RESUMEN
MicroRNAs (miRNAs) are endogenous oligoribonucleotides with exciting therapeutic potential. Early studies established a clear role for miRNAs in leukocyte biology. The first miRNA-based therapy, miravirsen, is now in phase 2 clinical trials, making the reality of these therapies undeniable. The capacity for miRNAs to fine-tune inflammatory signaling make them attractive treatment targets for immunological diseases. Nonetheless, the degree of redundancy among miRNAs, coupled with the promiscuity of miRNA binding sites in the transcriptome, require consideration when designing miRNA-directed interventions. Altered miRNA expression occurs across a range of inflammatory conditions, including inflammatory bowel disease, arthritis, and diabetes. However, very few studies successfully treated murine models of immunological diseases with miRNA-based approaches. While discussing recent studies targeting miRNAs to treat immunological conditions, we also reflect on the risks of miRNA targeting and showcase some newer delivery systems that may improve the pharmacological profile of this class of therapeutics.
Asunto(s)
Enfermedades del Sistema Inmune/tratamiento farmacológico , Enfermedades del Sistema Inmune/genética , Inflamación/tratamiento farmacológico , MicroARNs/antagonistas & inhibidores , Terapia Molecular Dirigida , Animales , Sitios de Unión/genética , Sistemas de Liberación de Medicamentos , Humanos , Enfermedades del Sistema Inmune/inmunología , Inflamación/genética , Inflamación/inmunología , Ratones , MicroARNs/genética , Oligonucleótidos/uso terapéuticoRESUMEN
Cytokines secreted at sites of inflammation impact the onset, progression, and resolution of inflammation. In this article, we investigated potential proresolving mechanisms of IFN-γ in models of inflammatory bowel disease. Guided by initial microarray analysis, in vitro studies revealed that IFN-γ selectively induced the expression of IL-10R1 on intestinal epithelia. Further analysis revealed that IL-10R1 was expressed predominantly on the apical membrane of polarized epithelial cells. Receptor activation functionally induced canonical IL-10 target gene expression in epithelia, concomitant with enhanced barrier restitution. Furthermore, knockdown of IL-10R1 in intestinal epithelial cells results in impaired barrier function in vitro. Colonic tissue isolated from murine colitis revealed that levels of IL-10R1 and suppressor of cytokine signaling 3 were increased in the epithelium and coincided with increased tissue IFN-γ and IL-10 cytokines. In parallel, studies showed that treatment of mice with rIFN-γ was sufficient to drive expression of IL-10R1 in the colonic epithelium. Studies of dextran sodium sulfate colitis in intestinal epithelial-specific IL-10R1-null mice revealed a remarkable increase in disease susceptibility associated with increased intestinal permeability. Together, these results provide novel insight into the crucial and underappreciated role of epithelial IL-10 signaling in the maintenance and restitution of epithelial barrier and of the temporal regulation of these pathways by IFN-γ.
Asunto(s)
Células Epiteliales/metabolismo , Interferón gamma/farmacología , Subunidad alfa del Receptor de Interleucina-10/biosíntesis , Interleucina-10/fisiología , Mucosa Intestinal/metabolismo , Animales , Línea Celular , Polaridad Celular , Colitis/inducido químicamente , Colitis/metabolismo , Citocinas/biosíntesis , Citocinas/genética , Sulfato de Dextran/toxicidad , Dextranos/farmacocinética , Células Epiteliales/efectos de los fármacos , Células Epiteliales/ultraestructura , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/farmacocinética , Regulación de la Expresión Génica , Humanos , Interferón gamma/biosíntesis , Interferón gamma/genética , Interferón gamma/fisiología , Subunidad alfa del Receptor de Interleucina-10/genética , Ratones , Ratones Endogámicos C57BL , Permeabilidad , Proteínas Recombinantes/farmacología , Factor de Transcripción STAT3/metabolismo , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/biosíntesis , Proteínas Supresoras de la Señalización de Citocinas/genéticaRESUMEN
IL-37, a newly described member of the IL-1 family, functions as a fundamental inhibitor of innate inflammation and immunity. In the present study, we examined a role for IL-37 during experimental colitis. A transgenic mouse strain was generated to express human IL-37 (hIL-37tg), and these mice were subjected to dextran sulfate sodium (DSS)-induced colitis. Despite the presence of a CMV promoter to drive expression of IL-37, mRNA transcripts were not present in colons at the resting state. Expression was observed only upon disruption of the epithelial barrier, with a six- to sevenfold increase (P = 0.02) on days 3 and 5 after continuous exposure to DSS. During the development of colitis, clinical disease scores were reduced by 50% (P < 0.001), and histological indices of colitis were one-third less in hIL-37tg mice compared with WT counterparts (P < 0.001). Reduced inflammation was associated with decreased leukocyte recruitment into the colonic lamina propria. In addition, release of IL-1ß and TNFα from ex vivo colonic explant tissue was decreased 5- and 13-fold, respectively, compared with WT (P ≤ 0.005), whereas IL-10 was increased sixfold (P < 0.001). However, IL-10 was not required for the anti-inflammatory effects of IL-37 because IL-10-receptor antibody blockade did not reverse IL-37-mediated protection. Mechanistically, IL-37 originating from hematopoietic cells was sufficient to exert anti-inflammatory effects because WT mice reconstituted with hIL-37tg bone marrow were protected from colitis. Thus, IL-37 emerges as key modulator of intestinal inflammation.
