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The currently observed high prevalence of allergic diseases has been associated with changes in microbial exposure in industrialized countries. Defined bacterial components represent a new strategy for modulating the allergic immune response. We show that intranasal administration of exopolysaccharide (EPS) isolated from Lacticaseibacillus (L.) rhamnosus LOCK900 induces TGF-ß1, IgA, and regulatory FoxP3+ T-cells in the lungs of naïve mice. Using the ovalbumin mouse model, we demonstrate that intranasal administration of EPS downregulates the development of allergic airway inflammation and the Th2 cytokine response in sensitized individuals. At the same time, EPS treatment of sensitized mice, similar to EPS-induced responses in naïve mice, significantly increased the level of total, OVA-specific, and also bacteria-specific IgA in bronchoalveolar lavage and the number of IgA-producing B-cells in the lung tissue of these mice. Thus, EPS derived from L. rhamnosus LOCK900 can be considered a safe candidate for preventing the development of allergic symptoms in the lungs of sensitized individuals upon exposure to an allergen.
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Hipersensibilidad , Lacticaseibacillus rhamnosus , Animales , Ratones , Lacticaseibacillus , Pulmón , Inflamación , Modelos Animales de Enfermedad , Inmunoglobulina A , Ovalbúmina , Ratones Endogámicos BALB C , Líquido del Lavado BronquioalveolarRESUMEN
Glucocorticoids (GCs) are hormones that are released in response to stressors and exhibit many activities, including immunomodulatory and anti-inflammatory activities. They are primarily synthesized in the adrenal gland but are also produced in peripheral tissues via regeneration of adrenal 11-oxo metabolites or by de novo synthesis from cholesterol. The present study investigated the influence of the microbiota on de novo steroidogenesis and regeneration of corticosterone in the intestine of germ-free (GF) and specific pathogen-free mice challenged with a physical stressor (anti-CD3 antibody i.p. injection). In the small intestine, acute immune stress resulted in increased mRNA levels of the proinflammatory cytokines IL1ß, IL6 and Tnfα and genes involved in de novo steroidogenesis (Stard3 and Cyp11a1), as well as in regeneration of active GCs from their 11-oxo metabolites (Hsd11b1). GF mice showed a generally reduced transcriptional response to immune stress, which was accompanied by decreased intestinal corticosterone production and reduced expression of the GC-sensitive marker Fkbp5. In contrast, the interaction between stress and the microbiota was not detected at the level of plasma corticosterone or the transcriptional response of adrenal steroidogenic enzymes. The results indicate a differential immune stress-induced intestinal response to proinflammatory stimuli and local corticosterone production driven by the gut microbiota.
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Corticosterona/metabolismo , Microbioma Gastrointestinal/fisiología , Intestino Delgado/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasas/genética , 11-beta-Hidroxiesteroide Deshidrogenasas/metabolismo , Animales , Masculino , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , Esteroides/metabolismo , Espectrometría de Masas en TándemRESUMEN
1. The underlying microbial metabolic activity toward xenobiotics is among the least explored factors contributing to the inter-individual variability in drug response. 2. Here, we analyzed the effect of microbiota on a non-steroidal anti-inflammatory drug nabumetone. 3. First, we cultivated the drug with the selected gut commensal and probiotic bacteria under both aerobic and anaerobic conditions and analyzed its metabolites by high-performance liquid chromatography (HPLC) with UV detection. To analyze the effect of microbiota on nabumetone pharmacokinetics in vivo, we administered a single oral dose of nabumetone to rodents with intentionally altered gut microbiome - either rats treated for three days with the antibiotic imipenem or to germ-free mice. Plasma levels of its main active metabolite 6 methoxy-2-naphthylacetic acid (6-MNA) were analyzed at pre-specified time intervals using HPLC with UV/fluorescence detection. 4. We found that nabumetone is metabolized by bacteria to its non-active metabolites and that this effect is stronger under anaerobic conditions. Although in vivo, none of the pharmacokinetic parameters of 6-MNA was significantly altered, there was a clear trend towards an increase of the AUC, Cmax and t1/2 in rats with reduced microbiota and germ-free mice.
