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BACKGROUND& AIMS: The protection provided by rotavirus (RV) vaccines is highly heterogeneous amongst individuals. We hypothesized that microbiota composition might influence RV vaccine efficacy. METHODS: First, we examined the potential of segmented filamentous bacteria (SFB) colonization to influence RV vaccine efficacy in mice. Next, we probed the Influence of human microbiomes on RV vaccination via administering mice fecal microbial transplants (FMT) from children with robust or minimal RV vaccine responsiveness. Post-FMT, mice were subjected to RV vaccination followed by RV challenge. RESULTS: SFB colonization induced a phenotype that was reminiscent of RV vaccine failure, i.e. failure to generate RV antigens and, consequently, anti-RV antibodies following RV vaccination resulting in proneness to RV challenge after SFB levels diminished. FMT from children to mice recapitulated donor vaccination phenotype. Specifically, mice receiving FMT from high-responsive vaccinees copiously shed RV antigens and robustly generated anti-RV antibodies following RV vaccination. Concomitantly, such mice were impervious to RV challenge. In contrast, mice receiving FMT from children who had not responded to RV vaccination exhibited only modest responses to RV vaccination and, concomitantly, remained prone to RV challenge. Microbiome analysis ruled out a role for SFB but suggested involvement of Clostridium perfringens. Oral administration of cultured C. perfringens to gnotobiotic mice partially recapitulated the RV vaccine non-responder phenotype. Analysis of published microbiome data found C. perfringens abundance in children modestly associated with RV vaccine failure. CONCLUSION: Microbiota composition influences RV vaccine efficacy with C. perfringens being one, perhaps of many, potential contributing taxa.
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Background & Aims: The extent to which live orally-administered rotavirus (RV) vaccines elicit protective immunity is highly heterogeneous. We hypothesized microbiota composition might influence vaccine efficacy. Methods: We tested this concept by examining extent to which colonizing mice with segmented filamentous bacteria (SFB) influenced RV vaccine efficacy.Influence of human microbiomes on RV vaccination was studied via administering germ-free mice fecal microbial transplants (FMT) from children with robust or minimal RV vaccine responsiveness. Post-FMT, mice were subjected to vaccination and challenge doses of RV. Results: SFB administration resulted in a phenotype reminiscent of RV vaccine failure, i.e. minimal generation of RV antigens and, consequently, lack of anti-RV antibodies resulting in proneness to RV challenge once SFB levels diminished. Transplant of microbiomes from children to mice recapitulated donor vaccination phenotype. Specifically, mice receiving FMT from high-responding children exhibited high levels of fecal RV antigen shedding and RV antibodies in response to RV vaccination and, concomitantly, were impervious to RV challenge. In contrast, mice receiving FMT from children who had not responded to RV vaccination exhibited only modest responses to RV challenge and, accordingly, remained prone to RV challenge. Microbiome analysis ruled out a role for SFB but suggested that RV vaccine failure might involve Clostridium perfringens . Oral administration of cultured C. perfringens to gnotobiotic mice partially recapitulated the RV vaccine non-responder phenotype. Analysis of previously-reported microbiome data found C. perfringens abundance in children associated with RV vaccine failure. Conclusion: Microbiota composition influences RV vaccine virus infection and, consequently, protective immunity. C. perfringens may be one, perhaps of many, bacterial species harbored in the intestine of RV-vaccine non-responders that influences RV vaccine outcomes.
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Prevention of rotavirus (RV) infection by gut-resident segmented filamentous bacteria (SFB) is an example of the influence of gut microbiota composition on enteric viral infection. Yet, the mechanism by which SFB prevents RV infection is poorly understood. A recent report that SFB colonization of germfree mice generates retinoic acid (RA) thus activating RA receptor (RAR) signaling, which protected against Citrobacter rodentium infection, prompted us to investigate whether this pathway might contribute to SFB's protection against RV infection. Colonization of conventional mice by SFB indeed increased intestinal RA levels and direct administration of RA partially mimicked the protection against RV infection conferred by SFB. Moreover, blockade of RAR signaling eliminated SFB's protection against RV infection. Blockade of RAR signaling did not impact RV infection in the absence of SFB, nor did it alter the protection against RV infection conferred by bacterial flagellin, which in contrast to SFB, is dependent upon IL-22 signaling. SFB/RA-mediated prevention of RV infection was associated with an RA-dependent increase in enterocyte migration, consistent with the notion that enhanced anoikis is the ultimate means by which SFB, IL-22, and RA impede RV infection.