Asunto(s)
Colitis/inmunología , Interleucina-1/metabolismo , Análisis de Varianza , Animales , Colitis/patología , Sulfato de Dextran , Citometría de Flujo , Humanos , Interleucina-1/inmunología , Interleucina-1beta/metabolismo , Ratones , Ratones Transgénicos , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: The earliest endoscopically-evident lesion in Crohn's disease is the aphthous ulcer, which develops over ectopic lymphoid tissues (ie, inducible lymphoid follicles (ILF), tertiary lymphoid tissue (TLT)) in the chronically inflamed intestine. ILF/TLT are induced within effector sites by homeostatic lymphoid chemokines, but their role in the development of intestinal ILF/TLT and in the pathogenesis of Crohn's disease is poorly understood. DESIGN: Using a mouse model of Crohn's-like ileitis (TNFARE) which develops florid induction of ILF/TLT within its terminal ileum, the contribution of the CCR7/CCL19/CCL21 chemokine axis during the development of TLT and its role in disease pathogenesis were assessed. RESULTS: Both CCL19 and CCL21 were increased within the inflamed ileum of TNFARE mice, which resulted in CCR7 internalisation and impaired T cell chemotaxis. ILF/TLT were a major source of CCL19 and CCL21 and increased local synthesis, augmented recruitment/retention of effector, naïve and central memory T cell subsets within the inflamed ileum. Immunoblockade of CCR7 resulted in further effector T cell retention and exacerbation of ileitis. CONCLUSIONS: Induction of ILF/TLT in the chronically inflamed intestine alters the homeostatic CCL19-CCL21 lymphoid-chemokine gradient and increases recruitment/retention of effector CCR7+ T cell subsets within the terminal ileum, contributing to the perpetuation of chronic inflammation. Thus, blockade of CCR7 or its ligands might result in deleterious consequences for subjects with chronic inflammatory diseases.
Asunto(s)
Quimiocina CCL19/metabolismo , Quimiocina CCL21/metabolismo , Coristoma/inmunología , Enfermedad de Crohn/inmunología , Ileítis/inmunología , Receptores CCR7/metabolismo , Subgrupos de Linfocitos T/metabolismo , Animales , Biomarcadores/metabolismo , Quimiotaxis de Leucocito , Coristoma/patología , Enfermedad de Crohn/patología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Ileítis/patología , Tejido Linfoide , Ratones , Ratones Mutantes , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The prevalence of inflammatory bowel disease (IBD) has increased dramatically in recent years, particularly in pediatric populations. Successful remission with current therapies is limited and often transient, leading patients to seek alternative therapies for symptom relief, including the use of medical marijuana (Cannabis sativa). However, chronic cannabis use among IBD patients is associated with increased risk for surgical interventions. Therefore, determining the direct impact of cannabis use on immune modulation in IBD patients is of critical importance. Peripheral blood mononuclear cells of cannabis using and non-using pediatric IBD patients were phenotyped by flow cytometry and functionally assessed for their cytokine production profile. A phospho-kinase array was also performed to better understand changes in immune responses. Results were then compared with serum phytocannabinoid profiles of each patient to identify cannabinoid-correlated changes in immune responses. Results demonstrated elevated levels of a myriad of pro-inflammatory cytokines in users versus non-users. Differences in signaling cascades of activated T cells between users and non-users were also observed. A number of anti-inflammatory cytokines were inversely correlated with serum phytocannabinoids. These results suggest that cannabis exposure, which can desensitize cannabinoid receptors, may prime pro-inflammatory pathways in pediatric IBD patients.
