RESUMO
Programmed death and shedding of epithelial cells is a powerful defense mechanism to reduce bacterial burden during infection but this activity cannot be indiscriminate because of the critical barrier function of the epithelium. We report that during cystitis, shedding of infected bladder epithelial cells (BECs) was preceded by the recruitment of mast cells (MCs) directly underneath the superficial epithelium where they docked and extruded their granules. MCs were responding to interleukin-1ß (IL-1ß) secreted by BECs after inflammasome and caspase-1 signaling. Upon uptake of granule-associated chymase (mouse MC protease 4 [mMCPT4]), BECs underwent caspase-1-associated cytolysis and exfoliation. Thus, infected epithelial cells require a specific cue for cytolysis from recruited sentinel inflammatory cells before shedding.
Assuntos
Quimases/imunologia , Citotoxinas/imunologia , Células Epiteliais/microbiologia , Mastócitos/imunologia , Infecções Urinárias/imunologia , Animais , Degranulação Celular/imunologia , Linhagem Celular , Grânulos Citoplasmáticos/química , Feminino , Imunofluorescência , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Mast cell (MC)-associated diseases, including allergy/anaphylaxis and neuroinflammatory pain disorders, exhibit a sex bias, with females at increase risk. While much attention has been directed toward adult sex hormones as drivers of sex differences, that female sex bias in MC-associated diseases is evident in prepubertal children, suggesting early-life origins of sex differences which have yet to be explored. Utilizing rodent models of MC-mediated anaphylaxis, our data here reveal that, 1) compared with females, males exhibit significantly reduced severity of MC-mediated anaphylactic responses that emerge prior to puberty and persist into adulthood, 2) reduced severity of MC-mediated anaphylaxis in males is linked with the naturally high level of perinatal androgens and can be recapitulated in females by perinatal exposure to testosterone proprionate, 3) perinatal androgen exposure guides bone marrow MC progenitors toward a masculinized tissue MC phenotype characterized by decreased concentration of prestored MC granule mediators (e.g., histamine, serotonin, and proteases) and reduced mediator release upon degranulation, and 4) engraftment of MC-deficient Kit W-sh/W-sh mice with adult male, female, or perinatally androgenized female MCs results in MC-mediated anaphylaxis response that reflects the MC sex and not host sex. Together, these data present evidence that sex differences in MC phenotype and resulting disease severity are established in early life by perinatal androgens. Thus, factors affecting levels of perinatal androgens could have a significant impact on MC development and MC-associated disease risk across the life span.
Assuntos
Anafilaxia , Androgênios/farmacologia , Mastócitos/efeitos dos fármacos , Fatores Sexuais , Animais , Modelos Animais de Doenças , Feminino , Inflamação , Masculino , Mastócitos/fisiologia , Camundongos , Camundongos Transgênicos , Testículo/citologia , Testículo/efeitos dos fármacosRESUMO
A 7 yr old female spayed mixed-breed dog was presented for a 1 wk history of neck pain and pelvic limb weakness. Examination revealed nonambulatory paraparesis and thoracolumbar hyperesthesia. MRI revealed extensive intramedullary T2-weighted/short tau inversion recovery hyperintensity and diffuse severe T1-post contrast meningeal enhancement of the thoracolumbar spinal cord. An L5-L6 cerebrospinal fluid sample revealed a suppurative pleocytosis (81% neutrophils, total protein 4362.5 mg/dL and nucleated cell count 352,000/µL). While awaiting the results of infectious disease testing, the dog was treated for suspected meningoencephalitis of unknown etiology with corticosteroids, cyclosporine, and a cytarabine arabinoside infusion. The dog neurologically declined and was started on broad-spectrum antibiotics. The dog continued to decline despite antibiotics, and infectious disease titers subsequently revealed serum antibody positivity for blastomycosis. The dog was then referred to a multispecialty referral hospital and was treated with amphotericin B followed by fluconazole. Prednisone was continued at anti-inflammatory doses. Urine blastomycosis antigen testing was submitted for subsequent disease monitoring but was negative. Five months after presentation the dog was clinically doing well with no identifiable neurologic deficits. This case demonstrates that neurologic blastomycosis may have negative urine antigen concentrations in some dogs and that other diagnostic modalities should be pursued when central nervous system fungal disease is suspected.
