Background and AimsThe clinical onset and severity of intestinal disorders in humans and animals can be profoundly impacted by early life stress. Here we investigated the impact of early weaning stress in pigs on intestinal physiology, clinical disease, and immune response to subsequent challenge with enterotoxigenic F18 E. coli (ETEC).MethodologyPigs weaned from their dam at 16 d, 18 d, and 20 d of age were given a direct oral challenge of F18 ETEC at 26 d of age. Pigs were monitored from days 0 to 4 post-infection for clinical signs of disease. On Day 4 post-ETEC challenge, ileal barrier function, histopathologic and inflammatory cytokine analysis were performed on ileal mucosa.ResultsEarly weaned pigs (16 d and 18 d weaning age) exhibited a more rapid onset and severity of diarrhea and reductions in weight gain in response to ETEC challenge compared with late weaned pigs (20 d weaning age). ETEC challenge induced intestinal barrier injury in early weaned pigs, indicated by reductions in ileal transepithelial electrical resistance (TER) and elevated FD4 flux rates, in early weaned pig ileum but not in late weaned pigs. ETEC-induced marked elevations in IL-6 and IL-8, neutrophil recruitment, and mast cell activation in late-weaned pigs; these responses were attenuated in early weaned pigs. TNF levels elevated in ETEC challenged ileal mucosa from early weaned pigs but not in other weaning age groups.ConclusionsThese data demonstrate the early weaning stress can profoundly alter subsequent immune and physiology responses and clinical outcomes to subsequent infectious pathogen challenge. Given the link between early life stress and gastrointestinal diseases of animals and humans, a more fundamental understanding of the mechanisms by which early life stress impacts subsequent pathophysiologic intestinal responses has implications for the prevention and management of important GI disorders in humans and animals.
Mast cell corticotropin-releasing factor subtype 2 suppresses mast cell degranulation and limits the severity of anaphylaxis and stress-induced intestinal permeability. Permalink https://escholarship.org/uc/item/8t3660m4 Journal Abstract 33 34 Background: Psychological stress and heightened MC activation are linked with important 35 immunological disorders including allergy, anaphylaxis, asthma, and functional bowel 36diseases, but the mechanisms remain poorly defined. We have previously demonstrated that 37 activation of the corticotropin releasing factor (CRF) system potentiates MC degranulation 38 responses during IgE-mediated anaphylaxis and psychological stress, via CRF receptor 39 subtype 1 (CRF 1 ) expressed on MCs. 40 41 Objective: In this study, we investigated the role of CRF receptor subtype 2 (CRF 2 ) as a 42 modulator of stress-induced MC degranulation and associated disease pathophysiology. 43 44 Methods: In vitro MC degranulation assays were performed with bone marrow derived MCs 45 (BMMCs) derived from WT and CRF 2 -deficient (CRF 2 -/-) mice and RBL-2H3 MCs transfected 46 with CRF 2 -overexpressing plasmid or CRF 2 -siRNA. In vivo MC responses and associated 47 pathophysiology in IgE-mediated passive systemic anaphylaxis (PSA) and acute 48 psychological restraint stress were measured in WT, CRF 2 -/-, and MC-deficient Kit W-sh/W-sh 49 knock-in mice. 50 51 Results: Compared with WT mice, CRF 2 -/exhibited heightened serum histamine levels and 52 exacerbated PSA-induced anaphylactic responses and colonic permeability. In addition, 53 CRF 2 -/mice exhibited increased serum histamine and colonic permeability following acute 54 restraint stress. Experiments with BMMCs and RBL-2H3 MCs demonstrated that CRF 2 55 expressed on MCs suppresses store-operated Ca 2+ entry (SOCE) signaling and MC 56 M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 3 degranulation induced by diverse MC stimuli. Experiments with MC-deficient Kit W-sh/W-sh mice 57 systemically engrafted with WT and CRF 2 -/-BMMCs demonstrated the functional importance 58 of MC-CRF 2 in modulating stress-induced pathophysiology. 59 60 Conclusions: MC CRF 2 is a negative, global modulator of stimuli-induced MC degranulation 61 and limits the severity of IgE-mediated anaphylaxis and stress-related disease pathogenesis. 62 63 Key messages 64 • Loss of CRF 2 function induces exacerbated MC degranulation, IgE-mediated 65 anaphylaxis and psychological stress-induced intestinal barrier dysfunction. 66 • MC-specific CRF 2 suppresses degranulation induced by diverse MC stimuli via 67 negative regulation of SOCE. 68 • Further characterization of the mechanisms by which CRF 2 negatively modulates MC 69 activation could lead to novel therapeutic approaches for stress-related immunological 70 disorders associated with MC hyperactivity. 71 72
