imunodeficiência Adquirida/patologia , Síndrome da Imunodeficiência Adquirida/terapia , Adulto , Humanos , Linfo-Histiocitose Hemofagocítica/sangue , Linfo-Histiocitose Hemofagocítica/patologia , Linfo-Histiocitose Hemofagocítica/terapia , Masculino , Reação Transfusional/sangue , Reação Transfusional/terapiaRESUMO
The defective intestinal epithelial tight junction (TJ) barrier has been postulated to be an important pathogenic factor contributing to intestinal inflammation. It has been shown that the proinflammatory cytokine IL-1ß causes an increase in intestinal permeability; however, the signaling pathways and the molecular mechanisms involved remain unclear. The major purpose of this study was to investigate the role of the p38 kinase pathway and the molecular processes involved. In these studies, the in vitro intestinal epithelial model system (Caco-2 monolayers) was used to delineate the cellular and molecular mechanisms, and a complementary in vivo mouse model system (intestinal perfusion) was used to assess the in vivo relevance of the in vitro findings. Our data indicated that the IL-1ß increase in Caco-2 TJ permeability correlated with an activation of p38 kinase. The activation of p38 kinase caused phosphorylation and activation of p38 kinase substrate, activating transcription factor (ATF)-2. The activated ATF-2 translocated to the nucleus where it attached to its binding motif on the myosin L chain kinase (MLCK) promoter region, leading to the activation of MLCK promoter activity and gene transcription. Small interfering RNA induced silencing of ATF-2, or mutation of the ATF-2 binding motif prevented the activation of MLCK promoter and MLCK mRNA transcription. Additionally, in vivo intestinal perfusion studies also indicated that the IL-1ß increase in mouse intestinal permeability required p38 kinase-dependent activation of ATF-2. In conclusion, these studies show that the IL-1ß-induced increase in intestinal TJ permeability in vitro and in vivo was regulated by p38 kinase activation of ATF-2 and by ATF-2 regulation of MLCK gene activity.
Assuntos
Fator 2 Ativador da Transcrição/metabolismo , Interleucina-1beta/metabolismo , Mucosa Intestinal/metabolismo , Quinase de Cadeia Leve de Miosina/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Fator 2 Ativador da Transcrição/genética , Animais , Western Blotting , Células CACO-2 , Permeabilidade da Membrana Celular/fisiologia , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica/fisiologia , Humanos , Imunidade nas Mucosas/fisiologia , Camundongos , Regiões Promotoras Genéticas , Transporte Proteico/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Junções Íntimas/genética , Junções Íntimas/metabolismo , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
The use of botanical products and vitamins in skin care creams and sunscreens is prevalent. Herein we conduct an evaluation of sunscreens to quantitatively assess how often sunscreens incorporate botanically derived products and vitamins. The most commonly used botanicals products and vitamins are identified and stratified based on the sunscreen sun protection factor (SPF). The overall prevalence for the use of botanical agents and vitamins was 62% and 79%, respectively. Aloe vera and licorice root extracts were the most common botanical agents used in sunscreens. Retinyl palmitate was the most common vitamin derivative utilized in sunscreens. The prices of sunscreens significantly increased when more than one botanical product was added. Botanical products and vitamins are widely utilized in sunscreens and more research is needed to assess how their inclusion may enhance or alter the function of sunscreens.
Assuntos
Extratos Vegetais/análise , Protetores Solares/química , Vitaminas/análise , Aloe/química , Análise Custo-Benefício , Diterpenos , Glycyrrhiza/química , Humanos , Ésteres de Retinil , Fator de Proteção Solar , Protetores Solares/economia , Vitamina A/análogos & derivados , Vitamina A/análiseRESUMO
Tumor necrosis factor (TNF-α) is a proinflammatory cytokine that plays a critical role in the pathogenesis of inflammatory bowel disease. TNF-α causes an increase in intestinal permeability; however, the signaling pathways and the molecular mechanisms involved remain unclear. The major purpose of this study was to investigate the role of MAP kinase pathways (ERK1/2 and p38 kinase) and the molecular processes involved. An in vitro intestinal epithelial model system consisting of Caco-2 monolayers and an in vivo mouse model system were used to delineate the cellular and molecular mechanisms involved in TNF-α effects on tight junction barrier. The TNF-α-induced increase in Caco-2 tight junction permeability was mediated by activation of the ERK1/2 signaling pathway, but not the p38 kinase pathway. Activation of the ERK1/2 pathway led to phosphorylation and activation of the ETS domain-containing transcription factor Elk-1. The activated Elk-1 translocated to the nucleus, where it bound to its binding motif on the myosin light chain kinase (MLCK) promoter region, leading to the activation of MLCK promoter activity and gene transcription. In addition, in vivo intestinal perfusion studies also indicated that the TNF-α-induced increase in mouse intestinal permeability requires ERK1/2-dependent activation of Elk-1. These studies provide novel insight into the cellular and molecular processes that regulate the TNF-α-induced increase in intestinal epithelial tight junction permeability.