Modulation of intestinal barrier function by glucocorticoids: Lessons from preclinical models

This work was supported by the "Centro de Investigacion Biomedica en Red de Enfermedades Hepaticas y Digestivas (CIBERehd)", belonging to Instituto de Salud Carlos III, Spain, and grants from: Ministry of Economy and Competitivity, partly with Fondo Europeo de Desarrollo Regional FEDER funds [SAF2017-88457-R, AGL2017-85270-R]; "Junta de Andalucia", Spain [CTS235, CTS164]; "Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III", Spain (PI19/00819), co-funded by European Regional Development Fund/European Social Fund, "Investing in your future"; "Junta de Castilla y Leon" (SA074P20),Spain; "Fundacio Marato TV3'' (201916-31), Spain; AECC Scientific Foundation (2017/2020), Spain; and "Centro Internacional sobre el Envejecimiento" (OLD-HEPAMARKER, 0348_CIE_6_E), Spain. MAA and MTG were supported by fellowships from the Ministry of Education. MA was supported by a postdoctoral contract with the CIBERehd. Funding for open access charge: Universidad de Granada/CBUA.Glucocorticoids (GCs) are widely used drugs for their anti-inflammatory and immunosuppressant effects, but they are associated with multiple adverse effects. Despite their frequent oral administration, relatively little attention has been paid to the effects of GCs on intestinal barrier function. In this review, we present a summary of the published studies on this matter carried out in animal models and cultured cells. In cultured intestinal epithelial cells, GCs have variable effects in basal conditions and generally enhance barrier function in the presence of inflammatory cytokines such as tumor necrosis factor (TNF). In turn, in rodents and other animals, GCs have been shown to weaken barrier function, with increased permeability and lower production of IgA, which may account for some features observed in stress models. When given to animals with experimental colitis, barrier function may be debilitated or strengthened, despite a positive anti-inflammatory activity. In sepsis models, GCs have a barrier-enhancing effect. These effects are probably related to the inhibition of epithelial cell proliferation and wound healing, modulation of the microbiota and mucus production, and interference with the mucosal immune system. The available information on underlying mechanisms is described and discussed."Centro de Investigacion Biomedica en Red de Enfermedades Hepaticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, SpainMinistry of Economy and CompetitivityEuropean Commission SAF2017-88457-R AGL2017-85270-RJunta de Andalucia European Commission CTS235 CTS164Instituto de Salud Carlos III PI19/00819European Regional Development Fund/European Social Fund, "Investing in your future"Junta de Castilla y Leon SA074P20Fundacio Marato TV3, Spain 201916-31AECC Scientific Foundation, Spain"Centro Internacional sobre el Envejecimiento" (OLD-HEPAMARKER), Spain 0348_CIE_6_EMinistry of EducationCIBERehdUniversidad de Granada/CBU

Mice carrying an epithelial deletion of the glucocorticoid receptor NR3C1 develop a higher tumor load in experimental colitis associated cancer

The glucocorticoid receptor NR3C1 is expressed in multiple cell types in the gut and elsewhere. Intestinal epithelial cells both produce and respond to glucocorticoids in different physiological and pathological contexts. In experimental colitis glucocorticoids have been shown to exert a dual role, dampening inflammation while producing a deterioration in animal status, including death. Mice with tamoxifen inducible, intestinal epithelial specific deletion of NR3C1 (NR3C1IEC mice) are protected against experimental colitis, suggesting glucocorticoid epithelial actions are deleterious. Since glucocorticoids modulate epithelial proliferation it follows that they may affect the development of colon cancer. In this study we set out to test this hypothesis using the dextran sulfate sodium - azoxymethane model of colitis-associated cancer. KO mice were found to exhibit a 2-fold higher tumor load but similar incidence and tumor size. Tumors had a higher trend to extend to the submucosal layer (36% vs. 0%) in NR3C1IEC mice, and overexpressed Lgr5, Egfr and Myc, consistent with increased proliferation and neoplastic transformation. Snai1 and Snai2 were upregulated specifically in tumors of NR3C1ΔIEC mice, suggesting enhanced epithelial to mesenchymal transition in the absence of the intestinal epithelial GC receptor. We conclude that endogenous GC epithelial signaling is involved in colitis associated cancer.This work was supported by funds from the Ministry of Economy and Competitivity, partly with Fondo Europeo de Desarrollo Regional (FEDER) funds [SAF2017-88457-R, AGL2017-85270-R, BFU2014-57736-P, AGL2014-58883-R] and by Junta de Andalucía [CTS235, CTS164]. MA and CJA were supported by the University of Granada (Contrato Puente Program - Plan Propio) and the Ministry of Education [Spain], respectively. CIBERehd is funded by Instituto de Salud Carlos III