Assuntos
Blastomicose , Doenças do Cão , Cães , Feminino , Animais , Blastomicose/diagnóstico , Blastomicose/tratamento farmacológico , Blastomicose/veterinária , Doenças do Cão/diagnóstico , Doenças do Cão/tratamento farmacológico , Doenças do Cão/cirurgia , Prednisona/uso terapêutico , Sistema Nervoso Central , Antibacterianos/uso terapêuticoRESUMO
Early-life adversity (ELA) is linked with the increased risk for inflammatory and metabolic diseases in later life, but the mechanisms remain poorly understood. Intestinal epithelial glucose transporters sodium-glucose-linked transporter 1 (SGLT1) and glucose transporter 2 (GLUT2) are the major route for intestinal glucose uptake but have also received increased attention as modulators of inflammatory and metabolic diseases. Here, we tested the hypothesis that early weaning (EW) in pigs, an established model of ELA, alters the development of epithelial glucose transporters and coincides with elevated markers of metabolic inflammation. The jejunum and ileum of 90-day-old pigs previously exposed to EW (16 days wean age), exhibited reduced SGLT1 activity (by â¼ 30%, P < 0.05) than late weaned (LW, 28 days wean age) controls. In contrast, GLUT2-mediated glucose transport was increased (P = 0.003) in EW pigs than in LW pigs. Reciprocal changes in SGLT1- and GLUT2-mediated transport coincided with transporter protein expression in the intestinal brush-border membranes (BBMs) that were observed at 90 days and 150 days of age. Ileal SGLT1-mediated glucose transport and BBM expression were inhibited by the ß-adrenergic receptor (ßAR) blocker propranolol in EW and LW pigs. In contrast, propranolol enhanced ileal GLUT2-mediated glucose transport (P = 0.015) and brush-border membrane vesicle (BBMV) abundance (P = 0.035) in LW pigs, but not in EW pigs. Early-weaned pigs exhibited chronically elevated blood glucose and C-reactive protein (CRP) levels, and adipocyte hypertrophy and upregulated adipogenesis-related gene expression in visceral adipose tissue. Altered development of intestinal glucose transporters by EW could underlie the increased risk for later life inflammatory and metabolic diseases.NEW & NOTEWORTHY These studies reveal that early-life adversity in the form of early weaning in pigs causes a developmental shift in intestinal glucose transport from SGLT1 toward GLUT2-mediated transport. Early weaning also induced markers of metabolic inflammation including persistent elevations in blood glucose and the inflammatory marker CRP, along with increased visceral adiposity. Altered intestinal glucose transport might contribute to increased risk for inflammatory and metabolic diseases associated with early-life adversity.
Assuntos
Glicemia , Propranolol , Animais , Glicemia/metabolismo , Feminino , Glucose/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Inflamação/metabolismo , Mucosa Intestinal/metabolismo , Transportador 1 de Glucose-Sódio/genética , Suínos , DesmameRESUMO
Increased knowledge of reproduction and health of domesticated animals is integral to sustain and improve global competitiveness of U.S. animal agriculture, understand and resolve complex animal and human diseases, and advance fundamental research in sciences that are critical to understanding mechanisms of action and identifying future targets for interventions. Historically, federal and state budgets have dwindled and funding for the United States Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA) competitive grants programs remained relatively stagnant from 1985 through 2010. This shortage in critical financial support for basic and applied research, coupled with the underappreciated knowledge of the utility of non-rodent species for biomedical research, hindered funding opportunities for research involving livestock and limited improvements in both animal agriculture and animal and human health. In 2010, the National Institutes of Health and USDA NIFA established an interagency partnership to promote the use of agriculturally important animal species in basic and translational research relevant to both biomedicine and agriculture. This interagency program supported 61 grants totaling over $107 million with 23 awards to new or early-stage investigators. This article will review the success of the 9-year Dual Purpose effort and highlight opportunities for utilizing domesticated agricultural animals in research.
Assuntos
Agricultura , Animais Domésticos , Animais , Gado , National Institutes of Health (U.S.) , Estados Unidos , United States Department of AgricultureRESUMO
Exposure to early life adversity (ELA) in the form of physical and/or psychological abuse or neglect increases the risk of developing psychiatric and inflammatory disorders later in life. It has been hypothesized that exposure to ELA results in persistent, low grade inflammation that leads to increased disease susceptibility by amplifying the crosstalk between stress-processing brain networks and the immune system, but the mechanisms remain largely unexplored. The meninges, a layer of three overlapping membranes that surround the central nervous system (CNS)- dura mater, arachnoid, and piamater - possess unique features that allow them to play a key role in coordinating immune trafficking between the brain and the peripheral immune system. These include a network of lymphatic vessels that carry cerebrospinal fluid from the brain to the deep cervical lymph nodes, fenestrated blood vessels that allow the passage of molecules from blood to the CNS, and a rich population of resident mast cells, master regulators of the immune system. Using a mouse model of ELA consisting of neonatal maternal separation plus early weaning (NMSEW), we sought to explore the effects of ELA on sucrose preference behavior, dura mater expression of inflammatory markers and mast cell histology in adult male and female C57Bl/6 mice. We found that NMSEW alone does not affect sucrose preference behavior in males or females, but it increases the dura mater expression of the genes coding for mast cell protease CMA1 (cma1) and the inflammatory cytokine TNF alpha (tnf alpha) in females. When NMSEW is combined with an adult mild stress (that does not affect behavior or gene expression in NH animals) females show reduced sucrose preference and even greater increases in meningeal cma1 levels. Interestingly, systemic administration of the mast cell stabilizer Ketotifen before exposure to adult stress prevents both, reduction in sucrose preference an increases in cma1 expression in NMSEW females, but facilitates stress-induced sucrose anhedonia in NMSEW males and NH females. Finally, histological analyses showed that, compared to males, females have increased baseline activation levels of mast cells located in the transverse sinus of the dura mater, where the meningeal lymphatics run along, and that, in males and females exposed to adult stress, NMSEW increases the number of mast cells in the interparietal region of the dura mater and the levels of mast cell activation in the sagittal sinus regions of the dura mater. Together, our results indicate that ELA induces long-term meningeal immune gene changes and heightened sensitivity to adult stress-induced behavioral and meningeal immune responses and that these effects could mediated via mast cells.