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Microbioma Gastrointestinal/efectos de los fármacos , Nabumetona/farmacocinética , Anaerobiosis , Animales , Antibacterianos/farmacología , Antiinflamatorios no Esteroideos/metabolismo , Antiinflamatorios no Esteroideos/farmacocinética , Disponibilidad Biológica , Microbioma Gastrointestinal/fisiología , Imipenem/farmacología , Masculino , Ratones Endogámicos BALB C , Nabumetona/metabolismo , Ácidos Naftalenoacéticos/metabolismo , Ácidos Naftalenoacéticos/farmacocinética , Ratas Wistar , Organismos Libres de Patógenos EspecíficosRESUMEN
The Lactobacillus casei strain, LOCK 0919, is intended for the dietary management of food allergies and atopic dermatitis (LATOPIC® BIOMED). The use of a probiotic to modulate immune responses is an interesting strategy for solving imbalance problems of gut microflora that may lead to various disorders. However, the exact bacterial signaling mechanisms underlying such modulations are still far from being understood. Here, we investigated variations in the chemical compositions and immunomodulatory properties of the polysaccharides (PS), L919/A and L919/B, which are produced by L. casei LOCK 0919. By virtue of their chemical features, such PS can modulate the immune responses to third-party antigens. Our results revealed that L919/A and L919/B could both modulate the immune response to Lactobacillus planatarum WCFS1, but only L919/B could alter the response of THP-1 cells (in terms of tumor necrosis factor alpha production) to L. planatarum WCFS1 and Escherichia coli Nissle 1917. The comprehensive immunochemical characterization is crucial for the understanding of the biological function as well as of the bacteria-host and bacteria-bacteria cross-talk.
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Factores Inmunológicos/química , Lacticaseibacillus casei/química , Polisacáridos/química , Escherichia coli/inmunología , Humanos , Inmunidad Celular/efectos de los fármacos , Factores Inmunológicos/inmunología , Factores Inmunológicos/uso terapéutico , Lacticaseibacillus casei/inmunología , Polisacáridos/inmunología , Probióticos/uso terapéuticoRESUMEN
Butyrate, a metabolite produced by gut bacteria, has demonstrated beneficial effects in the colon and has been used to treat inflammatory bowel diseases. However, the mechanism by which butyrate operates remains incompletely understood. Given that oral butyrate can exert either a direct impact on the gut mucosa or an indirect influence through its interaction with the gut microbiome, this study aimed to investigate three key aspects: (1) whether oral intake of butyrate modulates the expression of genes encoding short-chain fatty acid (SCFA) transporters (Slc16a1, Slc16a3, Slc16a4, Slc5a8, Abcg2) and receptors (Hcar2, Ffar2, Ffar3, Olfr78, Olfr558) in the colon, (2) the potential involvement of gut microbiota in this modulation, and (3) the impact of oral butyrate on the expression of colonic SCFA transporters and receptors during colonic inflammation. Specific pathogen-free (SPF) and germ-free (GF) mice with or without DSS-induced inflammation were provided with either water or a 0.5% sodium butyrate solution. The findings revealed that butyrate decreased the expression of Slc16a1, Slc5a8, and Hcar2 in SPF but not in GF mice, while it increased the expression of Slc16a3 in GF and the efflux pump Abcg2 in both GF and SPF animals. Moreover, the presence of microbiota was associated with the upregulation of Hcar2, Ffar2, and Ffar3 expression and the downregulation of Slc16a3. Interestingly, the challenge with DSS did not alter the expression of SCFA transporters, regardless of the presence or absence of microbiota, and the effect of butyrate on the transporter expression in SPF mice remained unaffected by DSS. The expression of SCFA receptors was only partially affected by DSS. Our results indicate that (1) consuming a relatively low concentration of butyrate can influence the expression of colonic SCFA transporters and receptors, with their expression being modulated by the gut microbiota, (2) the effect of butyrate does not appear to result from direct substrate-induced regulation but rather reflects an indirect effect associated with the gut microbiome, and (3) acute colon inflammation does not lead to significant changes in the transcriptional regulation of most SCFA transporters and receptors, with the effect of butyrate in the inflamed colon remaining intact.