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Microbioma Gastrointestinal , Infecciones por Rotavirus , Ratones , Animales , Infecciones por Rotavirus/prevención & control , Intestino Delgado , Bacterias , FlagelinaRESUMEN
BACKGROUND & AIMS: Nourishment of gut microbiota via consumption of fermentable fiber promotes gut health and guards against metabolic syndrome. In contrast, how dietary fiber impacts type 1 diabetes is less clear. METHODS: To examine impact of dietary fibers on development of type 1 diabetes in the streptozotocin (STZ)-induced and spontaneous non-obese diabetes (NOD) models, mice were fed grain-based chow (GBC) or compositionally defined diets enriched with a fermentable fiber (inulin) or an insoluble fiber (cellulose). Spontaneous (NOD mice) or STZ-induced (wild-type mice) diabetes was monitored. RESULTS: Relative to GBC, low-fiber diets exacerbated STZ-induced diabetes, whereas diets enriched with inulin, but not cellulose, strongly protected against or treated it. Inulin's restoration of glycemic control prevented loss of adipose depots, while reducing food and water consumption. Inulin normalized pancreatic function and markedly enhanced insulin sensitivity. Such amelioration of diabetes was associated with alterations in gut microbiota composition and was eliminated by antibiotic administration. Pharmacologic blockade of fermentation reduced inulin's beneficial impact on glycemic control, indicating a role for short-chain fatty acids (SCFA). Furthermore, inulin's microbiota-dependent anti-diabetic effect associated with SCFA-independent restoration of interleukin 22, which was necessary and sufficient to ameliorate STZ-induced diabetes. Inulin-enriched diets significantly delayed diabetes in NOD mice. CONCLUSIONS: Fermentable fiber confers microbiota-dependent increases in SCFA and interleukin 22 that, together, may have potential to prevent and/or treat type 1 diabetes.
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Bacterias/clasificación , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Fibras de la Dieta/administración & dosificación , Ácidos Grasos Volátiles/metabolismo , Interleucinas/metabolismo , Inulina/administración & dosificación , Animales , Bacterias/efectos de los fármacos , Bacterias/genética , Bacterias/aislamiento & purificación , Diabetes Mellitus Tipo 1/inducido químicamente , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/microbiología , Fibras de la Dieta/farmacología , Modelos Animales de Enfermedad , Microbioma Gastrointestinal/efectos de los fármacos , Hemoglobina Glucada/metabolismo , Inulina/farmacología , Masculino , Ratones , Ratones Endogámicos NOD , Tamaño de los Órganos/efectos de los fármacos , Páncreas/efectos de los fármacos , Páncreas/fisiopatología , Estreptozocina/efectos adversos , Resultado del Tratamiento , Interleucina-22RESUMEN
BACKGROUND: Bacterial flagellin is a major target of innate and adaptive immunity, both of which can promote and/or compensate for deficiencies in each other's function. METHODS: To investigate the role of innate immune detection of flagellin irrespective of adaptive immunity, we examined the consequences of loss of Toll-like receptor 5 (T5) and/or Nod-like receptor 4 (N4) upon a Rag1-deficient background. RESULTS: Mice lacking Toll-like receptor 5 and Rag1 (T5/Rag-DKO) exhibited frequent lethal Pasteurellaceae-containing abscesses that prevented breeding of these mice. Mice lacking Toll-like receptor 5, Nod-like receptor 4, and Rag1 (T5/N4/Rag-TKO) also resulted in sporadic lethal abdominal abscesses caused by similar Pasteurellaceae. In the absence of such infections, relative to Rag1-KO, T5/N4/Rag-TKO mice exhibited microbiota encroachment, low-grade inflammation, microbiota dysbiosis, and, moreover were highly prone to Citrobacter infection and developed severe colitis when adoptively transferred with colitogenic T cells. Relative proneness of T5/N4/Rag-TKO mice to T-cell colitis was ablated by antibiotics while fecal microbiota transplant from T5/N4/Rag-TKO mice to wild-type mice transferred proneness to Citrobacter infection, indicating that dysbiosis in T5/N4/Rag-TKO mice contributed to these phenotypes. CONCLUSIONS: These results demonstrate a critical role for innate immune detection of flagellin, especially in the intestinal tract and particularly in hosts deficient in adaptive immunity.