Assuntos
Anedonia , Mastócitos , Fatores Sexuais , Estresse Psicológico , Animais , Feminino , Masculino , Apresentação de Antígeno , Expressão Gênica , Privação Materna , Meninges , Sacarose , Fator de Necrose Tumoral alfa , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Early-life adversity contributes to the development of functional bowel disorders later in life through unresolved mechanisms. Here, we tested the hypothesis that early-life adversity alters anatomical and functional interactions between mast cells and enteric glia. The effects of early-life stress were studied using the neonatal maternal separation (NMS) stress mouse model. Anatomical relationships between mast cells and enteric glia were assessed using immunohistochemistry and mast cell reporter mice (Mcpt5Cre;GCaMP5g-tdT). Immunohistochemistry was used to assess the expression of histamine, histamine 1 (H1) receptors, and glial fibrillary acidic protein. Functional responses of glia to mast cell mediators were assessed in calcium imaging experiments using Sox10CreERT2;GCaMP5g-tdT mice and cultured human enteric glial cells. NMS increases mast cell numbers at the level of the myenteric plexus and their proximity to myenteric ganglia. Myenteric glia respond to mediators released by activated mast cells that are blocked by H1 receptor antagonists in mice and humans and by blocking neuronal activity with tetrodotoxin in mouse tissue. Histamine replicates the effects of mast cell supernatants on enteric glia, and NMS increases histamine production by mast cells. NMS reduces glial responses to mast cell mediators in mouse tissue, while potentiating responses in cultured human enteric glia. NMS increases myenteric glial fibrillary acidic protein expression and reduces glial process length but does not cause neurodegeneration. Histamine receptor expression is not altered by NMS and is localized to neurons in mice, but glia in humans. Early-life stress increases the potential for interactions between enteric glia and mast cells, and histamine is a potential mediator of mast cell-glial interactions through H1 receptors. We propose that glial-mast cell signaling is a mechanism that contributes to enteric neuroplasticity driven by early-life adversity.NEW & NOTEWORTHY Early-life adversity places an individual at risk for developing functional gastrointestinal disorders later in life through unknown mechanisms. Here, we show that interactions between mast cells and glia are disrupted by early-life stress in mice and that histamine is a potential mediator of mast cell-glial interactions.
Assuntos
Histamina/fisiologia , Acontecimentos que Mudam a Vida , Mastócitos/fisiologia , Neuroglia/fisiologia , Neurônios/fisiologia , Animais , Animais Recém-Nascidos , Contagem de Células , Células Cultivadas , Quimases/genética , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Antagonistas dos Receptores Histamínicos H1/farmacologia , Humanos , Privação Materna , Camundongos , Camundongos Endogâmicos C57BL , Plexo Mientérico/citologia , Plexo Mientérico/metabolismo , Gravidez , Receptores Histamínicos H1/metabolismo , Estresse Psicológico/fisiopatologiaRESUMO
Objective- Perivascular adipose tissue (PVAT) contains an independent adrenergic system that can take up, metabolize, release, and potentially synthesize the vasoactive catecholamine norepinephrine. Norepinephrine has been detected in PVAT, but the mechanism of its protection within this tissue is unknown. Here, we investigate whether PVAT adipocytes can store norepinephrine using VMAT (vesicular monoamine transporter). Approach and Results- High-performance liquid chromatography identified norepinephrine in normal male Sprague Dawley rat aortic, superior mesenteric artery, and mesenteric resistance vessel PVATs, and retroperitoneal fat. Real-time polymerase chain reaction revealed VMAT1 and VMAT2 mRNA expression in the adipocytes and stromal vascular fraction of mesenteric resistance vessel PVAT. Immunofluorescence demonstrated the presence of VMAT1 and VMAT2, and the colocalization of VMAT2 with norepinephrine, in the cytoplasm of adipocytes in mesenteric resistance vessel PVAT. A protocol was developed to capture real-time uptake of Mini 202-a functional and fluorescent VMAT probe-in live rat PVAT adipocytes. Mini 202 was taken up by freshly isolated and differentiated adipocytes from mesenteric resistance vessel PVAT and adipocytes from thoracic aortic and superior mesenteric artery PVATs. In adipocytes freshly isolated from mesenteric resistance vessel PVAT, addition of rose bengal (VMAT inhibitor), nisoxetine (norepinephrine transporter inhibitor), or corticosterone (organic cation 3 transporter inhibitor) significantly reduced Mini 202 signal. Immunofluorescence supports that neither VMAT1 nor VMAT2 is present in retroperitoneal adipocytes, suggesting that PVAT adipocytes may be unique in storing norepinephrine. Conclusions- This study supports a novel function of PVAT adipocytes in storing amines in a VMAT-dependent manner. It provides a foundation for future studies exploring the purpose and mechanisms of norepinephrine storage by PVAT in normal physiology and obesity-related hypertension.
