RESUMEN
While toll-like receptors (TLRs), which mediate innate immunity, are known to play an important role in host defense, recent work suggest their involvement in some integrated behaviors, including anxiety, depressive and cognitive functions. Here, we investigated the potential involvement of the flagellin receptor, TLR5, in anxiety, depression and cognitive behaviors using male TLR5 knock-out (KO) mice. We aobserved a specific low level of basal anxiety in TLR5 KO mice with an alteration of the hypothalamo-pituitary axis (HPA) response to acute restraint stress, illustrated by a decrease of both plasma corticosterone level and c-fos expression in the hypothalamic paraventricular nucleus where TLR5 was expressed, compared to WT littermates. However, depression and cognitive-related behaviors were not different between TLR5 KO and WT mice. Nor there were significant changes in the expression of some cytokines (IL-6, IL-10 and TNF-α) and other TLRs (TLR2, TLR3 and TLR4) in the prefrontal cortex, amygdala and hippocampus of TLR5 KO mice compared to WT mice. Moreover, mRNA expression of BDNF and glucocorticoid receptors in the hippocampus and amygdala, respectively, was not different. Finally, acute intracerebroventricular administration of flagellin, a specific TLR5 agonist, or chronic neomycin treatment did not exhibit a significant main effect, only a significant main effect of genotype was observed between TLR5 KO and WT mice. Together, those findings suggest a previously undescribed and specific role of TLR5 in anxiety and open original prospects in our understanding of the brain-gut axis function.
Asunto(s)
Ansiedad , Receptor Toll-Like 5 , Animales , Ansiedad/genética , Trastornos de Ansiedad , Corticosterona , Masculino , Ratones , Ratones Noqueados , Receptor Toll-Like 5/genéticaRESUMEN
Epithelia of the vertebrate intestinal tract characteristically maintain an inflammatory hyporesponsiveness toward the lumenal prokaryotic microflora. We report the identification of enteric organisms (nonvirulent Salmonella strains) whose direct interaction with model human epithelia attenuate synthesis of inflammatory effector molecules elicited by diverse proinflammatory stimuli. This immunosuppressive effect involves inhibition of the inhibitor kappaB/nuclear factor kappaB (IkappaB/NF-kappaB) pathway by blockade of IkappaB-alpha degradation, which prevents subsequent nuclear translocation of active NF-kappaB dimer. Although phosphorylation of IkappaB-alpha occurs, subsequent polyubiquitination necessary for regulated IkappaB-alpha degradation is completely abrogated. These data suggest that prokaryotic determinants could be responsible for the unique tolerance of the gastrointestinal mucosa to proinflammatory stimuli.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas I-kappa B , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , FN-kappa B/metabolismo , Salmonella/fisiología , Transactivadores , Núcleo Celular/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas del Citoesqueleto/metabolismo , Dimerización , Humanos , Mediadores de Inflamación/farmacología , Interleucina-8/genética , Interleucina-8/metabolismo , Leupeptinas/farmacología , Ligasas/metabolismo , Inhibidor NF-kappaB alfa , FN-kappa B/genética , Fosforilación , Salmonella/patogenicidad , Salmonella typhimurium/patogenicidad , Salmonella typhimurium/fisiología , Factor de Transcripción ReIA , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacología , Ubiquitina-Proteína Ligasas , Ubiquitinas/metabolismo , beta CateninaRESUMEN
Adenosine is formed in the intestinal lumen during active inflammation from neutrophil-derived 5' AMP. Using intestinal epithelial cell line T84, we studied the effect of adenosine on the secretion of IL-6, a proinflammatory cytokine involved in neutrophil degranulation and lymphocyte differentiation. Stimulation of T84 monolayers with either apical or basolateral adenosine induces A2b receptor-mediated increase in IL-6 secretion, which is polarized to the apical (luminal) compartment. In addition, Salmonella typhimurium, TNF-alpha, and forskolin, known inducers of IL-6 secretion in intestinal epithelial cells, also stimulate IL-6 secretion into the apical compartment. We show that IL6 promoter induction by adenosine occurs through cAMP-mediated activation of nuclear cAMP-responsive element-binding protein (CREB). We also show that IL-6 released in the luminal (apical) compartment achieves a sufficient concentration to activate neutrophils (from which the adenosine signal originates), since such IL-6 is found to induce an intracellular [Ca(++)] flux in neutrophils. We conclude that adenosine released in the intestinal lumen during active inflammation may induce IL-6 secretion, which is mediated by cAMP/CREB activation and occurs in an apically polarized fashion. This would allow sequential activation of neutrophil degranulation in the lumen -- a flow of events that would, in an epithelium-dependent fashion, enhance microbicidal activity of neutrophils as they arrive in the intestinal lumen.