Background Cardiac surgery, especially when employing cardiopulmonary bypass (CPB) and deep hypothermic circulatory arrest (DHCA), 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 (MC) are specialized immune cells that secrete various preformed effectors in response to cellular stress. We hypothesized that MCs are activated in a microenvironment shaped by intestinal ischemia/reperfusion (I/R), and investigated local and systemic consequences in a rat model of DHCA. Methods and Results Adult rats were cooled to 16-18°C on CPB before instituting DHCA for 45 minutes. Specimens were harvested following rewarming and 2 hours of recovery. Significant intestinal barrier disruption was found, together with macro- and microscopic evidence of I/R injury in ileum and colon but not in lungs or kidneys. Immunofluorescence and toluidine blue staining revealed MC hyperplasia and activation in the gut. Pretreatment with the MC stabilizer cromolyn sodium efficiently blocked MC degranulation and resulted in preserved intestinal morphology and barrier function following DHCA. Furthermore, cromolyn sodium treatment dramatically reduced intestinal neutrophil influx and systemic release of various proinflammatory cytokines. Conclusions Our data provides primary evidence that intestinal I/R is a leading pathophysiologic process in a rat model of DHCA and that intestinal injury and local and systemic inflammatory responses are critically dependent on MC activation. This identifies intestinal MCs as central players in DHCA-associated responses, and opens novel therapeutic possibilities for patients undergoing this procedure.
Life stress is a major risk factor in the onset and exacerbation of mast cell-associated diseases, including allergy/anaphylaxis, asthma, and irritable bowel syndrome. Although it is known that mast cells are highly activated upon stressful events, the mechanisms by which stress modulates mast cell function and disease pathophysiology remains poorly understood. Here, we investigated the role of corticotropin-releasing factor receptor subtype 1 (CRF) in mast cell degranulation and associated disease pathophysiology. In a mast cell-dependent model of IgE-mediated passive systemic anaphylaxis (PSA), prophylactic administration of the CRF-antagonist antalarmin attenuated mast cell degranulation and hypothermia. Mast cell-deficient mice engrafted with CRF bone marrow-derived mast cells (BMMCs) exhibited attenuated PSA-induced serum histamine, hypothermia, and clinical scores compared with wild-type BMMC-engrafted mice. mice engrafted with CRF BMMCs also exhibited suppressed in vivo mast cell degranulation and intestinal permeability in response to acute restraint stress. Genetic and pharmacologic experiments with murine BMMCs, rat RBL-2H3, and human LAD2 mast cells demonstrated that although CRF activation did not directly induce MC degranulation, CRF signaling potentiated the degranulation responses triggered by diverse mast cell stimuli and was associated with enhanced release of Ca from intracellular stores. Taken together, our results revealed a prominent role for CRF signaling in mast cells as a positive modulator of stimuli-induced degranulation and in vivo pathophysiologic responses to immunologic and psychologic stress.
Psychological stress triggers mast cell (MC) activation and is a major risk factor in the onset and exacerbation of MC-associated disorders including allergy/anaphylaxis, irritable bowel syndrome, and autoimmune diseases. The mechanisms by which stress modulates MC function and disease susceptibility is poorly understood. Our previous studies showed that corticotropin releasing factor receptor 1 (CRF1) signaling potentiated stress-induced MC degranulation. Here we utilized CRF2 deficient (CRF2 −/−) mice and MCs to define the in vivo and in vitro biological importance of CRF receptor subtype 2, CRF2. Compared with WT mice, CRF2 −/− mice exhibited greater serum histamine levels following restraint stress (RS) (by 143%) and IgE-mediated passive systemic anaphylaxis (PSA) (by 415%). The heightened response was greater in CRF2 −/− females compared with males. CRF2 −/− mice exhibited greater RS-induced and PSA-induced elevations in intestinal permeability compared to WT mice. Bone marrow derived MCs (BMMCs) derived from CRF2 −/− mice exhibited greater release of β-hexosaminidase and histamine and heightened Ca2+ mobilization from intracellular stores in response to diverse MC stimuli including IgE/DNP, c48/80, and A23187. MC-deficient mice engrafted with CRF2 −/− BMMCs exhibited greater RS-induced intestinal permeability and PSA-induced hypothermia compared with WT BMMC-engrafted mice. Together, these results demonstrate that MC-CRF2 receptor signaling exerts a global suppression of MC degranulation and pathophysiology. Elucidation of the mechanisms by which CRF2 dampens MC degranulation could reveal novel therapeutic targets for hyperactive MC disorders.
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