Assuntos
Adipócitos/metabolismo , Norepinefrina/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/fisiologia , Animais , Transporte Biológico , Células Cromafins/metabolismo , Feminino , Masculino , Artérias Mesentéricas/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Psychological stress and heightened mast cell (MC) activation are linked with important immunologic disorders, including allergy, anaphylaxis, asthma, and functional bowel diseases, but the mechanisms remain poorly defined. We have previously demonstrated that activation of the corticotropin-releasing factor (CRF) system potentiates MC degranulation responses during IgE-mediated anaphylaxis and psychological stress through corticotropin-releasing factor receptor subtype 1 (CRF1) expressed on MCs. OBJECTIVE: In this study we investigated the role of corticotropin-releasing factor receptor subtype 2 (CRF2) as a modulator of stress-induced MC degranulation and associated disease pathophysiology. METHODS: In vitro MC degranulation assays were performed with bone marrow-derived mast cells (BMMCs) derived from wild-type (WT) and CRF2-deficient (CRF2-/-) mice and RBL-2H3 MCs transfected with CRF2-overexpressing plasmid or CRF2 small interfering RNA. In vivo MC responses and associated pathophysiology in IgE-mediated passive systemic anaphylaxis and acute psychological restraint stress were measured in WT, CRF2-/-, and MC-deficient KitW-sh/W-sh knock-in mice. RESULTS: Compared with WT mice, CRF2-/- mice exhibited greater serum histamine levels and exacerbated IgE-mediated anaphylaxis and colonic permeability. In addition, CRF2-/- mice exhibited increased serum histamine levels and colonic permeability after acute restraint stress. Experiments with BMMCs and RBL-2H3 MCs demonstrated that CRF2 expressed on MCs suppresses store-operated Ca2+ entry signaling and MC degranulation induced by diverse MC stimuli. Experiments with MC-deficient KitW-sh/W-sh mice systemically engrafted with WT and CRF2-/- BMMCs demonstrated the functional importance of MC CRF2 in modulating stress-induced pathophysiology. CONCLUSIONS: MC CRF2 is a negative global modulator of stimuli-induced MC degranulation and limits the severity of IgE-mediated anaphylaxis and stress-related disease pathogenesis.
Assuntos
Anafilaxia/imunologia , Mucosa Intestinal/metabolismo , Mastócitos/fisiologia , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Estresse Psicológico/imunologia , Junções Íntimas/metabolismo , Animais , Degranulação Celular , Linhagem Celular , Modelos Animais de Doenças , Feminino , Liberação de Histamina/genética , Humanos , Imunoglobulina E/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos , Receptores de Hormônio Liberador da Corticotropina/genéticaRESUMO
BACKGROUND: Understanding the influence of dietary iron deficiency and dietary iron oversupplementation on intestinal health is important for both animal production and human health. OBJECTIVE: The aim of this study was to determine whether dietary iron concentration influences intestinal physiology, morphology, and inflammation in the porcine duodenum. METHODS: Twenty-four male pigs (21 d old) were fed diets containing either 20 mg Fe/kg [low dietary iron (L-Fe)], 120 mg Fe/kg [adequate dietary iron (A-Fe); control], or 520 mg Fe/kg [high dietary iron (H-Fe)] by FeSO4 supplement (dry matter basis). After 32-36 d, the duodenum was harvested from pigs and mounted in Ussing chambers for the measurement of transepithelial electrical resistance (TER), short-circuit current, and (3)H-mannitol permeability. Intestinal morphology and inflammation were assessed by histologic examination, and proinflammatory gene expression was assessed by real-time polymerase chain reaction. RESULTS: Compared with A-Fe-fed pigs, pigs fed L-Fe diets exhibited reduced TER (by 30%; P < 0.05). Compared with that of A-Fe-fed controls, the paracellular flux of (3)H-mannitol across the duodenal mucosa was higher (P < 0.05) in L-Fe-fed (>100%) and H-Fe-fed (â¼4-fold) pigs; the L-Fe-fed and H-Fe-fed groups did not differ significantly from one another. Compared with the L-Fe-fed pigs, the A-Fe-fed and H-Fe-fed pigs had malondialdehyde concentrations 1.4- and 2.5-fold higher in the duodenum and 4.4- and 6.6-fold higher in the liver, respectively (P < 0.05). Neutrophil counts were higher in both the L-Fe-fed (by 3-fold) and H-Fe-fed (by 3.3-fold) groups than in the A-Fe-fed group; the L-Fe-fed and H-Fe-fed groups did not significantly differ from one another. Duodenal mucosal tumor necrosis factor α (TNFA), interleukin (IL) 1ß, and IL6 relative gene expression was upregulated by 36%, 28%, and 45%, respectively, in H-Fe pigs (P < 0.05), but not in L-Fe pigs, compared with A-Fe pigs. CONCLUSION: These data suggest that adequate but not oversupplementation of dietary iron in pigs is required to maintain intestinal barrier health and function.
Assuntos
Dieta , Suplementos Nutricionais , Inflamação/etiologia , Mucosa Intestinal/fisiopatologia , Íons/metabolismo , Ferro da Dieta/administração & dosagem , Ferro/administração & dosagem , Animais , Transporte Biológico , Duodeno , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Interleucina-5/metabolismo , Ferro/metabolismo , Deficiências de Ferro , Ferro da Dieta/metabolismo , Fígado , Masculino , Malondialdeído/metabolismo , Neutrófilos/metabolismo , Estado Nutricional , Hipernutrição , Permeabilidade , Suínos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Early-life stress and adversity are major risk factors in the onset and severity of gastrointestinal (GI) disease in humans later in life. The mechanisms by which early-life stress leads to increased GI disease susceptibility in adult life remain poorly understood. Animal models of early-life stress have provided a foundation from which to gain a more fundamental understanding of this important GI disease paradigm. This review focuses on animal models of early-life stress-induced GI disease, with a specific emphasis on translational aspects of each model to specific human GI disease states. Early postnatal development of major GI systems and the consequences of stress on their development are discussed in detail. Relevant translational differences between species and models are highlighted.