Leptin-resistant Zucker rats with trinitrobenzene sulfonic acid colitis present a reduced inflammatory response but enhanced epithelial damage

The study was funded the following grants of the Ministerio de Economía y Competitividad and the Fondo Europeo de Desarrollo Regional FEDER (SAF2011-22922, SAF2011-22812, BFU2014- 57736-P, and AGL2014-58883-R) and Junta de Andalucía (CTS164, CTS235, and CTS6736). B. Rivero-Guti errez, R. Gámez-Belmonte, and M. Arredondo-Amador were supported by fellowships from the Ministerio de Educación.The role of leptin in the development of intestinal inflammation remains controversial, since proinflammatory and anti-inflammatory effects have been described. This study describes the effect of the absence of leptin signaling in intestinal inflammation. Experimental colitis was induced by intrarectal administration of trinitrobenzene sulfonic acid (TNBS) to lean and obese Zucker rats (n = 10). Effects on inflammation and mucosal barrier were studied. Bacterial translocation and LPS concentration were evaluated together with colonic permeability to 4-kDa FITC-dextran. Obese Zucker rats showed a lower intestinal myeloperoxidase and alkaline phosphatase activity, reduced alkaline phosphatase sensitivity to levamisole, and diminished colonic expression of Nos2, Tnf, and Il6, indicating attenuated intestinal inflammation, associated with attenuated STAT3, AKT, and ERK signaling in the colonic tissue. S100a8 and Cxcl1 mRNA levels were maintained, suggesting that in the absence of leptin signaling neutrophil activation rather than infiltration is hampered. Despite the lower inflammatory response, leptin resistance enhanced intestinal permeability, reflecting an increased epithelial damage. This was shown by augmented LPS presence in the portal vein of colitic obese Zucker rats, associated with induction of tissue nonspecific alkaline phosphatase, LPS-binding protein, and CD14 hepatic expression (involved in LPS handling). This was linked to decreased ZO-1 immunoreactivity in tight junctions and lower occludin expression. Our results indicate that obese Zucker rats present an attenuated inflammatory response to TNBS, but increased intestinal epithelial damage allowing the passage of bacterial antigens.Ministerio de Economía y Competitividad and the Fondo Europeo de Desarrollo Regional FEDER (SAF2011-22922, SAF2011-22812, BFU2014- 57736-P, and AGL2014-58883-R)Junta de Andalucía (CTS164, CTS235, and CTS6736)Ministerio de Educació

Epithelial deletion of the glucocorticoid receptor protects the mouse intestine against experimental inflammation