Asunto(s)
Adenosina/metabolismo , Interleucina-6/metabolismo , Mucosa Intestinal/metabolismo , Neutrófilos/metabolismo , Transducción de Señal/fisiología , Factores de Transcripción Activadores , Adenosina/farmacología , Animales , Proteínas Sanguíneas/metabolismo , Células COS , Línea Celular , Chlorocebus aethiops , Colforsina/metabolismo , Colforsina/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Células Epiteliales/citología , Células Epiteliales/metabolismo , Mucosa Intestinal/citología , Antagonistas de Receptores Purinérgicos P1 , Receptor de Adenosina A2B , Receptores Purinérgicos P1/metabolismo , Salmonella typhimurium/metabolismo , Factores de Tiempo , Factores de Transcripción/metabolismo , Transcripción Genética , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Interactions between the enteric pathogen Salmonella typhimurium and the luminal surface of the intestine provoke an acute inflammatory response, mediated in part by epithelial cell secretion of the chemokine IL-8 and other proinflammatory molecules. This study investigated the mechanism by which this pathogen induces IL-8 secretion in physiologically polarized model intestinal epithelia. IL-8 secretion induced by both the prototypical proinflammatory cytokine TNF-alpha and S. typhimurium was NF-kappaB dependent. However, NF-kappaB activation and IL-8 secretion induced by S. typhimurium, but not by TNF-alpha, was preceded by and required an increase in intracellular [Ca(2+)]. Additionally, agonists that increased intracellular [Ca(2+)] by receptor-dependent (carbachol) or independent (thapsigargin, ionomycin) means also induced IL-8 secretion. Furthermore, the ability of S. typhimurium mutants to induce IkappaB-alpha degradation, NF-kappaB translocation, and IL-8 transcription and secretion correlated precisely with their ability to induce an intracellular [Ca(2+)] increase in model intestinal epithelia, but not with their ability to invade these cells. Finally, S. typhimurium, but not TNF-alpha, induced a Ca(2+)-dependent phosphorylation of IkappaB-alpha. These results indicate that S. typhimurium-induced activation of NF-kappaB-dependent epithelial inflammatory responses proceeds by a Ca(2+)-mediated activation of an IkappaB-alpha kinase. These observations raise the possibility that pharmacologic intervention of the acute inflammatory response can be selectively matched to the specific class of initiating event.
Asunto(s)
Calcio/fisiología , Proteínas I-kappa B , Interleucina-8/biosíntesis , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , FN-kappa B/fisiología , Salmonella typhimurium/fisiología , Proteínas de Unión al ADN/fisiología , Humanos , Interleucina-1/farmacología , Inhibidor NF-kappaB alfa , Fosforilación , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Enteric pathogens induce intestinal epithelium to secrete chemokines that direct movement of polymorphonuclear leukocytes. Mechanisms that might downregulate secretion of these proinflammatory chemokines and thus contain intestinal inflammation have not yet been elucidated. The antiinflammatory activities exhibited by the arachidonate metabolite lipoxin A4 (LXA4) suggests that this eicosanoid, which is biosynthesized in vivo at sites of inflammation, might play such a role. We investigated whether chemokine secretion could be regulated by stable analogs of LXA4. Monolayers of T84 intestinal epithelial cells were infected with Salmonella typhimurium, which elicits secretion of distinct apical (pathogen-elicited epithelial chemoattractant) and basolateral (IL-8) chemokines. Stable analogs of LXA4 inhibited S. typhimurium-induced (but not phorbol ester-induced) secretion of both IL-8 and pathogen-elicited epithelial chemoattractant. LXA4 stable analogs did not alter bacterial adherence to nor internalization by epithelia, indicating that LXA4 stable analogs did not block all signals that Salmonella typhimurium activates in intestinal epithelia, but likely led to attenuation of signals that mediate chemokine secretion. Inhibition of S. typhimurium-induced IL-8 secretion by LXA4 analogs was concentration- (IC50 approximately 1 nM) and time-dependent (maximal inhibition approximately 1 h). As a result of these effects, LXA4 stable analogs inhibited the ability of bacteria-infected epithelia to direct polymorphonuclear leukocyte movement. These data suggest that LXA4 and its stable analogs may be useful in downregulating active inflammation at mucosal surfaces.