Assuntos
Gastroenteropatias/etiologia , Intestinos/fisiopatologia , Estresse Psicológico/complicações , Pesquisa Translacional Biomédica/métodos , Glândulas Suprarrenais/metabolismo , Glândulas Suprarrenais/fisiopatologia , Fatores Etários , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Sistema Nervoso Entérico/metabolismo , Sistema Nervoso Entérico/fisiopatologia , Gastroenteropatias/metabolismo , Gastroenteropatias/fisiopatologia , Gastroenteropatias/psicologia , Humanos , Sistema Hipotálamo-Hipofisário/metabolismo , Sistema Hipotálamo-Hipofisário/fisiopatologia , Mucosa Intestinal/metabolismo , Intestinos/inervação , Fatores de Risco , Especificidade da Espécie , Estresse Psicológico/metabolismo , Estresse Psicológico/fisiopatologia , Estresse Psicológico/psicologiaRESUMO
Research in the field of ischemia-reperfusion injury continues to be plagued by the inability to translate research findings to clinically useful therapies. This may in part relate to the complexity of disease processes that result in intestinal ischemia but may also result from inappropriate research model selection. Research animal models have been integral to the study of ischemia-reperfusion-induced intestinal injury. However, the clinical conditions that compromise intestinal blood flow in clinical patients ranges widely from primary intestinal disease to processes secondary to distant organ failure and generalized systemic disease. Thus models that closely resemble human pathology in clinical conditions as disparate as volvulus, shock, and necrotizing enterocolitis are likely to give the greatest opportunity to understand mechanisms of ischemia that may ultimately translate to patient care. Furthermore, conditions that result in varying levels of ischemia may be further complicated by the reperfusion of blood to tissues that, in some cases, further exacerbates injury. This review assesses animal models of ischemia-reperfusion injury as well as the knowledge that has been derived from each to aid selection of appropriate research models. In addition, a discussion of the future of intestinal ischemia-reperfusion research is provided to place some context on the areas likely to provide the greatest benefit from continued research of ischemia-reperfusion injury.
Assuntos
Intestinos/lesões , Isquemia/patologia , Traumatismo por Reperfusão/patologia , Pesquisa Translacional Biomédica , Animais , Modelos Animais de Doenças , Humanos , Intestinos/patologia , Isquemia/complicações , Oxigênio/metabolismo , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/metabolismo , Pesquisa Translacional Biomédica/métodosRESUMO
The objective of this study was to investigate the effect of increasing degrees of lipid peroxidation on structure and function of the small intestine of nursery pigs. A total of 216 pigs (mean body weight was 6·5 kg) were randomly allotted within weight blocks and sex and fed one of five experimental diets for 35 d (eleven pens per treatment with three to four pigs per pen). Treatments included a control diet without added lipid, and diets supplemented with 6 % soyabean oil that was exposed to heat (80°C) and constant oxygen flow (1 litre/min) for 0, 6, 9 and 12 d. Increasing lipid peroxidation linearly reduced feed intake (P<0·001) and weight gain (P=0·024). Apparent faecal digestibility of gross energy (P=0·001) and fat (P<0·001) decreased linearly as the degree of peroxidation increased. Absorption of mannitol (linear, P=0·097) and d-xylose (linear, P=0·089), measured in serum 2 h post gavage with a solution containing 0·2 g/ml of d-xylose and 0·3 g/ml of mannitol, tended to decrease progressively as the peroxidation level increased. Increasing peroxidation also resulted in increased villi height (linear, P<0·001) and crypt depth (quadratic, P=0·005) in the jejunum. Increasing peroxidation increased malondialdehyde concentrations (quadratic, P=0·035) and reduced the total antioxidant capacity (linear, P=0·044) in the jejunal mucosa. In conclusion, lipid peroxidation progressively diminished animal performance and modified the function and morphology of the small intestine of nursery pigs. Detrimental effects were related with the disruption of redox environment of the intestinal mucosa.
Assuntos
Gorduras na Dieta/administração & dosagem , Intestino Delgado/efeitos dos fármacos , Peróxidos/química , Animais , Gorduras na Dieta/farmacologia , Digestão , Relação Dose-Resposta a Droga , Comportamento Alimentar , Feminino , Absorção Intestinal , Mucosa Intestinal/metabolismo , Intestino Delgado/fisiologia , Masculino , Manitol/administração & dosagem , Manitol/metabolismo , Óleo de Soja/metabolismo , Suínos/crescimento & desenvolvimento , Xilose/administração & dosagemRESUMO
BACKGROUND: Previous work showed that weaning stress causes gut barrier dysfunction partly by triggering the release of corticotropin releasing factor (CRF) and thereby inducing the degranulation of intestinal mast cell (MC). This study investigated the hypothesis that attenuating the weaning-induced activation of the CRF-MC axis via administration of a MC stabilizing agent (cromolyn) may improve gut permeability and piglet performance after weaning. RESULTS: To test the hypothesis twenty piglets were weaned (20 ± 1.0 d of age; 6.4 ± 0.4 kg of BW) and injected intraperitoneally with saline (control, n = 10) or 20 mg/kg BW of sodium cromolyn (cromolyn, n = 10) at - 0.5, 8 and 16 h relative to weaning. Piglets were housed individually and fed ad libitum a pre-starter diet from one to 15 d post-weaning followed by a starter diet until the end of the study on d 36. Cromolyn improved intestinal permeability as indicated by the reduced recovery of cobalt and mannitol in plasma samples. Cromolyn treated pigs consumed more feed (369 vs. 313 g/d; P < 0.009), gained more BW (283 vs. 238 g/d; P < 0.006), and grew more efficiently (0.60 vs. 0.40; P < 0.042) than their control counterparts. As a result, cromolyn treated pigs were 1.4 kg heavier than those in the control group by d 36 after weaning (16.5 vs. 17.9 kg; P < 0.002). CONCLUSIONS: In agreement with our hypothesis, present data indicate that the cromolyn-mediated improvement of intestinal permeability is associated with enhanced pig performance after weaning.