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Microbiota plays a role in shaping the HPA-axis response to psychological stressors. To examine the role of microbiota in response to acute immune stressor, we stimulated the adaptive immune system by anti-CD3 antibody injection and investigated the expression of adrenal steroidogenic enzymes and profiling of plasma corticosteroids and their metabolites in specific pathogen-free (SPF) and germ-free (GF) mice. Using UHPLC-MS/MS, we showed that 4 hours after immune challenge the plasma levels of pregnenolone, progesterone, 11-deoxycorticosterone, corticosterone (CORT), 11-dehydroCORT and their 3α/ß-, 5α-, and 20α-reduced metabolites were increased in SPF mice, but in their GF counterparts, only CORT was increased. Neither immune stress nor microbiota changed the mRNA and protein levels of enzymes of adrenal steroidogenesis. In contrast, immune stress resulted in downregulated expression of steroidogenic genes (Star, Cyp11a1, Hsd3b1, Hsd3b6) and upregulated expression of genes of the 3α-hydroxysteroid oxidoreductase pathway (Akr1c21, Dhrs9) in the testes of SPF mice. In the liver, immune stress downregulated the expression of genes encoding enzymes with 3ß-hydroxysteroid dehydrogenase (HSD) (Hsd3b2, Hsd3b3, Hsd3b4, Hsd3b5), 3α-HSD (Akr1c14), 20α-HSD (Akr1c6, Hsd17b1, Hsd17b2) and 5α-reductase (Srd5a1) activities, except for Dhrs9, which was upregulated. In the colon, microbiota downregulated Cyp11a1 and modulated the response of Hsd11b1 and Hsd11b2 expression to immune stress. These data underline the role of microbiota in shaping the response to immune stressor. Microbiota modulates the stress-induced increase in C21 steroids, including those that are neuroactive that could play a role in alteration of HPA axis response to stress in GF animals.
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Sistema Hipotálamo-Hipofisario , Microbiota , Masculino , Ratones , Animales , Sistema Hipotálamo-Hipofisario/metabolismo , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Espectrometría de Masas en Tándem , Sistema Hipófiso-Suprarrenal/metabolismo , Esteroides/metabolismo , Corticosterona/metabolismoRESUMEN
PRRSV is capable of evading the effective immune response, thus persisting in piglets and throughout the swine herd. We show here that PRRSV invades the thymus and causes depletion of T-cell precursors and alteration of the TCR repertoire. Developing thymocytes are affected during negative selection when they transit from the triple-negative to triple-positive stages at the corticomedullary junction just before entering the medulla. The restriction of repertoire diversification occurs in both helper and cytotoxic αß-T cells. As a result, critical viral epitopes are tolerated, and infection becomes chronic. However, not all viral epitopes are tolerated. Infected piglets develop antibodies capable of recognizing PRRSV, but these are not virus neutralizing. Further analysis showed that the lack of an effective immune response against the critical viral structures results in the absence of a germinal center response, overactivation of T and B cells in the periphery, robust production of useless antibodies of all isotypes, and the inability to eliminate the virus. Overall, the results show how a respiratory virus that primarily infects and destroys myelomonocytic cells has evolved strategies to disrupt the immune system. These mechanisms may be a prototype for how other viruses can similarly modulate the host immune system.
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Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Porcinos , Animales , Anticuerpos Antivirales , Epítopos , Linfocitos BRESUMEN
Introduction: Porcine reproductive and respiratory syndrome virus (PRRSV) emerged about 30 years ago and continues to cause major economic losses in the pork industry. The lack of effective modified live vaccines (MLV) allows the pandemic to continue. Background and objective: We have previously shown that wild strains of PRRSV affect the nascent T cell repertoire in the thymus, deplete T cell clones recognizing viral epitopes essential for neutralization, while triggering a chronic, robust, but ineffective antibody response. Therefore, we hypothesized that the current MLV are inappropriate because they cause similar damage and fail to prevent viral-induced dysregulation of adaptive immunity. Methods: We tested three MLV strains to demonstrate that all have a comparable negative effect on thymocytes in vitro. Further in vivo studies compared the development of T cells in the thymus, peripheral lymphocytes, and antibody production in young piglets. These three MLV strains were used in a mixture to determine whether at least some of them behave similarly to the wild virus type 1 or type 2. Results: Both the wild and MLV strains cause the same immune dysregulations. These include depletion of T-cell precursors, alteration of the TCR repertoire, necrobiosis at corticomedullary junctions, low body weight gain, decreased thymic cellularity, lack of virus-neutralizing antibodies, and production of non-neutralizing anti-PRRSV antibodies of different isotypes. Discussion and conclusion: The results may explain why the use of current MLV in young animals may be ineffective and why their use may be potentially dangerous. Therefore, alternative vaccines, such as subunit or mRNA vaccines or improved MLV, are needed to control the PRRSV pandemic.