Asunto(s)
Quimiocinas/metabolismo , Ácidos Hidroxieicosatetraenoicos/química , Ácidos Hidroxieicosatetraenoicos/farmacología , Mucosa Intestinal/efectos de los fármacos , Lipoxinas , Salmonella typhimurium/patogenicidad , Células Cultivadas , Factores Quimiotácticos/farmacología , Quimiotaxis de Leucocito/efectos de los fármacos , Estabilidad de Medicamentos , Interleucina-8/metabolismo , Mucosa Intestinal/citología , Neutrófilos/inmunología , Salmonella typhimurium/inmunología , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Intestinal epithelial cells express hPepT1, an apical transporter responsible for the uptake of a broad array of small peptides. As these could conceivably include n-formyl peptides, we examined whether hPepT1 could transport the model n-formylated peptide fMLP and, if so, whether such cellular uptake of fMLP influenced neutrophil-epithelial interactions. fMLP uptake into oocytes was enhanced by hPepT1 expression. In addition, fMLP competitively inhibited uptake of a known hPepT1 substrate (glycylsarcosine) in hPepT1 expressing oocytes. hPepT1 peptide uptake was further examined in a polarized human intestinal epithelial cell line (Caco2-BBE) known to express this transporter. Epithelial monolayers internalized apical fMLP in a fashion that was competitively inhibited by other hPepT1 recognized solutes, but not by related solutes that were not transported by hPepT1. Fluorescence analyses of intracellular pH revealed that fMLP uptake was accompanied by cytosolic acidification, consistent with the known function of hPepT1 as a peptide H+ cotransporter. Lumenal fMLP resulted in directed movement of neutrophils across epithelial monolayers. Solutes that inhibit hPepT1-mediated fMLP transport decreased neutrophil transmigration by approximately 50%. Conversely, conditions that enhanced the rate of hPepT1-mediated fMLP uptake (cytosolic acidification) enhanced neutrophil-transepithelial migration by approximately 70%. We conclude that hPepT1 transports fMLP and uptake of these peptide influences neutrophil-epithelial interactions. These data (a) emphasize the importance of hPepT1 in mediating intestinal inflammation, (b) raise the possibility that modulating hPepT1 activity could influence states of intestinal inflammation, and (c) provide the first evidence of a link between active transepithelial transport and neutrophil-epithelial interactions.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Portadoras/fisiología , Factores Quimiotácticos/metabolismo , Células Epiteliales/efectos de los fármacos , Escherichia coli/metabolismo , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Simportadores , Animales , Calcio/metabolismo , Quimiotaxis de Leucocito/efectos de los fármacos , Neoplasias del Colon/patología , Dipéptidos/metabolismo , Células Epiteliales/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Transporte Iónico/efectos de los fármacos , Proteínas de Neoplasias/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Transportador de Péptidos 1 , Células Tumorales Cultivadas , Xenopus laevisRESUMEN
This study investigated whether soluble paracrine factors mediated Salmonella-induced IL-8 expression in polarized model intestinal epithelia. We found that the basolateral media of model epithelia that had been apically infected with Salmonella typhimurium for a short period (10 minutes) could activate IL-8 secretion in virgin model epithelia, demonstrating that a proinflammatory factor (PIF) was indeed present. Initial characterization found that PIF was a heat-stable protein with a molecular mass of about 50 kDa that acts on the basolateral, but not apical, surface of model intestinal epithelia to elicit IL-8 secretion. PIF was not present in the media of model epithelia stimulated with other inducers of IL-8 secretion (TNF-alpha or carbachol) but was present in S. typhimurium supernatants, indicating PIF is of bacterial origin. PIF was purified from bacterial culture supernatants by anion/cation exchange chromatography and SDS-PAGE and found by using microsequencing to be the protein flagellin. In support of this finding, flagellin-deficient S. typhimurium mutants did not secrete detectable levels of PIF (i.e., a bioactivity that induced IL-8 secretion when placed basolaterally on model epithelia). Furthermore, viable flagellin-deficient mutant organisms (fliC/fljB and flhD) failed to elicit IL-8 secretion when added apically to model intestinal epithelia. These findings indicate that translocation of flagellin across epithelia, subsequent to apical epithelial-S. typhimurium interaction, is likely a major means of activating a mucosal inflammatory response.