Assuntos
Cromolina Sódica/farmacologia , Intestinos/efeitos dos fármacos , Intestinos/fisiologia , Suínos/fisiologia , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal , Animais , Dieta/veterinária , Ingestão de Alimentos/efeitos dos fármacos , Estresse Fisiológico , Suínos/crescimento & desenvolvimento , Desmame , Aumento de Peso/efeitos dos fármacosRESUMO
BACKGROUND: Organic acids, such as citric and sorbic acid, and pure plant-derived constituents, like monoterpens and aldehydes, have a long history of use in pig feeding as alternatives to antibiotic growth promoters. However, their effects on the intestinal barrier function and inflammation have never been investigated. Therefore, aim of this study was to assess the impact of a microencapsulated mixture of citric acid and sorbic acid (OA) and pure botanicals, namely thymol and vanillin, (PB) on the intestinal integrity and functionality of weaned pigs and in vitro on Caco-2 cells. In the first study 20 piglets were divided in 2 groups and received either a basal diet or the basal diet supplemented with OA + PB (5 g/kg) for 2 weeks post-weaning at the end of which ileum and jejunum samples were collected for Ussing chambers analysis of trans-epithelial electrical resistance (TER), intermittent short-circuit current (I SC), and dextran flux. Scrapings of ileum mucosa were also collected for cytokine analysis (n = 6). In the second study we measured the effect of these compounds directly on TER and permeability of Caco-2 monolayers treated with either 0.2 or 1 g/l of OA + PB. RESULTS: Pigs fed with OA + PB tended to have reduced I SC in the ileum (P = 0.07) and the ileal gene expression of IL-12, TGF-ß, and IL-6 was down regulated. In the in vitro study on Caco-2 cells, TER was increased by the supplementation of 0.2 g/l at 4, 6, and 14 days of the experiment, whereas 1 g/l increased TER at 10 and 12 days of treatment (P < 0.05). Dextran flux was not significantly affected though a decrease was observed at 7 and 14 days (P = 0.10 and P = 0.09, respectively). CONCLUSIONS: Overall, considering the results from both experiments, OA + PB improved the maturation of the intestinal mucosa by modulating the local and systemic inflammatory pressure ultimately resulting in a less permeable intestine, and eventually improving the growth of piglets prematurely weaned.
Assuntos
Benzaldeídos/farmacologia , Ácido Cítrico/farmacologia , Inflamação/veterinária , Ácido Sórbico/farmacologia , Suínos , Timol/farmacologia , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal , Animais , Benzaldeídos/administração & dosagem , Células CACO-2 , Ácido Cítrico/administração & dosagem , Citocinas/genética , Citocinas/metabolismo , Dieta/veterinária , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/prevenção & controle , Intestinos/efeitos dos fármacos , Ácido Sórbico/administração & dosagem , Timol/administração & dosagemRESUMO
BACKGROUND & AIMS: Heme oxygenase-1 (HO-1) and its metabolic by-product, carbon monoxide (CO), protect against intestinal inflammation in experimental models of colitis, but little is known about their intestinal immune mechanisms. We investigated the interactions among CO, HO-1, and the enteric microbiota in mice and zebrafish. METHODS: Germ-free, wild-type, and interleukin (Il)10(-/-) mice and germ-free zebrafish embryos were colonized with specific pathogen-free (SPF) microbiota. Germ-free or SPF-raised wild-type and Il10(-/-) mice were given intraperitoneal injections of cobalt(III) protoporphyrin IX chloride (CoPP), which up-regulates HO-1, the CO-releasing molecule Alfama-186, or saline (control). Colitis was induced in wild-type mice housed in SPF conditions by infection with Salmonella typhimurium. RESULTS: In colons of germ-free, wild-type mice, SPF microbiota induced production of HO-1 via activation of nuclear factor erythroid 2-related factor 2-, IL-10-, and Toll-like receptor-dependent pathways; similar observations were made in zebrafish. SPF microbiota did not induce HO-1 in colons of germ-free Il10(-/-) mice. Administration of CoPP to Il10(-/-) mice before transition from germ-free to SPF conditions reduced their development of colitis. In Il10(-/-) mice, CO and CoPP reduced levels of enteric bacterial genomic DNA in mesenteric lymph nodes. In mice with S typhimurium-induced enterocolitis, CoPP reduced the numbers of live S typhimurium recovered from the lamina propria, mesenteric lymph nodes, spleen, and liver. Knockdown of HO-1 in mouse macrophages impaired their bactericidal activity against E coli, E faecalis, and S typhimurium, whereas exposure to CO or overexpression of HO-1 increased their bactericidal activity. HO-1 induction and CO increased acidification of phagolysosomes. CONCLUSIONS: Colonic HO-1 prevents colonic inflammation in mice. HO-1 is induced by the enteric microbiota and its homeostatic function is mediated, in part, by promoting bactericidal activities of macrophages.