Intestinal epithelium glucocorticoid receptor knockout mice (NR3C1 IEC) were treated with dextran sulfate sodium (DSS, 2.5%) to induce colitis. Inflammatory status was assessed by morphological and biochemical methods and corticoid production was measured in colonic explants. Key Results. After 7 days of DSS NR3C1 mice exhibited lower weight loss and tissue damage, reduced colonic expression of S100A9, attenuated phosphorylation of STAT3 and a better overall state compared with WT. Ki67 immunoreactivity was also shifted, indicating an effect on epithelial proliferation. A subgroup of mice were treated with budesonide and showed completely prevented budesonide induced weight loss. Epithelial deletion of the glucocorticoid receptor also protected mice in a protracted colitis protocol. Conversely knockout mice presented a worse status compared to the control group at 1 day post DSS, as shown by blood in feces and increased inflammatory parameters. In a separate experiment colonic corticosterone production was shown to be significantly increased in knockout mice at 7 days of colitis but not at earlier stages. Conclusions and Implications. The intestinal epithelial glucocorticoid receptor has deleterious effects in experimental colitis induced by dextran sodium sulfate, probably related to inhibition of epithelial proliferative responses leading to impaired wound healing and reduced endogenous corticosterone production.Ministry of Economy and Competitivity, partly with Fondo Europeo de Desarrollo Regional FEDER funds [BFU2014-57736-P, AGL2014-58883-R, SAF2017-88457-R, AGL2017-85270-R]Junta de Andalucía [CTS235, CTS164]Ministry of EducationCIBERehd is funded by Instituto de Salud Carlos II

Implicación del receptor de glucocorticoides NR3C1 del epitelio intestinal en la respuesta a esteroides, la esteroidogénesis y la neoplasia

Conclusions. 1. The intestinal epithelial glucocorticoid receptor is basally activated in the mouse colon in vivo. Its loss provokes transitory colonic inflammation, presumably due to alterations in the intestinal permeability and/or a higher proinflammatory signaling by the epithelium and infiltrated TCRγδ+ lymphocytes, where the intestinal microbiota seems to play an essential role, as this response is not present in the small intestine. 2. The intestinal epithelial glucocorticoid receptor has a dual role in DSS-induced experimental colitis. Initially, it exerts a deleterious effect, as its absence worsens animal status in the early phase of exposure to DSS. However, as the inflammatory response is established, the deletion of the receptor is clearly protective, to the point of preventing 48% of animal deaths in a chronic colitis protocol. 3. Budesonide treatment of experimental colitis induced by DSS at supratherapeutic doses is strengthened by the absence of the intestinal epithelial glucocorticoid receptor. This effect is observed only in female mice. The mechanism may be related to a specific interaction of the receptor, or simply to the fact that inflammation is more pronounced in females. These results suggest that the clinical response of intestinal inflammation to glucocorticoids could be improved in the absence of epithelial actions. 4. The intestinal epithelial NR3C1 collaborates in the regulation of the novo corticosterone synthesis, possibly through a negative feedback. The resulting increased intestinal corticosterone synthesis is correlated to the protection against colitis, and therefore could be mechanistically involved. 5. In mice, intestinal steroidogenesis is sufficient to the maintenance of the local levels of corticosterone, while in the skin a partial dependence is observed. 6. The deletion of intestinal epithelial NR3C1 increases cellular proliferation and causes a higher tumor load in the AOM/DSS induced experimental colitis-associated colorectal cancer model, although these tumors are less invasive.1. El receptor epitelial de glucocorticoides se encuentra activado de forma basal en las células epiteliales intestinales en el ratón. Su pérdida genera una inflamación colónica transitoria debida a alteraciones en la permeabilidad intestinal y/o a una mayor señalización proinflamatoria por parte del epitelio y de linfocitos infiltrados, donde la microbiota parece tener un papel fundamental, en la medida en que dicha respuesta no se manifiesta en el intestino delgado. 2. El receptor epitelial intestinal de glucocorticoides tiene un papel dual en la respuesta a la colitis experimental inducida por DSS. Inicialmente ejerce un efecto perjudicial, ya que su deleción potencia el daño en la fase temprana de la exposición a DSS. Sin embargo, a medida que se desarrolla la respuesta inflamatoria la deleción del receptor es netamente protectora, hasta el punto de evitar la muerte del 48% de los animales en un protocolo crónico de colitis. 3. El tratamiento de la colitis experimental inducida por DSS con dosis supraterapéuticas de budesonida se ve potenciado por la ausencia del receptor epitelial intestinal de glucocorticoides. Este efecto se observa únicamente en hembras, lo que puede deberse a una interacción receptorial específica o bien simplemente a la mayor gravedad de la inflamación en las mismas. Estos resultados sugieren que la respuesta clínica de la inflamación intestinal a glucocorticoides podría ser mejorada en ausencia de acciones a nivel epitelial. 4. El GR del epitelio intestinal participa en la regulación de la síntesis de corticosterona de novo, posiblemente a través de una retroalimentación negativa. La potenciación de la síntesis intestinal de corticosterona se correlaciona con la protección frente a la colitis, y por tanto puede estar implicada desde el punto de vista mecanístico. 5. En el ratón la esteroidogénesis intestinal es suficiente para el mantenimiento de los niveles locales de corticosterona, mientras que en la piel existe una dependencia parcial. 6. La deleción del GR del epitelio intestinal incrementa la proliferación celular y provoca una mayor carga tumoral en el cáncer colorrectal asociado a colitis experimental inducido por AOM/DSS, aunque estos tumores son menos invasivos.Tesis Univ. Granada