RESUMEN
The heat shock response is a critical component of the inflammatory cascade that prevents misfolding of new proteins and regulates immune responses. Activation of clusters of differentiation (CD)4+ T cells causes an upregulation of heat shock transcription factor, heat shock factor 1 (HSF1). We hypothesized that HSF1 promotes a pro-regulatory phenotype during inflammation. To validate this hypothesis, we interrogated cell-specific HSF1 knockout mice and HSF1 transgenic mice using in vitro and in vivo techniques. We determined that while HSF1 expression was induced by anti-CD3 stimulation alone, the combination of anti-CD3 and transforming growth factor ß, a vital cytokine for regulatory T cell (Treg) development, resulted in increased activating phosphorylation of HSF1, leading to increased nuclear translocation and binding to heat shock response elements. Using chromatin immunoprecipitation (ChIP), we demonstrate the direct binding of HSF1 to foxp3 in isolated murine CD4+ T cells, which in turn coincided with induction of FoxP3 expression. We defined that conditional knockout of HSF1 decreased development and function of Tregs and overexpression of HSF1 led to increased expression of FoxP3 along with enhanced Treg suppressive function. Adoptive transfer of CD45RBHigh CD4 colitogenic T cells along with HSF1 transgenic CD25+ Tregs prevented intestinal inflammation when wild-type Tregs did not. Finally, overexpression of HSF1 provided enhanced barrier function and protection from murine ileitis. This study demonstrates that HSF1 promotes Treg development and function and may represent both a crucial step in the development of induced regulatory T cells and an exciting target for the treatment of inflammatory diseases with a regulatory T-cell component. SIGNIFICANCE STATEMENT: The heat shock response (HSR) is a canonical stress response triggered by a multitude of stressors, including inflammation. Evidence supports the role of the HSR in regulating inflammation, yet there is a paucity of data on its influence in T cells specifically. Gut homeostasis reflects a balance between regulatory clusters of differentiation (CD)4+ T cells and pro-inflammatory T-helper (Th)17 cells. We show that upon activation within T cells, heat shock factor 1 (HSF1) translocates to the nucleus, and stimulates Treg-specific gene expression. HSF1 deficiency hinders Treg development and function and conversely, HSF1 overexpression enhances Treg development and function. While this work, focuses on HSF1 as a novel therapeutic target for intestinal inflammation, the findings have significance for a broad range of inflammatory conditions.
Asunto(s)
Inflamación , Linfocitos T Reguladores , Animales , Ratones , Factores de Transcripción Forkhead/genética , Factores de Transcripción del Choque Térmico/genética , Respuesta al Choque Térmico , Ratones Noqueados , Ratones TransgénicosRESUMEN
Tissues of the mucosa are lined by an epithelium that provides barrier and transport functions. It is now appreciated that inflammatory responses in inflammatory bowel diseases are accompanied by striking shifts in tissue metabolism. In this paper, we examined global metabolic consequences of mucosal inflammation using both in vitro and in vivo models of disease. Initial analysis of the metabolic signature elicited by inflammation in epithelial models and in colonic tissue isolated from murine colitis demonstrated that levels of specific metabolites associated with cellular methylation reactions are significantly altered by model inflammatory systems. Furthermore, expression of enzymes central to all cellular methylation, S-adenosylmethionine synthetase and S-adenosylhomocysteine hydrolase, are increased in response to inflammation. Subsequent studies showed that DNA methylation is substantially increased during inflammation and that epithelial NF-κB activity is significantly inhibited following treatment with a reversible S-adenosylhomocysteine hydrolase inhibitor, DZ2002. Finally, these studies demonstrated that inhibition of cellular methylation in a murine model of colitis results in disease exacerbation while folate supplementation to promote methylation partially ameliorates the severity of murine colitis. Taken together, these results identify a global change in methylation, which during inflammation, translates to an overall protective role in mucosal epithelia.