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Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Animales , Porcinos , Síndrome Respiratorio y de la Reproducción Porcina/prevención & control , Anticuerpos Antivirales , Vacunas Atenuadas , Sistema InmunológicoRESUMEN
The intestinal microbiota is known to influence postnatal growth. We previously found that a strain of Lactiplantibacillus plantarum (strain LpWJL) buffers the adverse effects of chronic undernutrition on the growth of juvenile germ-free mice. Here, we report that LpWJL sustains the postnatal growth of malnourished conventional animals and supports both insulin-like growth factor-1 (IGF-1) and insulin production and activity. We have identified cell walls isolated from LpWJL, as well as muramyl dipeptide and mifamurtide, as sufficient cues to stimulate animal growth despite undernutrition. Further, we found that NOD2 is necessary in intestinal epithelial cells for LpWJL-mediated IGF-1 production and for postnatal growth promotion in malnourished conventional animals. These findings indicate that, coupled with renutrition, bacteria cell walls or purified NOD2 ligands have the potential to alleviate stunting.
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Microbioma Gastrointestinal , Crecimiento , Intestinos , Lactobacillaceae , Desnutrición , Proteína Adaptadora de Señalización NOD2 , Animales , Ratones , Pared Celular/química , Células Epiteliales/microbiología , Células Epiteliales/fisiología , Microbioma Gastrointestinal/fisiología , Vida Libre de Gérmenes , Trastornos del Crecimiento/fisiopatología , Trastornos del Crecimiento/terapia , Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Mucosa Intestinal/microbiología , Mucosa Intestinal/fisiología , Intestinos/microbiología , Intestinos/fisiología , Lactobacillaceae/fisiología , Desnutrición/fisiopatología , Desnutrición/terapia , Proteína Adaptadora de Señalización NOD2/metabolismo , Crecimiento/efectos de los fármacos , Crecimiento/fisiología , Acetilmuramil-Alanil-Isoglutamina/farmacología , Acetilmuramil-Alanil-Isoglutamina/uso terapéuticoRESUMEN
The development of inflammatory bowel disease (IBD) is associated with alterations in the gut microbiota. There is currently no universal treatment for this disease, thus emphasizing the importance of developing innovative therapeutic approaches. Gut microbiome-derived metabolite butyrate with its well-known anti-inflammatory effect in the gut is a promising candidate. Due to increased intestinal permeability during IBD, butyrate may also reach the liver and influence liver physiology, including hepatic drug metabolism. To get an insight into this reason, the aim of this study was set to clarify not only the protective effects of the sodium butyrate (SB) administration on colonic inflammation but also the effects of SB on hepatic drug metabolism in experimental colitis induced by dextran sodium sulfate (DSS) in mice. It has been shown here that the butyrate pre-treatment can alleviate gut inflammation and reduce the leakiness of colonic epithelium by restoration of the assembly of tight-junction protein Zonula occludens-1 (ZO-1) in mice with DSS-induced colitis. In this article, butyrate along with inflammation has also been shown to affect the expression and enzyme activity of selected cytochromes P450 (CYPs) in the liver of mice. In this respect, CYP3A enzymes may be very sensitive to gut microbiome-targeted interventions, as significant changes in CYP3A expression and activity in response to DSS-induced colitis and/or butyrate treatment have also been observed. With regard to medications used in IBD and microbiota-targeted therapeutic approaches, it is important to deepen our knowledge of the effect of gut inflammation, and therapeutic interventions were followed concerning the ability of the organism to metabolize drugs. This gut-liver axis, mediated through inflammation as well as microbiome-derived metabolites, may affect the response to IBD therapy.