Epithelial deletion of the glucocorticoid receptor ( Nr3c1

Intestinal epithelium glucocorticoid receptor knockout mice (NR3C1 IEC) were treated with dextran sulfate sodium (DSS, 2.5%) to induce colitis. Inflammatory status was assessed by morphological and biochemical methods and corticoid production was measured in colonic explants. Key Results. After 7 days of DSS NR3C1 mice exhibited lower weight loss and tissue damage, reduced colonic expression of S100A9, attenuated phosphorylation of STAT3 and a better overall state compared with WT. Ki67 immunoreactivity was also shifted, indicating an effect on epithelial proliferation. A subgroup of mice were treated with budesonide and showed completely prevented budesonide induced weight loss. Epithelial deletion of the glucocorticoid receptor also protected mice in a protracted colitis protocol. Conversely knockout mice presented a worse status compared to the control group at 1 day post DSS, as shown by blood in feces and increased inflammatory parameters. In a separate experiment colonic corticosterone production was shown to be significantly increased in knockout mice at 7 days of colitis but not at earlier stages. Conclusions and Implications. The intestinal epithelial glucocorticoid receptor has deleterious effects in experimental colitis induced by dextran sodium sulfate, probably related to inhibition of epithelial proliferative responses leading to impaired wound healing and reduced endogenous corticosterone production.Ministry of Economy and Competitivity, partly with Fondo Europeo de Desarrollo Regional FEDER funds [BFU2014-57736-P, AGL2014-58883-R, SAF2017-88457-R, AGL2017-85270-R]Junta de Andalucía [CTS235, CTS164]Ministry of EducationCIBERehd is funded by Instituto de Salud Carlos II

Calprotectin protects against experimental colonic inflammation in mice.

Calprotectin is a heterodimer composed of two myeloid-related proteins, S100A8 and S100A9, that is abundant in neutrophils and monocytes/macrophages. Faecal levels of calprotectin are used routinely to monitor inflammatory bowel disease activity. We aimed to assess the role of calprotectin in intestinal inflammation, using the dextran sulfate sodium model of colitis in mice. Calprotectin was administered (50 or 100 μg·day-1 ) by the intrarectal or by i.p. injection (50 μg·day-1 only). The condition of the mice was characterized by morphological and biochemical methods. Intrarectal calprotectin protected significantly against colitis, as shown by lower levels of macroscopic and microscopic damage, colonic myeloperoxidase activity and decreased expression of TNFα and toll-like receptor 4. IL-17 production by spleen and mesenteric lymph node cells was reduced. Calprotectin had no effect on body weight loss or colonic thickening. There were no effects of calprotectin after i.p. injection. Calprotectin had virtually no effects in control, non-colitic mice. Calprotectin had almost no effect on the colonic microbiota but enhanced barrier function. Treatment of rat IEC18 intestinal epithelial cells in vitro with calprotectin induced output of the chemokines CXL1 and CCL2, involving the receptor for advanced glycation end products- and NFκB. Calprotectin exerted protective effects in experimental colitis when given by the intrarectal route, by actions that appear to involve effects on the epithelium

Modulation of intestinal barrier function by glucocorticoids: Lessons from preclinical models.