Asunto(s)
Colitis/metabolismo , Inflamación/metabolismo , Mucosa Intestinal/metabolismo , Metabolómica/métodos , Adenina/análogos & derivados , Adenina/farmacología , Adenosilhomocisteinasa/genética , Adenosilhomocisteinasa/metabolismo , Animales , Western Blotting , Butiratos/farmacología , Línea Celular Tumoral , Colitis/genética , Colon/efectos de los fármacos , Colon/metabolismo , Colon/patología , Metilación de ADN/efectos de los fármacos , Sulfato de Dextran/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Perfilación de la Expresión Génica/métodos , Células HeLa , Humanos , Inflamación/genética , Interferón gamma/metabolismo , Interferón gamma/farmacología , Mucosa Intestinal/patología , Espectroscopía de Resonancia Magnética , Metionina Adenosiltransferasa/genética , Metionina Adenosiltransferasa/metabolismo , Metilación/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Mucositis/genética , Mucositis/metabolismo , FN-kappa B/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
UNLABELLED: BACKGROUND; Imprinting an effector or regulatory phenotype on naïve T cells requires education at induction sites by dendritic cells (DC). Objectives To analyse the effect of inflammation on the frequency of mononuclear phagocytes (MP) and the effect of altering their frequency by administration of Flt3-L in chronic ileitis. METHODS: Using a tumour necrosis factor (TNF) driven model of ileitis (ie, TNFΔARE) that recapitulates many features of Crohn's disease (CD), dynamic changes in the frequency and functional state of MP within the inflamed ileum were assessed by flow cytometry, immunofluorescence and real-time reverse-transcription PCR and by generating CX(3)CR1 GFP-reporter TNFΔARE mice. The effect of Flt3-L supplementation on the severity of ileitis, and the frequency of CD103(+) DC and of FoxP3(+) regulatory T cells was also studied in TNFΔARE mice. RESULTS: CD11c(Hi)/MHCII(+) MP accumulated in inflamed ilea, predominantly mediated by expansion of the CX(3)CR1(+) MP subpopulation. This coincided with a decreased pro-regulatory CD103(+) DC. The phenotype of these MP was that of activated cells, as they expressed increased CD80 and CD86 on their surface. Flt3-ligand administration resulted in a preferential expansion of CD103(+) DC that attenuated the severity of ileitis in 20-week-old TNFΔARE mice, mediated by increased CD4(+)/CD25(+)/FoxP3(+) regulatory T cells. CONCLUSIONS: Results support a role for Flt3-L as a potential therapeutic agent in Crohn's-like ileitis.
Asunto(s)
Antígenos CD/biosíntesis , Enfermedad de Crohn/tratamiento farmacológico , Células Dendríticas/inmunología , Factores de Transcripción Forkhead/biosíntesis , Ileítis/tratamiento farmacológico , Cadenas alfa de Integrinas/biosíntesis , Linfocitos T Reguladores/inmunología , Tirosina Quinasa 3 Similar a fms/farmacología , Animales , Antígenos CD/genética , Enfermedad de Crohn/genética , Enfermedad de Crohn/inmunología , Células Dendríticas/patología , Modelos Animales de Enfermedad , Citometría de Flujo , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica , Ileítis/genética , Ileítis/inmunología , Íleon/inmunología , Íleon/metabolismo , Íleon/patología , Cadenas alfa de Integrinas/genética , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T Reguladores/patología , Resultado del TratamientoRESUMEN
BACKGROUND: Inflammatory bowel diseases, encompassing Crohn's disease and ulcerative colitis, are characterised by persistent leucocyte tissue infiltration leading to perpetuation of an inappropriate inflammatory cascade. The neuronal guidance molecule netrin-1 has recently been implicated in the orchestration of leucocyte trafficking during acute inflammation. We therefore hypothesised that netrin-1 could modulate leucocyte infiltration and disease activity in a model of inflammatory bowel disease. DESIGN: DSS-colitis was performed in mice with partial genetic netrin-1 deficiency (Ntn-1(+/-) mice) or wild-type mice treated with exogenous netrin-1 via osmotic pump to examine the role of endogenous and therapeutically administered netrin-1. These studies were supported by in vitro models of transepithelial migration and intestinal epithelial barrier function. RESULTS: Consistent with our hypothesis, we observed induction of netrin-1 during intestinal inflammation in vitro or in mice exposed to experimental colitis. Moreover, mice with partial netrin-1 deficiency demonstrated an exacerbated course of DSS-colitis compared to littermate controls, with enhanced weight loss and colonic shortening. Conversely, mice treated with exogenous mouse netrin-1 experienced attenuated disease severity. Importantly, permeability studies and quantitative assessment of apoptosis reveal that netrin-1 signalling events do not alter mucosal permeability or intestinal epithelial cell apoptosis. In vivo studies of leucocyte transmigration demonstrate suppression of neutrophil trafficking as a key function mediated by endogenous or exogenously administered netrin-1. Finally, genetic studies implicate the A2B adenosine receptor in netrin-1-mediated protection during DSS-colitis. CONCLUSIONS: The present study identifies a previously unrecognised role for netrin-1 in attenuating experimental colitis through limitation of neutrophil trafficking.