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Microbiome is now considered as a significant metabolic organ with an immense potential to influence overall human health. A number of diseases that are associated with pharmacotherapy interventions was linked with altered gut microbiota. Moreover, it has been reported earlier that gut microbiome modulates the fate of more than 30 commonly used drugs and, vice versa, drugs have been shown to affect the composition of the gut microbiome. The molecular mechanisms of this mutual relationship, however, remain mostly elusive. Recent studies indicate an indirect effect of the gut microbiome through its metabolites on the expression of biotransformation enzymes in the liver. The aim of this study was to analyse the effect of gut microbiome on the fate of metronidazole in the mice through modulation of system of drug metabolizing enzymes, namely by alteration of the expression and activity of selected cytochromes P450 (CYPs). To assess the influence of gut microbiome, germ-free mice (GF) in comparison to control specific-pathogen-free (SPF) mice were used. First, it has been found that the absence of microbiota significantly affected plasma concentration of metronidazole, resulting in higher levels (by 30%) of the parent drug in murine plasma of GF mice. Further, the significant interaction between presence/absence of the gut microbiome and effect of metronidazole application, which together influence mRNA expression of CAR, PPARα, Cyp2b10 and Cyp2c38 was determined. Administration of metronidazole itself influenced significantly mRNA expression of Cyp1a2, Cyp2b10, Cyp2c38 and Cyp2d22. Finally, GF mice have shown lower level of enzyme activity of CYP2A and CYP3A than their SPF counterparts. The results hence have shown that, beside direct bacterial metabolism, different expression and enzyme activity of hepatic CYPs in the presence/absence of gut microbiota may be responsible for the altered metronidazole metabolism.
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Microbioma Gastrointestinal , Animales , Hígado , Metronidazol , RatonesRESUMEN
The classical definition of probiotics states that bacteria must be alive to be beneficial for human organism. However, recent reports show that inactivated bacteria or their effector molecules can also possess such properties. In this study, we investigated the physical and immunomodulatory properties of four Bifidobacterium strains in the heat-treated (HT) and untreated (UN) forms. We showed that temperature treatment of bacteria changes their size and charge, which affects their interaction with epithelial and immune cells. Based on the in vitro assays, we observed that all tested strains reduced the level of OVA-induced IL-4, IL-5, and IL-13 in the spleen culture of OVA-sensitized mice. We selected Bifidobacterium longum ssp. longum CCM 7952 (Bl 7952) for further analysis. In vivo experiments confirmed that untreated Bl 7952 exhibited allergy-reducing properties when administered intranasally to OVA-sensitized mice, which manifested in significant suppression of airway inflammation. Untreated Bl 7952 decreased local and systemic levels of Th2 related cytokines, OVA-specific IgE antibodies and simultaneously inhibited airway eosinophilia. In contrast, heat-treated Bl 7952 was only able to reduce IL-4 levels in the lungs and eosinophils in bronchoalveolar lavage, but increased neutrophil and macrophage numbers. We demonstrated that the viability status of Bl 7952 is a prerequisite for the beneficial effects of bacteria, and that heat treatment reduces but does not completely abolish these properties. Further research on bacterial effector molecules to elucidate the beneficial effects of probiotics in the prevention of allergic diseases is warranted.
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Bifidobacterium , Supervivencia Celular , Hipersensibilidad , Inflamación , Probióticos , Animales , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos BALB CRESUMEN
Mucosal surfaces are colonized by highly diverse commensal microbiota. Coating with secretory IgA (SIgA) promotes the survival of commensal bacteria while it inhibits the invasion by pathogens. Bacterial coating could be mediated by antigen-specific SIgA recognition, polyreactivity, and/or by the SIgA-associated glycans. In contrast to many in vitro studies, only a few reported the effect of SIgA glycans in vivo. Here, we used a germ-free antibody-free newborn piglets model to compare the protective effect of SIgA, SIgA with enzymatically removed N-glycans, Fab, and Fc containing the secretory component (Fc-SC) during oral necrotoxigenic E. coli O55 challenge. SIgA, Fab, and Fc-SC were protective, whereas removal of N-glycans from SIgA reduced SIgA-mediated protection as demonstrated by piglets' intestinal histology, clinical status, and survival. In vitro analyses indicated that deglycosylation of SIgA did not reduce agglutination of E. coli O55. These findings highlight the role of SIgA-associated N-glycans in protection. Further structural studies of SIgA-associated glycans would lead to the identification of those involved in the species-specific inhibition of attachment to corresponding epithelial cells.