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Colitis , Flagelina/inmunología , Proteínas de Homeodominio , Inmunidad Innata , Proteínas NLR , Receptor Toll-Like 5 , Inmunidad Adaptativa , Animales , Disbiosis , Proteínas de Homeodominio/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas NLR/genética , Receptor Toll-Like 5/genéticaRESUMEN
Bacterial flagellin can elicit production of TLR5-mediated IL-22 and NLRC4-mediated IL-18 cytokines that act in concert to cure and prevent rotavirus (RV) infection. This study investigated the mechanism by which these cytokines act to impede RV. Although IL-18 and IL-22 induce each other's expression, we found that IL-18 and IL-22 both impeded RV independently of one another and did so by distinct mechanisms that involved activation of their cognate receptors in intestinal epithelial cells (IEC). IL-22 drove IEC proliferation and migration toward villus tips, which resulted in increased extrusion of highly differentiated IEC that serve as the site of RV replication. In contrast, IL-18 induced cell death of RV-infected IEC thus directly interrupting the RV replication cycle, resulting in spewing of incompetent virus into the intestinal lumen and causing a rapid drop in the number of RV-infected IEC. Together, these actions resulted in rapid and complete expulsion of RV, even in hosts with severely compromised immune systems. These results suggest that a cocktail of IL-18 and IL-22 might be a means of treating viral infections that preferentially target short-lived epithelial cells.
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Anoicis/inmunología , Interleucina-18/metabolismo , Interleucinas/metabolismo , Mucosa Intestinal/patología , Infecciones por Rotavirus/inmunología , Animales , Movimiento Celular/inmunología , Proliferación Celular , Modelos Animales de Enfermedad , Células Epiteliales/inmunología , Células Epiteliales/patología , Células Epiteliales/virología , Femenino , Humanos , Interleucina-18/genética , Interleucina-18/inmunología , Interleucina-18/uso terapéutico , Interleucinas/genética , Interleucinas/inmunología , Interleucinas/uso terapéutico , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Mucosa Intestinal/virología , Masculino , Ratones , Ratones Noqueados , Rotavirus/inmunología , Infecciones por Rotavirus/tratamiento farmacológico , Infecciones por Rotavirus/virología , Transducción de Señal/inmunología , Interleucina-22RESUMEN
Segmented filamentous bacteria (SFB) are well appreciated for eliciting Th17 cell immune responses. Here, we report the genome sequence of a murine isolate of SFB, which confers strong protection against rotavirus infection independent of acquired immunity.
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Rotavirus (RV) encounters intestinal epithelial cells amidst diverse microbiota, opening possibilities of microbes influencing RV infection. Although RV clearance typically requires adaptive immunity, we unintentionally generated RV-resistant immunodeficient mice, which, we hypothesized, reflected select microbes protecting against RV. Accordingly, such RV resistance was transferred by co-housing and fecal transplant. RV-protecting microbiota were interrogated by heat, filtration, and antimicrobial agents, followed by limiting dilution transplant to germ-free mice and microbiome analysis. This approach revealed that segmented filamentous bacteria (SFB) were sufficient to protect mice against RV infection and associated diarrhea. Such protection was independent of previously defined RV-impeding factors, including interferon, IL-17, and IL-22. Colonization of the ileum by SFB induced changes in host gene expression and accelerated epithelial cell turnover. Incubation of RV with SFB-containing feces reduced infectivity in vitro, suggesting direct neutralization of RV. Thus, independent of immune cells, SFB confer protection against certain enteric viral infections and associated diarrheal disease.