Asunto(s)
Flagelina/metabolismo , Inflamación/etiología , Mucosa Intestinal/microbiología , Salmonella typhimurium/patogenicidad , Línea Celular , Epitelio/inmunología , Epitelio/microbiología , Flagelina/genética , Humanos , Inflamación/inmunología , Inflamación/microbiología , Mediadores de Inflamación/metabolismo , Interleucina-8/metabolismo , Mucosa Intestinal/inmunología , Modelos Biológicos , Mutación , Salmonella typhimurium/genética , Salmonella typhimurium/fisiologíaRESUMEN
Regulatory and effector T helper (Th) cells are abundant at mucosal surfaces, especially in the intestine, where they control the critical balance between tolerance and inflammation. However, the key factors that reciprocally dictate differentiation along these specific lineages remain incompletely understood. Here we report that the interleukin-1 (IL-1) family member IL-36γ signals through IL-36 receptor, myeloid differentiation primary response gene 88, and nuclear factor-κBp50 in CD4+ T cells to potently inhibit Foxp3-expressing induced regulatory T cell (Treg) development, while concomitantly promoting the differentiation of Th9 cells via a IL-2-STAT5- (signal transducer and activator of transcription factor 5) and IL-4-STAT6-dependent pathway. Consistent with these findings, mice deficient in IL-36γ were protected from Th cell-driven intestinal inflammation and exhibited increased colonic Treg cells and diminished Th9 cells. Our findings thus reveal a fundamental contribution for the IL-36/IL-36R axis in regulating the Treg-Th9 cell balance with broad implications for Th cell-mediated disorders, such as inflammatory bowel diseases and particularly ulcerative colitis.
Asunto(s)
Colitis Ulcerosa/inmunología , Colon/inmunología , Receptores de Interleucina-1/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Reguladores/inmunología , Animales , Benzofuranos , Diferenciación Celular , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/metabolismo , Humanos , Interleucina-2/metabolismo , Interleucina-9/metabolismo , Ratones , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Quinolinas , Receptores de Interleucina-1/genética , Factores de Transcripción STAT/metabolismo , Factor de Transcripción STAT5/metabolismo , Transducción de Señal , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismoRESUMEN
Specific components of the intestinal microbiota are capable of influencing immune responses such that a mutualistic relationship is established. In mice, colonization with segmented filamentous bacteria (SFB) induces T-helper-17 (Th17) cell differentiation in the intestine, yet the effector functions of interleukin (IL)-17A in response to SFB remain incompletely understood. Here we report that colonization of mice with SFB-containing microbiota induced IL-17A- and CXCR2-dependent recruitment of neutrophils to the ileum. This response required adaptive immunity, as Rag-deficient mice colonized with SFB-containing microbiota failed to induce IL-17A, CXCL1 and CXCL2, and displayed defective neutrophil recruitment to the ileum. Interestingly, neutrophil depletion in wild-type mice resulted in significantly augmented Th17 responses and SFB expansion, which correlated with impaired expression of IL-22 and antimicrobial peptides. These data provide novel insight into a dynamic IL-17A-CXCR2-neutrophil axis during acute SFB colonization and demonstrate a central role for neutrophils in limiting SFB expansion.
Asunto(s)
Bacterias/inmunología , Microbioma Gastrointestinal/inmunología , Íleon/inmunología , Interleucina-17/metabolismo , Neutrófilos/inmunología , Receptores de Interleucina-8B/metabolismo , Células Th17/inmunología , Inmunidad Adaptativa/genética , Animales , Péptidos Catiónicos Antimicrobianos/metabolismo , Bacterias/crecimiento & desarrollo , Diferenciación Celular , Movimiento Celular/genética , Células Cultivadas , Proteínas de Homeodominio/genética , Íleon/microbiología , Interleucinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Interleucina-22RESUMEN
Secretory IgA (SIgA) directed against gut resident bacteria enables the mammalian mucosal immune system to establish homeostasis with the commensal gut microbiota after weaning. Germinal centers (GCs) in Peyer's patches (PPs) are the principal inductive sites where naive B cells specific for bacterial antigens encounter their cognate antigens and receive T-cell help driving their differentiation into IgA-producing plasma cells. We investigated the role of antigen sampling by intestinal M cells in initiating the SIgA response to gut bacteria by developing mice in which receptor activator of nuclear factor-κB ligand (RANKL)-dependent M-cell differentiation was abrogated by conditional deletion of Tnfrsf11a in the intestinal epithelium. Mice without intestinal M cells had profound delays in PP GC maturation and emergence of lamina propria IgA plasma cells, resulting in diminished levels of fecal SIgA that persisted into adulthood. We conclude that M-cell-mediated sampling of commensal bacteria is a required initial step for the efficient induction of intestinal SIgA.