Glucocorticoids (GCs) are widely used drugs for their anti-inflammatory and immunosuppressant effects, but they are associated with multiple adverse effects. Despite their frequent oral administration, relatively little attention has been paid to the effects of GCs on intestinal barrier function. In this review, we present a summary of the published studies on this matter carried out in animal models and cultured cells. In cultured intestinal epithelial cells, GCs have variable effects in basal conditions and generally enhance barrier function in the presence of inflammatory cytokines such as tumor necrosis factor (TNF). In turn, in rodents and other animals, GCs have been shown to weaken barrier function, with increased permeability and lower production of IgA, which may account for some features observed in stress models. When given to animals with experimental colitis, barrier function may be debilitated or strengthened, despite a positive anti-inflammatory activity. In sepsis models, GCs have a barrier-enhancing effect. These effects are probably related to the inhibition of epithelial cell proliferation and wound healing, modulation of the microbiota and mucus production, and interference with the mucosal immune system. The available information on underlying mechanisms is described and discussed

Cu-Doped ZnO Thin Films Deposited by a Sol-Gel Process Using Two Copper Precursors: Gas-Sensing Performance in a Propane Atmosphere

A study on the propane gas-sensing properties of Cu-doped ZnO thin films is presented in this work. The films were deposited on glass substrates by sol-gel and dip coating methods, using zinc acetate as a zinc precursor, copper acetate and copper chloride as precursors for doping. For higher sensitivity values, two film thickness values are controlled by the six and eight dippings, whereas for doping, three dippings were used, irrespective of the Cu precursor. The film structure was analyzed by X-ray diffractometry, and the analysis of the surface morphology and film composition was made through scanning electron microscopy (SEM) and secondary ion mass spectroscopy (SIMS), respectively. The sensing properties of Cu-doped ZnO thin films were then characterized in a propane atmosphere, C3H8, at different concentration levels and different operation temperatures of 100, 200 and 300 °C. Cu-doped ZnO films doped with copper chloride presented the highest sensitivity of approximately 6 × 104, confirming a strong dependence on the dopant precursor type. The results obtained in this work show that the use of Cu as a dopant in ZnO films processed by sol-gel produces excellent catalysts for sensing C3H8 gas

Circadian Rhythms in Hormone-sensitive Lipase in Human Adipose Tissue: Relationship to Meal Timing and Fasting Duration.

Fat mobilization in adipose tissue (AT) has a specific timing. However, circadian rhythms in the activity of the major enzyme responsible for fat mobilization, hormone-sensitive lipase (HSL), have not been demonstrated in humans. To analyze in a cross-sectional study whether there is an endogenous circadian rhythm in HSL activity in human AT ex vivo and whether rhythm characteristics are related to food timing or fasting duration. Abdominal AT biopsies were obtained from 18 severely obese participants (age: 46 ± 11 years; body mass index 42 ± 6 kg/m2) who underwent laparoscopic gastric bypass. Twenty-four-hour rhythms of HSL activity and LIPE (HSL transcript in humans) expression in subcutaneous AT were analyzed together with habitual food timing and night fasting duration. HSL activity exhibited a circadian rhythm (P = .023) and reached the maximum value at circadian time 16 (CT) that corresponded to around midnight (relative local clock time. Similarly, LIPE displayed a circadian rhythm with acrophase also at night (P = .0002). Participants with longer night fasting duration >11.20 hours displayed almost double the amplitude (1.91 times) in HSL activity rhythm than those with short duration (P = .013); while habitual early diners (before 21:52 hours) had 1.60 times higher amplitude than late diners (P = .035). Our results demonstrate circadian rhythms in HSL activity and may lead to a better understanding of the intricate relationships between food timing, fasting duration and body fat regulation