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Inmunidad Adaptativa/genética , Diarrea/microbiología , Mucosa Intestinal/microbiología , Infecciones por Rotavirus/microbiología , Animales , Antiinfecciosos/farmacología , Bacterias/genética , Bacterias/metabolismo , Diarrea/prevención & control , Diarrea/virología , Heces/microbiología , Regulación de la Expresión Génica/genética , Humanos , Íleon/microbiología , Íleon/patología , Íleon/virología , Interferones/genética , Interleucina-17/genética , Interleucinas/genética , Mucosa Intestinal/patología , Mucosa Intestinal/virología , Ratones , Microbiota/genética , Rotavirus/patogenicidad , Infecciones por Rotavirus/prevención & control , Infecciones por Rotavirus/virología , Interleucina-22RESUMEN
Most viruses first encounter host cells at mucosal surfaces, which are typically colonized by a complex ecosystem of microbes collectively referred to as the microbiota. Recent studies demonstrate the microbiota plays an important role in mediating host-viral interactions and determining the outcomes of these encounters. This review outlines recently described examples of how bacteria and viruses impact each other particularly during infectious processes. Mechanistically, these effects can be broadly categorized as reflecting direct bacterial-viral interactions and/or involving microbial impacts upon innate and/or adaptive immunity.
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Infecciones Bacterianas/inmunología , Fenómenos Fisiológicos Bacterianos , Interacciones Huésped-Patógeno/inmunología , Interacciones Microbianas/inmunología , Virosis/inmunología , Fenómenos Fisiológicos de los Virus , Animales , Antibiosis , Microbioma Gastrointestinal , Humanos , Sistema Inmunológico/citología , Sistema Inmunológico/inmunología , Sistema Inmunológico/metabolismoRESUMEN
Activators of innate immunity may have the potential to combat a broad range of infectious agents. We report that treatment with bacterial flagellin prevented rotavirus (RV) infection in mice and cured chronically RV-infected mice. Protection was independent of adaptive immunity and interferon (IFN, type I and II) and required flagellin receptors Toll-like receptor 5 (TLR5) and NOD-like receptor C4 (NLRC4). Flagellin-induced activation of TLR5 on dendritic cells elicited production of the cytokine interleukin-22 (IL-22), which induced a protective gene expression program in intestinal epithelial cells. Flagellin also induced NLRC4-dependent production of IL-18 and immediate elimination of RV-infected cells. Administration of IL-22 and IL-18 to mice fully recapitulated the capacity of flagellin to prevent or eliminate RV infection and thus holds promise as a broad-spectrum antiviral agent.