Asunto(s)
Colitis/metabolismo , Mucosa Intestinal/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Infiltración Neutrófila , Proteínas Supresoras de Tumor/metabolismo , Enfermedad Aguda , Animales , Biomarcadores/metabolismo , Línea Celular , Colitis/inmunología , Modelos Animales de Enfermedad , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/metabolismo , Mucosa Intestinal/inmunología , Ratones , Ratones Endogámicos C57BL , Factores de Crecimiento Nervioso/administración & dosificación , Netrina-1 , Permeabilidad , Migración Transendotelial y Transepitelial , Proteínas Supresoras de Tumor/administración & dosificaciónRESUMEN
BACKGROUND & AIMS: Retinoic acid (RA), produced by intestinal epithelial cells (IECs) and dendritic cells (DCs) promotes the induction of regulatory T cells (Tregs) and decreases the induction of T-helper (Th)17 cells. METHODS: We studied the roles of RA in mice that overproduce tumor necrosis factor (TNF) and develop chronic ileitis (TNF_ARE mice). We assessed the frequency and function of CD103+ DCs, Th17 cells, and Tregs by flow cytometry, and we measured expression of cytokines and retinaldehyde dehydrogenase (RALDH) enzymes in ileum samples, DCs, and IECs by real-time polymerase chain reaction. We quantified RA by electrochemical analysis and examined the effect of RA supplementation on TNF-induced ileitis using histologic, coculture, and suppression assays and flow cytometry. RESULTS: Numbers of CD103+ DCs decreased in the inflamed ilea of mice with chronic disease; RA synthetic machinery (RALDH1,2) was down-regulated. Nevertheless, the proportion of CD4+, CD25+, FoxP3+ Tregs increased, indicating an alternate source for RA. IECs responded to reduced levels of RA by up-regulating RALDH3 in vivo and in vitro. Net tissue levels of RA remained lower in TNF+ARE than wild-type mice, indicating that epithelial up-regulation of RALDH3 could not maintain adequate concentrations of RA, probably because of loss of IEC mass. RA supplementation significantly attenuated disease by increasing the number and function of CD103+ DCs and Tregs and reducing Th17 cells. CONCLUSIONS: Reduced levels of RA appear to induce IECs to up-regulate synthesis of RA. RA supplementation attenuates ileitis through its effects on CD103+ DCs, Tregs, and Th17 cells. RA supplementation might offer therapeutic benefit in Crohn's disease.
Asunto(s)
Ileítis/tratamiento farmacológico , Ileítis/patología , Linfocitos T Reguladores/patología , Células Th17/patología , Tretinoina/uso terapéutico , Animales , Antígenos CD/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Citocinas/metabolismo , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Células Dendríticas/patología , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/metabolismo , Cadenas alfa de Integrinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/metabolismo , Células Th17/efectos de los fármacos , Tretinoina/farmacologíaRESUMEN
BACKGROUND: The mucus gel layer (MGL) lining the colon is integral to exclusion of bacteria and maintaining intestinal homeostasis in health and disease. Some MGL defects allowing bacteria to directly contact the colonic surface are commonly observed in ulcerative colitis (UC). The major macromolecular component of the colonic MGL is the secreted gel-forming mucin MUC2, whose expression is essential for homeostasis in health. In UC, another gel-forming mucin, MUC5AC, is induced. In mice, Muc5ac is protective during intestinal helminth infection. Here we tested the expression and functional role of MUC5AC/Muc5ac in UC biopsies and murine colitis. METHODS: We measured MUC5AC/Muc5ac expression in UC biopsies and in dextran sulfate sodium (DSS) colitis. We performed DSS colitis in mice deficient in Muc5ac (Muc5ac-/-) to model the potential functional role of Muc5ac in colitis. To assess MGL integrity, we quantified bacterial-epithelial interaction and translocation to mesenteric lymph nodes. Antibiotic treatment and 16S rRNA gene sequencing were performed to directly investigate the role of bacteria in murine colitis. RESULTS: Colonic MUC5AC/Muc5ac mRNA expression increased significantly in active UC and murine colitis. Muc5ac-/- mice experienced worsened injury and inflammation in DSS colitis compared with control mice. This result was associated with increased bacterial-epithelial contact and translocation to the mesenteric lymph nodes. However, no change in microbial abundance or community composition was noted. Antibiotic treatment normalized colitis severity in Muc5ac-/- mice to that of antibiotic-treated control mice. CONCLUSIONS: MUC5AC/Muc5ac induction in the acutely inflamed colon controls injury by reducing bacterial breach of the MGL.