Assuntos
Translocação Bacteriana/fisiologia , Monóxido de Carbono/farmacologia , Colite/prevenção & controle , Heme Oxigenase-1/metabolismo , Salmonella typhimurium/fisiologia , Animais , Translocação Bacteriana/efeitos dos fármacos , Western Blotting , Colite/tratamento farmacológico , Colite/microbiologia , Modelos Animais de Doenças , Escherichia coli/patogenicidade , Gentamicinas/farmacologia , Heme Oxigenase-1/biossíntese , Macrófagos/citologia , Macrófagos/fisiologia , Metagenoma , Camundongos , Camundongos Endogâmicos C57BL , Distribuição Aleatória , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Irritable bowel syndrome and related disorders of gut-brain interaction (DGBI) are common and exhibit a complex, poorly understood etiology that manifests as abnormal gut motility and pain. Risk factors such as biological sex, stressors during critical periods, and inflammation are thought to influence DGBI vulnerability by reprogramming gut-brain circuits, but the specific cells affected are unclear. Here, we used a model of early life stress to understand cellular mechanisms in the gut that produce DGBIs. Our findings identify enteric glia as a key cellular substrate in which stress and biological sex converge to dictate DGBI susceptibility. Enteric glia exhibit sexual dimorphism in genes and functions related to cellular communication, inflammation, and disease susceptibility. Experiencing early life stress has sex-specific effects on enteric glia that cause a phenotypic switch in male glia toward a phenotype normally observed in females. This phenotypic transformation is followed by physiological changes in the gut, mirroring those observed in DGBI in humans. These effects are mediated, in part, by alterations to glial prostaglandin and endocannabinoid signaling. Together, these data identify enteric glia as a cellular integration site through which DGBI risk factors produce changes in gut physiology and suggest that manipulating glial signaling may represent an attractive target for sex-specific therapeutic strategies in DGBIs.
RESUMO
Objectives were to examine the temporal pattern of intestinal mast cell dynamics and the effects of a mast cell stabilizer (ketotifen [Ket]) during acute heat stress (HS) in growing pigs. Crossbred barrows (nâ =â 42; 32.3â ±â 1.9 kg body weight [BW]) were randomly assigned to 1 of 7 environmental-therapeutic treatments: (1) thermoneutral (TN) control (TNCon; nâ =â 6), (2) 2 h HS control (2 h HSCon; nâ =â 6), (3) 2 h HSâ +â Ket (2 h HSKet; nâ =â 6); (4) 6 h HSCon (nâ =â 6), (5) 6 h HSKet (nâ =â 6), (6) 12 h HSCon (nâ =â 6), or (7) 12 h HSKet (nâ =â 6). Following 5 d of acclimation to individual pens, pigs were enrolled in two experimental periods (P). During P1 (3 d), pigs were housed in TN conditions (21.5â ±â 0.8 °C) for the collection of baseline measurements. During P2, TNCon pigs remained in TN conditions for 12 h, while HS pigs were exposed to constant HS (38.1â ±â 0.2 °C) for either 2, 6, or 12 h. Pigs were euthanized at the end of P2, and blood and tissue samples were collected. Regardless of time or therapeutic treatment, pigs exposed to HS had increased rectal temperature, skin temperature, and respiration rate compared to their TNCon counterparts (1.9 °C, 6.9° C, and 119 breaths/min; Pâ <â 0.01). As expected, feed intake and BW gain markedly decreased in HS pigs relative to their TNCon counterparts (Pâ <â 0.01). Irrespective of therapeutic treatment, circulating corticotropin-releasing factor decreased from 2 to 12 h of HS relative to TNCon pigs (Pâ <â 0.01). Blood cortisol increased at 2 h of HS (2-fold; Pâ =â 0.04) and returned to baseline by 6 h. Plasma histamine (a proxy of mast cell activation) remained similar across thermal treatments and was not affected by Ket administration (Pâ >â 0.54). Independent of Ket or time, HS increased mast cell numbers in the jejunum (94%; Pâ <â 0.01); however, no effects of HS on mast cell numbers were detected in the ileum or colon. Jejunum and ileum myeloperoxidase area remained similar among treatments (Pâ >â 0.58) but it tended to increase (12%; Pâ =â 0.08) in the colon in HSCon relative to TNCon pigs. Circulating lymphocytes and basophils decreased in HSKet relative to TN and HSCon pigs (Pâ ≤â 0.06). Blood monocytes and eosinophils were reduced in HS pigs relative to their TNCon counterparts (Pâ <â 0.01). In summary, HS increased jejunum mast cell numbers and altered leukocyte dynamics and proinflammatory biomarkers. However, Ket administration had no effects on mast cell dynamics measured herein.