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Flagelina/administración & dosificación , Inmunidad Innata , Interleucina-18/inmunología , Interleucinas/inmunología , Infecciones por Rotavirus/prevención & control , Receptor Toll-Like 5/fisiología , Animales , Diarrea/inmunología , Diarrea/terapia , Diarrea/virología , Modelos Animales de Enfermedad , Heces/virología , Flagelina/inmunología , Proteínas de Homeodominio/genética , Interleucina-18/administración & dosificación , Interleucina-18/genética , Interleucinas/administración & dosificación , Interleucinas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Mutación , Infecciones por Rotavirus/inmunología , Infecciones por Rotavirus/terapia , Receptor Toll-Like 5/genética , Esparcimiento de Virus , Interleucina-22RESUMEN
The Kir4.1 channel is expressed in the brainstem, retina and kidney where it acts on K(+) transportation and pH-dependent membrane potential regulation. Its heteromerization with Kir5.1 leads to K(+) currents with distinct properties such as single-channel conductance, rectification, pH sensitivity and phosphorylation modulation. Here we show that Kir5.1 also enables S-glutathionylation to the heteromeric channel. Expressed in HEK cells, an exposure to the oxidant H(2)O(2) or diamide produced concentration-dependent inhibitions of the whole-cell Kir4.1-Kir5.1 currents. In inside-out patches, currents were inhibited strongly by a combination of diamide/GSH or H(2)O(2)/GSH but not by either alone. The currents were also suppressed by GSSG and the thiol oxidants pyridine disulfides (PDSs), suggesting S-glutathionylation. In contrast, none of the exposures had significant effects on the homomeric Kir4.1 channel. Cys158 in the TM2 helix of Kir5.1 was critical for the S-glutathionylation, which was accessible to intracellular but not extracellular oxidants. Site-directed mutagenesis of this residue (C158A or C158T) abolished the Kir4.1-Kir5.1 current modulation by oxidants, and eliminated almost completely the biochemical interaction of Kir5.1 with GSH. In tandem Kir4.1-Kir5.1 constructs, the channel with a single Cys158 was inhibited to the same degree as the wild-type channel, suggesting that one glutathione moiety is sufficient to block the channel. Consistent with the location of Cys158, GSSG inhibited the channel only when the channel was open, indicating that the channel inhibition was state dependent. The finding that the heteromeric Kir4.1-Kir5.1 channel but not the homomeric Kir4.1 is subject to the S-glutathionylation thus suggests a novel Kir4.1-Kir5.1 channel modulation mechanism that is likely to occur in oxidative stress.
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Glutatión/fisiología , Estrés Oxidativo/fisiología , Canales de Potasio de Rectificación Interna/fisiología , Células HEK293 , Humanos , Oxidantes/farmacología , Canales de Potasio de Rectificación Interna/antagonistas & inhibidores , Canales de Potasio de Rectificación Interna/química , Conformación Proteica , Compuestos de Sulfhidrilo/farmacologíaRESUMEN
BACKGROUND AND PURPOSE: Rosiglitazone is an anti-diabetic drug acting as an insulin sensitizer. We recently found that rosiglitazone also inhibits the vascular isoform of ATP-sensitive K(+) channels and compromises vasodilatory effects of ß-adrenoceptor activation and pinacidil. As its potency for the channel inhibition is in the micromolar range, rosiglitazone may be used as an effective K(ATP) channel inhibitor for research and therapeutic purposes. Therefore, we performed experiments to determine whether other isoforms of K(ATP) channels are also sensitive to rosiglitazone and what their sensitivities are. EXPERIMENTAL APPROACH: K(IR) 6.1/SUR2B, K(IR) 6.2/SUR1, K(IR) 6.2/SUR2A, K(IR) 6.2/SUR2B and K(IR) 6.2ΔC36 channels were expressed in HEK293 cells and were studied using patch-clamp techniques. KEY RESULTS: Rosiglitazone inhibited all isoforms of K(ATP) channels in excised patches and in the whole-cell configuration. Its IC(50) was 10 µmol·L(-1) for the K(IR) 6.1/SUR2B channel and â¼45 µmol·L(-1) for K(IR) 6.2/SURx channels. Rosiglitazone also inhibited K(IR) 6.2ΔC36 channels in the absence of the sulphonylurea receptor (SUR) subunit, with potency (IC(50) = 45 µmol·L(-1) ) almost identical to that for K(IR) 6.2/SURx channels. Single-channel kinetic analysis showed that the channel inhibition was mediated by augmentation of the long-lasting closures without affecting the channel open state and unitary conductance. In contrast, rosiglitazone had no effect on K(IR) 1.1, K(IR) 2.1 and K(IR) 4.1 channels, suggesting that the channel inhibitory effect is selective for K(IR) 6.x channels. CONCLUSIONS AND IMPLICATIONS: These results suggest a novel K(ATP) channel inhibitor that acts on the pore-forming K(IR) 6.x subunit, affecting the channel gating.