Asunto(s)
Linfocitos B/inmunología , Microbioma Gastrointestinal/inmunología , Centro Germinal/inmunología , Inmunoglobulina A Secretora/metabolismo , Mucosa Intestinal/fisiología , Ganglios Linfáticos Agregados/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Presentación de Antígeno , Antígenos Bacterianos/inmunología , Diferenciación Celular , Células Cultivadas , Homeostasis , Inmunidad Humoral , Inmunidad Mucosa , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor Activador del Factor Nuclear kappa-B/genética , SimbiosisRESUMEN
Neutrophils phagocytize high-valency immune complexes (HIC) by an Fc gamma receptor-mediated mechanism, activating an oxidative burst and initiating degranulation. In contrast, neutrophils exhibit chemotaxis to N-formylated peptides [e.g., N-formyl-methionyl-leucyl-phenylalanine (fMLP)] and secrete far fewer oxidants or granule contents than neutrophils activated by HIC. However, if neutrophils are treated with cytochalasin B (CB) or permeabilized with streptolysin O, chemoattractant-induced neutrophil secretion is increased to a level beyond that observed in response to HIC. Because priming neutrophils with CB, or permeabilizing them, also augments activation of phospholipase D (PLD) in response to fMLP, we reasoned that, in intact (i.e., nonpermeabilized) unprimed neutrophils, PLD may participate in a signaling pathway specific to phagocytic stimuli such as HIC and hence may contribute to degranulation control. PLD activity in response to HIC and fMLP correlated closely with stimulus-induced azurophilic degranulation under a wide variety of experimental conditions, including compounds that abrogated or augmented stimulus-induced PLD action. PLD activation preceded, and appeared to be necessary for, azurophilic degranulation. These results suggest that PLD may play a central role in controlling azurophilic degranulation and provide signaling specificity between pathways activated by fMLP and HIC in intact neutrophils.
Asunto(s)
Complejo Antígeno-Anticuerpo/fisiología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Activación Neutrófila/fisiología , Neutrófilos/fisiología , Fosfolipasa D/fisiología , Receptores de IgG/fisiología , Transducción de Señal/fisiología , Degranulación de la Célula/efectos de los fármacos , Degranulación de la Célula/fisiología , Citocalasina B/farmacología , Activación Enzimática , Humanos , Activación Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Fosfolipasa D/metabolismo , Sensibilidad y Especificidad , Transducción de Señal/efectos de los fármacos , Factores de TiempoRESUMEN
Neutrophils phagocytize high-valency immune complexes (HIC) by an Fc receptor-mediated mechanism. Engaging Fc receptors in this manner induces PMN to generate superoxide and release the contents of both their specific and azurophilic granules. Signaling events that precede and accompany PMN secretion include activation of phospholipase D (PLD), as well as changes in cytoplasmic [Ca2+] (delta[Ca2+]in) and pH (delta pHin). Although the role of PLD and delta[Ca2+]in in mediating Fc receptor-mediated PMN secretion has been studied, whether pHin plays a regulatory role has not yet been defined. HIC-stimulated PMN undergo an intracellular acidification followed by a prolonged Na+/H+ antiport-mediated alkalinization. To investigate the role of the pH transient in controlling degranulation, the Na+/H+ antiport was inhibited either with 100 microM dimethylamiloride (DMA) or by substituting N-methyl-glucamine for extracellular sodium. Blocking the antiport with DMA led to hyperacidified PMN, which exhibited an increase in degranulation, but did not affect generation of superoxide. DMA did not alter the ability of neutrophils to phagocytose and oxidize dichlorodihydrofluoresceinated HIC, suggesting the increase in degranulation was not the result of failed phagocytosis. Investigation into whether the observed increase in degranulation when the antiport was blocked was mediated by PLD or delta[Ca2+]in revealed that blocking the antiport increased HIC-induced PLD activity but had no effect on HIC-induced delta[Ca2+]in. Blocking the Na+/H+ antiport by ion substitution caused similar effects on PMN signaling and secretion as was seen with DMA. These results indicate that Na+/H+ antiport activity is not necessary for degranulation or superoxide release in HIC-stimulated PMN and that hyperacidification of the cytoplasm can modulate degranulation. Therefore, pHin, via its effect on PLD, may be a control point of degranulation and may represent one way that neutrophils achieve differential control of their antibacterial products.