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Infecciones por Escherichia coli/inmunología , Escherichia coli/fisiología , Inmunoglobulina A Secretora/metabolismo , Fragmentos Fab de Inmunoglobulinas/metabolismo , Polisacáridos/metabolismo , Anticuerpos de Cadena Única/metabolismo , Aglutinación , Animales , Animales Recién Nacidos , Resistencia a la Enfermedad , Femenino , Vida Libre de Gérmenes , Glicosilación , Embarazo , PorcinosRESUMEN
Sexual differences and the composition/function of the gut microbiome are not considered the most important players in the drug metabolism field; however, from the recent data it is obvious that they may significantly affect the response of the patient to therapy. Here, we evaluated the effect of microbial colonization and sex differences on mRNA expression and the enzymatic activity of hepatic cytochromes P450 (CYPs) in germ-free (GF) mice, lacking the intestinal flora, and control specific-pathogen-free (SPF) mice. We observed a significant increase in the expression of Cyp3a11 in female SPF mice compared to the male group. However, the sex differences were erased in GF mice, and the expression of Cyp3a11 was about the same in both sexes. We have also found higher Cyp2c38 gene expression in female mice compared to male mice in both the SPF and GF groups. Moreover, these changes were confirmed at the level of enzymatic activity, where the female mice exhibit higher levels of functional CYP2C than males in both groups. Interestingly, we observed the same trend as with CYP3A enzymes: a diminished difference between the sexes in GF mice. The presented data indicate that the mouse gut microbiome plays an important role in sustaining sexual dimorphism in terms of hepatic gene expression and metabolism.
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The gut microbiota is involved in a number of different metabolic processes of the host organism, including the metabolism of xenobiotics. In our study, we focused on liver cytochromes P450 (CYPs), which can metabolize a wide range of exo- and endogenous molecules. We studied changes in mRNA expression and CYP enzyme activities, as well as the mRNA expression of transcription factors that have an important role in CYP expression, all in stressed germ-free (GF) and stressed specific-pathogen-free (SPF) mice. Besides the presence of the gut microbiota, we looked at the difference between acute and chronic stress. Our results show that stress has an impact on CYP mRNA expression, but it is mainly chronic stress that has a significant effect on enzyme activities along with the gut microbiome. In acutely stressed mice, we observed significant changes at the mRNA level, however, the corresponding enzyme activities were not influenced. Our study suggests an important role of the gut microbiota along with chronic psychosocial stress in the expression and activity of CYPs, which can potentially lead to less effective drug metabolism and, as a result, a harmful impact on the organism.
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Sistema Enzimático del Citocromo P-450/metabolismo , Microbioma Gastrointestinal/fisiología , Hígado/enzimología , ARN Mensajero/metabolismo , Estrés Psicológico , Xenobióticos/metabolismo , Animales , Sistema Enzimático del Citocromo P-450/genética , Regulación Enzimológica de la Expresión Génica , Hígado/microbiología , Masculino , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The gut microbiota play an important role in shaping brain functions and behavior, including the activity of the hypothalamus-pituitary-adrenocortical (HPA) axis. However, little is known about the effect of the microbiota on the distinct structures (hypothalamus, pituitary, and adrenals) of the HPA axis. In the present study, we analyzed the influence of the microbiota on acute restraint stress (ARS) response in the pituitary, adrenal gland, and intestine, an organ of extra-adrenal glucocorticoid synthesis. Using specific pathogen-free (SPF) and germ-free (GF) male BALB/c mice, we showed that the plasma corticosterone response to ARS was higher in GF than in SPF mice. In the pituitary, stress downregulated the expression of the gene encoding CRH receptor type 1 (Crhr1), upregulated the expression of the Fkbp5 gene regulating glucocorticoid receptor sensitivity and did not affect the expression of the proopiomelanocortin (Pomc) and glucocorticoid receptor (Gr) genes. In contrast, the microbiota downregulated the expression of pituitary Pomc and Crhr1 but had no effect on Fkbp5 and Gr. In the adrenals, the steroidogenic pathway was strongly stimulated by ARS at the level of the steroidogenic transcriptional regulator Sf-1, cholesterol transporter Star and Cyp11a1, the first enzyme of steroidogenic pathway. In contrast, the effect of the microbiota was significantly detected at the level of genes encoding steroidogenic enzymes but not at the level of Sf-1 and Star. Unlike adrenal Sf-1, the expression of the gene Lrh-1, which encodes the crucial transcriptional regulator of intestinal steroidogenesis, was modulated by the microbiota and ARS and this effect differed between the ileum and colon. The findings demonstrate that gut microbiota have an impact on the response of the pituitary, adrenals and intestine to ARS and that the interaction between stress and the microbiota during activation of glucocorticoid steroidogenesis differs between organs. The results suggest that downregulated expression of pituitary Pomc and Crhr1 in SPF animals might be an important factor in the exaggerated HPA response of GF mice to stress.