Asunto(s)
Colitis Ulcerosa/genética , Colitis/genética , Colon/metabolismo , Mucosa Intestinal/metabolismo , Mucina 5AC/metabolismo , Animales , Bacterias/genética , Colitis/inducido químicamente , Colitis/microbiología , Colitis Ulcerosa/microbiología , Colitis Ulcerosa/patología , Colon/microbiología , Sulfato de Dextran , Modelos Animales de Enfermedad , Homeostasis , Humanos , Mucosa Intestinal/microbiología , Ratones , Factores Protectores , ARN Ribosómico 16SRESUMEN
BACKGROUND & AIMS: Lymphocyte recruitment to sites of inflammation requires the sequential engagement of adhesion molecules and chemokine receptors. In the current studies we analyzed the role of CD44 for the development of chronic small-intestinal inflammatory infiltrates in vivo. METHODS: By using a tumor necrosis factor (TNF)-driven model of chronic ileitis (ie, B6.129P-TNF(DeltaAU-rich element [ARE])) that recapitulates many features of Crohn's disease, we noticed dynamic changes in the expression and functional state of CD44 and its ligand hyaluronan via enzyme-linked immunosorbent assay, real-time reverse-transcription polymerase chain reaction, immunohistochemistry, and flow cytometry. In addition, we assessed the role of lymphocyte populations during induction of ileitis through adoptive transfer studies, and generated CD44-deficient TNFDeltaARE mice to assess the role of CD44 for development of ileitis. RESULTS: Soluble hyaluronan levels and expression of hyaluronan synthase-1 were increased in TNFDeltaARE mice. This coincided with increased expression of CD44 (including variant 7) and reactivity towards hyaluronan on CD4(+) T cells. CD44 was spatially colocalized with the gut-homing integrin alpha(4)beta(7), spatially linking lymphocyte rolling with arrest. These cells had an effector phenotype because they lacked L-selectin and a higher proportion in diseased mice produced TNF and interleukin-2 compared with wild-type littermates. Lastly, CD4(+) but not CD8(+) T cells conferred ileitis to RAG(-/-) recipients and deficiency of one or both alleles of the CD44 gene resulted in attenuation of the severity of ileitis in TNFDeltaARE mice. CONCLUSIONS: Our findings support an important role of CD44 expressed by CD4(+) and CD8(+) for development of ileitis mediated by TNF overproduction.
Asunto(s)
Receptores de Hialuranos/metabolismo , Ileítis/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Enfermedad Crónica , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Expresión Génica , Receptores de Hialuranos/genética , Receptores de Hialuranos/inmunología , Ileítis/metabolismo , Ileítis/patología , Íleon/metabolismo , Íleon/patología , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Pro-inflammatory cytokine TNFα antagonizes regulatory T cell (Treg) suppressive function with a measurable reduction of IL-10 protein secretion. Tregs are critical to suppress excessive immune activation, particularly within the intestine where high antigenic loads elicit chronic subclinical immune activation. Employing a TNFα-driven murine inflammatory bowel disease (IBD) model (TNFΔARE/+), which mirrors the Treg expansion and transmural ileitis seen in Crohn's disease, we demonstrate that the TNFα-mediated loss of Treg suppressive function coincides with induction of a specific miRNA, miR-106a in both humans and mice, via NFκB promoter binding to suppress post-transcriptional regulation of IL-10 release. Elevation of miR-106a and impaired Treg function in this model recapitulate clinical data from IBD patients. MiR-106a deficiency promotes Treg induction, suppressive function and IL-10 production in vitro. MiR-106a knockout attenuated chronic murine ileitis, whereas T cell restricted deficiency of miR-106a attenuated adoptive transfer colitis. In both models, attenuated inflammation coincided with suppression of both Th1 and Th17 cell subset expansion within the intestinal lamina propria. Collectively, our data demonstrate impaired Treg suppressive function in a murine IBD model consistent with human disease and support the potential for inhibition of miR-106a as a future therapeutic approach to treat chronic inflammatory conditions including IBD.
Asunto(s)
Inflamación/genética , Enfermedades Inflamatorias del Intestino/genética , Mucosa Intestinal/inmunología , MicroARNs/genética , Linfocitos T Reguladores/inmunología , Células TH1/inmunología , Células Th17/inmunología , Animales , Hidrolasas de Éster Carboxílico/genética , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Inflamación/inmunología , Enfermedades Inflamatorias del Intestino/inmunología , Interleucina-10/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/genética , Regiones Promotoras Genéticas/genética , Procesamiento Proteico-Postraduccional , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
BACKGROUND: Epidural analgesia with levobupivacaine and bupivacaine is a common and effective method of labor pain relief. However, its use is associated with an increased instrumental delivery rate. One of the mechanisms postulated to account for this unwanted effect is the direct effect of local anesthetics on myometrial contractility. We determined the effects of bupivacaine and levobupivacaine on the amplitude and frequency of contractions of human term myometrium. METHODS: Uterine specimens were obtained from nonlaboring parturients scheduled for elective lower-segment cesarean delivery at term. Longitudinal muscle strips were prepared and mounted vertically in tissue chambers, and changes in the amplitude (peak force) and the frequency of contractions were recorded. Spontaneous contractions commenced after a period of application of 1 g (9.81 mN) of tension to the myometrial strips. No uterotonic drugs were used. The muscle strips were then exposed to cumulative concentrations of bupivacaine and levobupivacaine and dose-response curves were generated. RESULTS: Both bupivacaine and levobupivacaine decreased the amplitude of contractions in human myometrium in a concentration-dependent manner, reaching significance at 1x10(-4) M for both bupivacaine and levobupivacaine compared with the internal control amplitude. With both drugs, the decrease in amplitude was accompanied by an increase in the frequency of contractions reaching significance at 3x10(-5) M for both bupivacaine and levobupivacaine compared with the internal control frequency. CONCLUSIONS: The concentrations required for the effects on amplitude are much higher (33 fold) than the clinically relevant plasma concentrations of these drugs after epidural administration, and are unlikely to be significant in the setting of low-dose epidural analgesia in labor.