Asunto(s)
Degranulación de la Célula/fisiología , Neutrófilos/enzimología , Neutrófilos/fisiología , Fosfolipasa D/metabolismo , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Amilorida/análogos & derivados , Amilorida/farmacología , Complejo Antígeno-Anticuerpo/farmacología , Degranulación de la Célula/efectos de los fármacos , Células Cultivadas , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/enzimología , Activación Enzimática , Humanos , Concentración de Iones de Hidrógeno , Neutrófilos/efectos de los fármacos , Superóxidos/metabolismoRESUMEN
Receptor-mediated activation of neutrophils (PMN) initiates possibly interdependent events, including a rapid transient increase in [Ca2+]i, implicated as a second messenger. To investigate whether this transient is required for eventual degranulation, PMN were incubated with an intracellular Ca2+ chelator (BAPTA), then exposed to chemotactic peptide [N-formyl-methionyl-leucyl-phenylalanine (fMLP)l with or without cytochalasin B (CB) or to high-valency immune complexes (HIC); delta[Ca2+]i, delta(pH)i, oxidative burst, and elastase release were then evaluated (plus or minus EGTA 15 s before stimulation) after 2 and 15 min incubation in 0.9 mM Ca2+. With either fMLP plus CB or HIC stimulation, BAPTA-treated cells were unable to achieve a Ca2+ transient with a 2-min incubation, whereas a 15-min incubation allowed the BAPTA-treated cells to recover a portion of the delta[Ca2+]i. Even though BAPTA-treated cells were unable to mount a delta[Ca2+]i at 2 min, HIC-stimulated BAPTA-treated cells were able to elicit an oxidative burst (33% of control) and degranulation (67% of control). Therefore, we conclude that delta[Ca2+]i modulates but is not required for oxidative burst or degranulation.
Asunto(s)
Complejo Antígeno-Anticuerpo , Calcio/metabolismo , Degranulación de la Célula , Neutrófilos/fisiología , Estallido Respiratorio , Citocalasina B/farmacología , Citosol/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Elastasa de Leucocito/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacología , Receptores de Formil Péptido , Receptores de IgG/fisiología , Receptores Inmunológicos/fisiología , Receptores de Péptidos/fisiología , Transducción de SeñalRESUMEN
Genetics Institute has developed and launched oprelvekin (rhIL-11; Neumega), a recombinant form of human IL-11. In November 1997, the FDA cleared oprelvekin for the prevention of severe thrombocytopenia and the reduction of the need for platelet transfusions following myelosuppressive chemotherapy in susceptible patients with non-myeloid malignancies 12703021. The product was launched at the end of 1997 [312556]. By December 1999, phase III trials for Crohn's disease (CD) were underway [363007]. Genetics Institute had commenced a 150-patient phase II trial for mild-to-moderate CD and mucositis and the company planned to file regulatory procedures for the indication of CD in 1999 [271210]. An oral formulation for this indication has been developed. Oprelvekin is also undergoing phase I clinical trials for colitis [396157], phase II clinical trials for rheumatoid arthritis [413835] and clinical trials for psoriasis [299644]. In March 1997, Wyeth-Ayerst became the licensee for Europe, Africa, Latin America and Asia (with the exception of Japan). Genetics Institute holds marketing rights for North America [239273]. In Japan, oprelvekin is being developed by Genetics Institute and Yamanouchi; phase III trials have commenced [295049] and were ongoing in May 2001 [411763]. In April 1996, analysts at Yamaichi estimated launch in 2001 and maximum annual sales of over yen 10 billion [215896]. In January 1998, Morgan Stanley Dean Witter predicted Yamanouchi's share of sales to be yen 1 billion in 2001, rising to yen 2 billion in 2002 [315458]. Sales of oprelvekin were US $34 million for Genetics institute in fiscal 2000 while, in July 2001, Credit Suisse First Boston estimated that this figure will be US $30 million and US $34 million in 2001 and 2002, respectively [416883].
Asunto(s)
Artritis Reumatoide/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Interleucina-11/uso terapéutico , Proteínas Recombinantes/uso terapéutico , Trombocitopenia/tratamiento farmacológico , Animales , Ensayos Clínicos Fase I como Asunto , Ensayos Clínicos Fase II como Asunto , Humanos , Interleucina-11/efectos adversos , Interleucina-11/metabolismo , Interleucina-11/farmacología , Interleucina-11/toxicidad , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/toxicidad , Relación Estructura-ActividadRESUMEN
Alicaforsen (ISIS-2302) is an RNase H-dependent antisense inhibitor of the intercellular adhesion molecule ICAM-1 under development by Isis Pharmaceuticals, for the potential treatment of a variety of inflammatory disorders [175741]. As of April 1997 it was in phase III trials for Crohn's disease (CD); however, the trial failed and, in December 1999, the company suspended development for this indication [352801]. In October 2000, the company re-initiated development in CD [384820] and new phase III trials had begin by May 2001 [409704]. In August 2000, phase II studies of alicaforsen in an enema formulation for ulcerative colitis and a topical formulation for psoriasis were ongoing [378715]. Development of the compound for the potential treatment of rheumatoid arthritis (RA) was discontinued in 1999 [347579]. By the end of 1998, alicaforsen was in phase II trials for kidney transplant rejection. At this time, these trials were expected to finish in mid-1999 [343460]. However, they were ongoing in September 1999, although no further development has been reported for this indication since that time [338672]. In February 1995, Isis Pharmaceuticals and Boehringer Ingelheim (BI) signed a collaborative agreement on cell adhesion inhibitors, including alicaforsen [174111]. By early 1999, Isis and BI were to decide on the next developmental step for alicaforsen following further analyses of its performance against CD [292915], [315439]. Their joint development agreement was terminated in 1999; Isis regained rights to the product and by September 1999 was in talks to license alicaforsen to another partner for CD [338672]. In June 2000, Cytogenix entered into a sponsored research agreement with Baylor College of Medicine at the Texas Medical Center Houston for the use of its ssDNA expression system for the development of antisense strategies directed against intercellular adhesion molecules for the purpose of reducing lung inflammation and injury in disease states and conditions [369677]. US-05514788, and other patents, cover antisense cell adhesion molecule inhibitors [212289], [234792].