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Microbioma Gastrointestinal , Sistema Hipotálamo-Hipofisario , Sistema Hipófiso-Suprarrenal , Restricción Física , Estrés Psicológico/microbiología , Glándulas Suprarrenales/metabolismo , Animales , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Colon/metabolismo , Colon/microbiología , Corticosterona/sangre , Regulación de la Expresión Génica , Íleon/metabolismo , Íleon/microbiología , Masculino , Ratones Endogámicos BALB C , Fosfoproteínas/genética , Hipófisis/metabolismo , Proopiomelanocortina/genética , Receptores de Hormona Liberadora de Corticotropina/genética , Factor Esteroidogénico 1/genética , Estrés Psicológico/sangreRESUMEN
Background: Mucosal mast cells (MC) are key players in IgE-mediated food allergy (FA). The evidence on the interaction between gut microbiota, MC and susceptibility to FA is contradictory. Objective: We tested the hypothesis that commensal bacteria are essential for MC migration to the gut and their maturation impacting the susceptibility to FA. Methods: The development and severity of FA symptoms was studied in sensitized germ-free (GF), conventional (CV), and mice mono-colonized with L. plantarum WCFS1 or co-housed with CV mice. MC were phenotypically and functionally characterized. Results: Systemic sensitization and oral challenge of GF mice with ovalbumin led to increased levels of specific IgE in serum compared to CV mice. Remarkably, despite the high levels of sensitization, GF mice did not develop diarrhea or anaphylactic hypothermia, common symptoms of FA. In the gut, GF mice expressed low levels of the MC tissue-homing markers CXCL1 and CXCL2, and harbored fewer MC which exhibited lower levels of MC protease-1 after challenge. Additionally, MC in GF mice were less mature as confirmed by flow-cytometry and their functionality was impaired as shown by reduced edema formation after injection of degranulation-provoking compound 48/80. Co-housing of GF mice with CV mice fully restored their susceptibility to develop FA. However, this did not occur when mice were mono-colonized with L. plantarum. Conclusion: Our results demonstrate that microbiota-induced maturation and gut-homing of MC is a critical step for the development of symptoms of experimental FA. This new mechanistic insight into microbiota-MC-FA axis can be exploited in the prevention and treatment of FA in humans.
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Hipersensibilidad a los Alimentos/etiología , Hipersensibilidad a los Alimentos/metabolismo , Mastocitos/inmunología , Mastocitos/metabolismo , Microbiota , Animales , Biomarcadores , Degranulación de la Célula/genética , Degranulación de la Célula/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Femenino , Hipersensibilidad a los Alimentos/patología , Microbioma Gastrointestinal , Vida Libre de Gérmenes , Metagenoma , Metagenómica/métodos , Ratones , Microbiota/inmunología , ARN Ribosómico 16SRESUMEN
Germ-free animals have been used to define the vital role of commensal bacteria on the maturation of the host immune system. However, the role of bacterial residues in diet in this setting is poorly understood. Here we investigated the effect of bacterial contamination in sterile diet on the level of allergic sensitization in germ-free mice. Sterile grain-based diets ST1 and R03 were tested for the level of bacterial contamination. ST1 contained higher amount of bacterial DNA, approximately ten times more endotoxin, and induced higher, TLR4-dependent, cytokine production in dendritic cells compared to R03. In a germ-free mouse model of sensitization to the major birch pollen allergen Bet v 1, feeding on ST1 for at least two generations was associated with decreased production of allergen-specific IgE and IgG1 antibodies in sera in comparison to R03. Furthermore, reduced levels of allergen-specific and ConA-induced cytokines IL-4, IL-5 and IL-13 accompanied by increased levels of IFN-γ were detected in splenocytes cultures of these mice. Our results show that contamination of experimental diet with bacterial residues, such as endotoxin, significantly affects the development of allergic sensitization in germ-free mice. Therefore, careful selection of sterile food is critical for the outcomes of germ-free or gnotobiotic experimental models of immune-deviated diseases.