Heat stress (HS) affects various physiological, metabolic, and endocrine parameters, ostensibly due to reduced intestinal barrier integrity and the ensuing immune response. Evidence indicates that generalized "stress" may be a critical component of HS-induced leaky gut, a mechanism likely mediated by mast cells. Mast cell activation has been extensively associated with various stress-related intestinal inflammatory conditions; however, its contribution to intestinal barrier dysfunction during HS remains unclear. Thus, this study was designed to evaluate mast cell dynamics during an acute HS challenge and to assess the effects a mast cell stabilizer on biomarkers of intestinal inflammation. Herein, HS induced a rapid increase in circulating cortisol, increased jejunum mast cell numbers, and altered metabolism, leukocyte dynamics, and proinflammatory biomarkers. Contrary to our hypothesis, HS did not alter circulating histamine (a biomarker of mast cell activation), and mast cell stabilization did not affect mast cell numbers nor altered histamine concentrations. Altogether, our observations support a connection between HS and intestinal mast cell infiltration that may contribute to the pathophysiology of intestinal dysfunction during a heat load.
Assuntos
Transtornos de Estresse por Calor , Doenças dos Suínos , Suínos , Animais , Dieta , Mastócitos , Resposta ao Choque Térmico , Temperatura Cutânea , Reto , Temperatura Alta , Transtornos de Estresse por Calor/veterináriaRESUMO
Mast cells are innate immune cells that play a crucial role in numerous physiological processes across tissues by releasing pre-stored and newly synthesized mediators in response to stimuli, an activity largely driven by changes in gene expression. Given their widespread influence, dysfunction in mast cells can contribute to a variety of pathologies including allergies, long COVID, and autoimmune and neuroinflammatory disorders. Despite this, the specific transcriptional mechanisms that control mast cell mediator release remain poorly understood, significantly hindering the development of effective therapeutic strategies. We found that the two proteins encoded by the transcription factor FosB, FOSB and the highly stable variant ΔFOSB, are robustly expressed upon stimulation in both murine and human mast cell progenitors. Motivated by these findings, we generated a novel mouse model with targeted ablation of FosB gene expression specifically in mast cells (MC FosB- ) by crossing a mast cell-specific Cre reporter line (Mcpt5-Cre) with a Cre-dependent floxed FosB mouse lines. We found that mast cell progenitors derived from MC FosB- mice, compared to wild types (WT), exhibit baseline increased histamine content and vesicle numbers. Additionally, they show enhanced calcium mobilization, degranulation, and histamine release following allergy-related IgE-mediated stimulation, along with heightened IL-6 release in response to infection-like LPS stimulation. In vivo experiments with IgE- mediated and LPS challenges revealed that MC FosB- mice experience greater drops in body temperature, heightened activation of tissue-resident mast cells, and increased release of pro-inflammatory mediators compared to their WT counterparts. These findings suggest that FosB products play a crucial regulatory role in moderating stimulus-induced mast cell activation in response to both IgE and LPS stimuli. Lastly, by integrating CUT&RUN and RNAseq data, we identified several genes targeted by ΔFOSB that could mediate these observed effects, including Mir155hg, CLCF1, DUSP4, and Trib1. Together, this study provides the first evidence that FOSB/ΔFOSB modulate mast cell functions and provides a new possible target for therapeutic interventions aimed at ameliorating mast cell-related diseases.
RESUMO
OBJECTIVE: Cardiac surgery, especially when employing cardiopulmonary bypass and deep hypothermic circulatory arrest, is associated with systemic inflammatory responses that significantly affect morbidity and mortality. Intestinal perfusion abnormalities have been implicated in such responses, but the mechanisms linking local injury and systemic inflammation remain unclear. Intestinal mast cells are specialized immune cells that secrete various preformed effectors in response to cellular stress. We hypothesized that mast cells are activated in a microenvironment shaped by intestinal ischemia/reperfusion, and investigated local and systemic consequences. DESIGN: Rat model of deep hypothermic circulatory arrest. SETTING: University research laboratory. SUBJECTS: Twelve- to 14-week-old male Sprague-Dawley rats. INTERVENTIONS: Rats were anesthetized and cooled to 16°C to 18°C on cardiopulmonary bypass before instituting deep hypothermic circulatory arrest for 45 minutes. Specimens were harvested following rewarming and 2 hours of recovery. MEASUREMENTS AND MAIN RESULTS: Significant intestinal barrier disruption was found, together with macro- and microscopic evidence of ischemia/reperfusion injury in ileum and colon, but not in the lungs or kidneys. Immunofluorescence and toluidine blue staining revealed increased numbers of mast cells and their activation in the gut. In animals pretreated with the mast cell stabilizer, cromolyn sodium, mast cell degranulation was blocked, and intestinal morphology and barrier function were preserved following deep hypothermic circulatory arrest. Furthermore, cromolyn sodium treatment was associated with reduced intestinal neutrophil influx and blunted systemic release of proinflammatory cytokines. CONCLUSION: Our data provide primary evidence that intestinal ischemia/reperfusion is a leading pathophysiologic process in a rat model of deep hypothermic circulatory arrest, and that intestinal injury, and local and systemic inflammatory responses are critically dependent on mast cell activation. This identifies intestinal mast cells as central players in deep hypothermic circulatory arrest-associated responses, and opens novel therapeutic possibilities for patients undergoing this procedure.