Asunto(s)
Artritis Reumatoide/tratamiento farmacológico , Asma/tratamiento farmacológico , Colitis Ulcerosa/tratamiento farmacológico , Enfermedad de Crohn/tratamiento farmacológico , Fármacos Gastrointestinales/uso terapéutico , Rechazo de Injerto/prevención & control , Inmunosupresores/uso terapéutico , Oligodesoxirribonucleótidos Antisentido/farmacología , Oligodesoxirribonucleótidos Antisentido/uso terapéutico , Psoriasis/tratamiento farmacológico , Tionucleótidos/farmacología , Tionucleótidos/uso terapéutico , Animales , Ensayos Clínicos Fase I como Asunto , Ensayos Clínicos Fase II como Asunto , Ensayos Clínicos Fase III como Asunto , Fármacos Gastrointestinales/efectos adversos , Fármacos Gastrointestinales/metabolismo , Fármacos Gastrointestinales/farmacología , Fármacos Gastrointestinales/toxicidad , Humanos , Inmunosupresores/efectos adversos , Inmunosupresores/metabolismo , Inmunosupresores/farmacología , Inmunosupresores/toxicidad , Oligodesoxirribonucleótidos Antisentido/efectos adversos , Oligodesoxirribonucleótidos Antisentido/metabolismo , Oligodesoxirribonucleótidos Antisentido/toxicidad , Oligonucleótidos Fosforotioatos , Relación Estructura-Actividad , Tionucleótidos/efectos adversos , Tionucleótidos/metabolismo , Tionucleótidos/toxicidadRESUMEN
Bacterial flagellin is a dominant innate immune activator of the intestine. Therefore, we examined the role of the intracellular flagellin receptor, NLRC4, in protecting the gut and/or driving inflammation. In accordance with NLRC4 acting through transcription-independent pathways, loss of NLRC4 did not reduce the rapid robust changes in intestinal gene expression induced by flagellin administration. Loss of NLRC4 did not alter basal intestinal homeostasis nor predispose mice to development of colitis upon administration of an anti-interleukin (IL)-10R monoclonal antibody. However, epithelial injury induced by dextran sulfate sodium in mice lacking NLRC4 resulted in a more severe disease, indicating a role for NLRC4 in protecting the gut. Moreover, loss of NLRC4 resulted in increased mortality in response to flagellate, but not aflagellate Salmonella infection. Thus, despite not being involved in rapid intestinal gene remodeling upon detection of flagellin, NLRC4-mediated inflammasome activation results in production of IL-1ß and IL-18, two cytokines that protect mice from mucosal and systemic challenges.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de Unión al Calcio/metabolismo , Colitis/inmunología , Flagelina/metabolismo , Mucosa Intestinal/inmunología , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/inmunología , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/inmunología , Células Cultivadas , Colitis/inducido químicamente , Citocinas/genética , Citocinas/metabolismo , Sulfato de Dextran/administración & dosificación , Modelos Animales de Enfermedad , Flagelina/genética , Flagelina/inmunología , Humanos , Inmunidad Innata/genética , Ratones , Ratones Noqueados , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/inmunologíaRESUMEN
Toll-like receptor-5 (TLR5)-mediated detection of flagellin induces nuclear factor (NF)-κB-mediated transcription of host defense gene expression, whereas recognition of intracellular flagellin by interleukin (IL)-1-converting enzyme protease-activation factor (IPAF) results in maturation/secretion of the inflammasome cytokine IL-1ß. The potent effects of IL-1ß are counter-regulated by secretory IL-1 receptor antagonist (sIL-1Ra). We studied the roles of flagellin receptors in regulating the expression of IL-1ß and sIL-1Ra and their subsequent roles in inflammation. Flagellin induced sIL-1Ra in intestinal epithelia and macrophages in a dose- and time-dependent manner, whereas IL-1ß was only induced in macrophages. In vivo, flagellin-induced sIL-1Ra, but not IL-1ß, was absolutely dependent upon TLR5 expressed on non-hemopioetic cells. Thus, loss of TLR5 increased the IL-1ß/sIL-1Ra ratio on flagellin treatment, which correlated with increased inflammatory pathology in response to this product. Furthermore, the flagellin/TLR5 interaction was important for the induction of sIL-1Ra and limiting inflammatory pathology on Salmonella infection. Finally, reduced sIL-1Ra levels in TLR5KO mice correlated with spontaneous colitis. Taken together, we demonstrate that intestinal epithelia, despite not expressing IL-1ß, secrete sIL-1Ra in a TLR5-dependent manner suggesting that loss of TLR5 may promote inflammation by increasing IL-1ß activity. Thus, optimizing the balance between inflammasome cytokines and their endogenous inhibitors might prove a useful strategy to treat inflammatory disorders.