Asunto(s)
Dieta , Endotoxinas/toxicidad , Hipersensibilidad/inmunología , Inmunización , Animales , Antígenos de Plantas/inmunología , Cruzamiento , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Citocinas/biosíntesis , Contaminación de ADN , ADN Bacteriano/análisis , Células Dendríticas/efectos de los fármacos , Sistema Digestivo/inmunología , Epítopos/inmunología , Vida Libre de Gérmenes , Células HEK293 , Humanos , Hipersensibilidad/patología , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , Ligandos , Ratones Endogámicos BALB C , Mitógenos/farmacología , Bazo/patología , Receptor Toll-Like 4/metabolismoRESUMEN
Here, we compared the abilities of polysaccharides L900/2 and L900/3, which were previously isolated from Lactobacillus rhamnosus LOCK 0900, to modulate the immune response to bystander antigens in a mouse model of ovalbumin (OVA) sensitization. In vivo, both polysaccharides reduced the levels of OVA-specific IgE, IgE-dependent basophil degranulation and IgG2a antibodies, but had no effect on the levels of OVA-specific IgA or IgG1. Interestingly, both polysaccharides triggered recall cellular responses with distinct properties. L900/3 significantly suppressed the OVA-induced upregulations of IL-4, IL-5, IL-10 and IL-13 in re-stimulated spleen cells and mesenteric lymph nodes. Our findings support and expand on our previous in vitro studies by demonstrating that polymer L900/3 might modulate the Th1/Th2 balance and could be a promising candidate molecule for preventing allergic sensitization.
Asunto(s)
Alérgenos/inmunología , Inmunización , Factores Inmunológicos/administración & dosificación , Lacticaseibacillus rhamnosus/química , Polisacáridos Bacterianos/administración & dosificación , Serpinas/inmunología , Animales , Basófilos/inmunología , Degranulación de la Célula , Citocinas/biosíntesis , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Factores Inmunológicos/aislamiento & purificación , Ratones , Polisacáridos Bacterianos/aislamiento & purificaciónRESUMEN
Increasing numbers of clinical trials and animal experiments have shown that probiotic bacteria are promising tools for allergy prevention. Here, we analyzed the immunomodulatory properties of three selected lactobacillus strains and the impact of their mixture on allergic sensitization to Bet v 1 using a gnotobiotic mouse model. We showed that Lactobacillus (L.) rhamnosus LOCK0900, L. rhamnosus LOCK0908 and L. casei LOCK0919 are recognized via Toll-like receptor 2 (TLR2) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) receptors and stimulate bone marrow-derived dendritic cells to produce cytokines in species- and strain-dependent manners. Colonization of germ-free (GF) mice with a mixture of all three strains (Lmix) improved the intestinal barrier by strengthening the apical junctional complexes of enterocytes and restoring the structures of microfilaments extending into the terminal web. Mice colonized with Lmix and sensitized to the Bet v 1 allergen showed significantly lower levels of allergen-specific IgE, IgG1 and IgG2a and an elevated total IgA level in the sera and intestinal lavages as well as an increased transforming growth factor (TGF)-ß level compared with the sensitized GF mice. Splenocytes and mesenteric lymph node cells from the Lmix-colonized mice showed the significant upregulation of TGF-ß after in vitro stimulation with Bet v 1. Our results show that Lmix colonization improved the gut epithelial barrier and reduced allergic sensitization to Bet v 1. Furthermore, these findings were accompanied by the increased production of circulating and secretory IgA and the regulatory cytokine TGF-ß. Thus, this mixture of three lactobacillus strains shows potential for use in the prevention of increased gut permeability and the onset of allergies in humans.