Asunto(s)
Anestésicos Locales/farmacología , Bupivacaína/farmacología , Miometrio/efectos de los fármacos , Contracción Uterina/efectos de los fármacos , Bupivacaína/análogos & derivados , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Técnicas In Vitro , Levobupivacaína , EmbarazoRESUMEN
BACKGROUND AND AIMS: Inflammatory Bowel Diseases [IBDs] are chronic intestinal inflammatory conditions in part mediated by CD4+ T cells. Anti-inflammatory Foxp3+ regulatory T cells [Tregs] maintain immune homeostasis and protect against IBD development via multiple mechanisms, including cytokine secretion and cell-cell interaction. CCAAT enhancer binding protein-beta [C/EBPß] is a stress-responsive transcription factor linked with IBD susceptibility. Whole-body C/EBPß deficiency induces CD4+ T cell-predominant hyperproliferation, and we hypothesize that this may be due to impaired Treg function. METHODS: We used the C/EBPß-/- mice in the CD45RBHigh adoptive transfer model, to assess C/EBPß-/- CD4+ T cells for their colitiogenic potential, and C/EBPß-/- CD4+ Foxp3+ Tregs for their ability to inhibit colitis. We assessed Tregs from the C/EBPß-/- mice for expression of Treg functional genes and proteins. RESULTS: Naïve C/EBPß-/- CD4+ T cells are more colitogenic in vivo. The exacerbated colitis does not appear to reflect impaired Treg development, however, as C/EBPß-/- mice displayed more, rather than fewer intestinal CD4+Foxp3+ Tregs in vivo. Instead, this reflects impaired Treg function as seen by the reduced capacity to suppress T cell proliferation in vitro, along with decreased secretion of the anti-inflammatory cytokine IL-10. These findings were corroborated in vivo by additional adoptive co-transfer studies in which wildtype Tregs prevented colitis but C/EBPß-/- Tregs did not. CONCLUSION: C/EBPß deficiency impairs Treg function and potentiates T cell-mediated colitis. A clearer understanding of the function of this transcription factor may provide a novel therapeutic strategy for IBD.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Linfocitos T CD4-Positivos/inmunología , Colitis , Inflamación , Enfermedades Inflamatorias del Intestino , Linfocitos T Reguladores/inmunología , Animales , Colitis/inmunología , Colitis/patología , Citocinas/metabolismo , Eliminación de Gen , Inflamación/inmunología , Inflamación/patología , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/patología , Ratones , Transducción de SeñalRESUMEN
Intestinal epithelial barrier repair is vital for remission in inflammatory bowel disease (IBD). Extracellular adenosine signaling has been implicated in promoting restoration of epithelial barrier function. Currently, no clinically approved agents target this pathway. Adenosine signaling is terminated by uptake from the extracellular space via equilibrative nucleoside transporters (ENTs). We hypothesized that ENT inhibition could dampen intestinal inflammation. Initial studies demonstrated transcriptional repression of ENT1 and ENT2 in IBD biopsies or in murine IBD models. Subsequent studies in mice with global Ent1 or Ent2 deletion revealed selective protection of Ent2-/- mice. Elevated intestinal adenosine levels in conjunction with abolished protection following pharmacologic blockade of A2B adenosine receptors implicate adenosine signaling as the mechanism of gut protection in Ent2-/- mice. Additional studies in mice with tissue-specific deletion of Ent2 uncovered epithelial Ent2 as the target. Moreover, intestinal protection provided by a selective Ent2 inhibitor was abolished in mice with epithelium-specific deletion of Ent2 or the A2B adenosine receptor. Taken together, these findings indicate that increased mucosal A2B signaling following repression or deletion of epithelial Ent2 coordinates the resolution of intestinal inflammation. This study suggests the presence of a targetable purinergic network within the intestinal epithelium designed to limit tissue inflammation.