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Flagelina/inmunología , Inflamación/inmunología , Proteína Antagonista del Receptor de Interleucina 1/metabolismo , Interleucina-1beta/genética , Receptor Toll-Like 5/inmunología , Animales , Caspasa 1/metabolismo , Línea Celular , Colitis/inmunología , Ensayo de Inmunoadsorción Enzimática , Expresión Génica , Regulación de la Expresión Génica , Inflamasomas , Proteína Antagonista del Receptor de Interleucina 1/genética , Interleucina-1beta/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/genética , FN-kappa B/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Infecciones por Salmonella/inmunología , Receptor Toll-Like 5/metabolismoRESUMEN
Rotavirus (RV), a leading cause of severe diarrhea, primarily infects intestinal epithelial cells (IECs) causing self-limiting illness. To better understand innate immunity to RV, we sought to define the extent to which IEC activation of anti-viral responses required viral replication or could be recapitulated by inactivated RV or its components. Using model human intestinal epithelia, we observed that RV-induced activation of signaling events and gene expression typically associated with viral infection was largely mimicked by administration of ultraviolet (UV)-inactivated RV. Use of anti-interferon (IFN) neutralizing antibodies revealed that such replication-independent anti-viral gene expression required type I IFN signaling. In contrast, RV-induction of nuclear factor-κB-mediated interleukin-8 expression was dependent on viral replication. The anti-viral gene expression induced by UV-RV was not significantly recapitulated by RV RNA or RV virus-like particles although the latter could enter IEC. Together, these results suggest that RV proteins mediate viral entry into epithelial cells leading to intracellular detection of RV RNA that generates an anti-viral response.
Asunto(s)
Interferón Tipo I/metabolismo , Mucosa Intestinal/metabolismo , FN-kappa B/metabolismo , Infecciones por Rotavirus/inmunología , Rotavirus/fisiología , Anticuerpos Bloqueadores/farmacología , Línea Celular , Regulación Viral de la Expresión Génica/inmunología , Humanos , Inmunidad Innata , Interferón Tipo I/inmunología , Interleucina-8/biosíntesis , Interleucina-8/genética , Interleucina-8/inmunología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Mucosa Intestinal/virología , FN-kappa B/inmunología , ARN Viral/inmunología , Rotavirus/patogenicidad , Infecciones por Rotavirus/virología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Rayos Ultravioleta , Virión/inmunología , Inactivación de Virus , Replicación ViralAsunto(s)
Antiinflamatorios no Esteroideos/farmacología , Ácidos Hidroxieicosatetraenoicos/farmacología , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Lipoxinas , Animales , Humanos , Ácidos Hidroxieicosatetraenoicos/biosíntesis , Técnicas In Vitro , Enfermedades Inflamatorias del Intestino/etiología , Enfermedades Inflamatorias del Intestino/fisiopatologíaRESUMEN
The mucosal immune system is charged with defending the host's vast interfaces with the outside world from the enormous and diverse group of microbes that colonizes these surfaces. A key means by which the mucosal immune system protects the host from such diverse microbes is using germ-line-encoded receptors that target structurally conserved motifs that mediate important bacterial functions. This review focuses on one embodiment of this notion, namely, the mucosal innate immune targeting of flagellin, the primary structural component of flagella, which afford bacteria the ability of directed locomotion. Specifically, we discuss the mechanisms by which flagellin is recognized by the innate immune system, their role in host defense, chronic inflammatory disease, and potential approaches to pharmacologically manipulate these pathways to benefit the host. Discussion will focus on the intestinal tract but will also incorporate key findings in other mucosal surfaces.