Asunto(s)
Colitis Ulcerosa/patología , Enfermedad de Crohn/patología , Transportador Equilibrativo 2 de Nucleósido/metabolismo , Mucosa Intestinal/patología , Receptor de Adenosina A2B/metabolismo , Adenosina/metabolismo , Antagonistas del Receptor de Adenosina A2/administración & dosificación , Animales , Biopsia , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/inmunología , Colon/efectos de los fármacos , Colon/inmunología , Colon/patología , Enfermedad de Crohn/inmunología , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Tranportador Equilibrativo 1 de Nucleósido/genética , Tranportador Equilibrativo 1 de Nucleósido/metabolismo , Transportador Equilibrativo 2 de Nucleósido/antagonistas & inhibidores , Transportador Equilibrativo 2 de Nucleósido/genética , Femenino , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/inmunología , Masculino , Ratones , Ratones Noqueados , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunologíaRESUMEN
BACKGROUND: Inflammatory bowel disease (IBD) is a lifelong disease of the gastrointestinal tract whose annual incidence and prevalence is on the rise. Current immunosuppressive therapies available for treatment of IBD offer limited benefits and lose effectiveness, exposing a significant need for the development of novel therapies. In the clinical setting, cannabis has been shown to provide patients with IBD symptomatic relief, although the underlying mechanisms of their anti-inflammatory effects remain unclear. METHODS: This review reflects our current understanding of how targeting the endocannabinoid system, including cannabinoid receptors 1 and 2, endogenous cannabinoids anandamide and 2-arachidonoylglycerol, atypical cannabinoids, and degrading enzymes including fatty acid amide hydrolase and monoacylglycerol lipase, impacts murine colitis. In addition, the impact of cannabinoids on the human immune system is summarized. RESULTS: Cannabinoid receptors 1 and 2, endogenous cannabinoids, and atypical cannabinoids are upregulated in inflammation, and their presence and stimulation attenuate murine colitis, whereas cannabinoid receptor antagonism and cannabinoid receptor deficient models reverse these anti-inflammatory effects. In addition, inhibition of endocannabinoid degradation through monoacylglycerol lipase and fatty acid amide hydrolase blockade can also attenuate colitis development, and is closely linked to cannabinoid receptor expression. CONCLUSIONS: Although manipulation of the endocannabinoid system in murine colitis has proven to be largely beneficial in attenuating inflammation, there is a paucity of human study data. Further research is essential to clearly elucidate the specific mechanisms driving this anti-inflammatory effect for the development of therapeutics to target inflammatory disease such as IBD.
Asunto(s)
Antiinflamatorios/farmacología , Colitis/tratamiento farmacológico , Colitis/fisiopatología , Endocannabinoides/fisiología , Marihuana Medicinal/farmacología , Animales , Cannabinoides/metabolismo , Humanos , Inflamación , Ratones , Receptores de Cannabinoides/metabolismoRESUMEN
BACKGROUND AND AIMS: Inflammatory bowel disease [IBD] is characterised by a disruption of immune homeostasis, which is tightly regulated to protect against harmful pathogens yet not react to commensal antigens. Animal studies indicate that regulatory T cells [Treg] modulate the immune response to prevent IBD development. Lactoferrin [LF] is an endogenous anti-inflammatory pleiotropic protein secreted at high concentrations in colostrum and at mucosal sites. However, the effect of LF on specific T lymphocyte populations has not been studied. Here, we identify a novel mechanism by which a recombinant human LF, VEN-120, regulates T cell populations in health and disease. METHODS: Two murine models of intestinal inflammation, the dextran sodium sulphate colitis model and the TNFΔARE/+ model of ileitis, were used to study the anti-inflammatory and T cell modulating ability of VEN-120. Flow cytometry was used to evaluate T cell populations within the lamina propria and mesenteric lymph nodes, and to evaluate the effect of VEN-120 on CD4+ T cells in vitro. RESULTS: VEN-120 reduced inflammation in both models of IBD, accompanied by increased Tregs in the intestinal lamina propria. Treatment of CD4+ T cells in vitro resulted in an upregulation of Treg genes and skewing towards a Treg population. This in vitro T cell skewing translated to an increase of Treg homing to the intestinal lamina propria and associated lymph tissue in healthy mice. CONCLUSIONS: These data provide a novel immunological mechanism by which VEN-120 modulates T cells to restrict inflammatory T cell-driven disease.