Influencia del eje interleucina-10/p38 MAPK en el epitelio intestinal sobre la respuesta al tratamiento con glucocorticoides en la colitis ulcerosa

La enfermedad inflamatoria intestinal (EII) está constituida por dos entidades principales: la colitis ulcerosa y la enfermedad de Crohn. La EII es una incurable y compleja patología con una incidencia creciente en nuestro país. Los glucocorticoides son la primera línea terapéutica en la colitis ulcerosa activa, aunque distintos estudios han demostrado que hasta un 60% de los pacientes no responden de forma adecuada al tratamiento esteroidal. Así que el fracaso terapéutico a los glucocorticoides constituye una de las principales complicaciones en el tratamiento de la EII, con una grave repercusión en la evolución del paciente. Estudios previos de nuestro grupo han asociado la presencia de Interleucina (IL)-10 en biopsias intestinales de pacientes con enfermedad de Crohn activa con una buena respuesta a los glucocorticoides. Por otra parte, la deficiencia de esta misma citocina inmunoreguladora en modelos animales se ha relacionado con un aumento de la permeabilidad intestinal. Asimismo, la IL-10 es capaz de reducir el estrés del retículo endoplasmático en células epiteliales del intestino producido por TNF-α. Además, estudios propios y de otros grupos sugieren un importante protagonismo patológico del epitelio intestinal en la colitis ulcerosa. En conjunto, las premisas anteriores sugieren un papel clave de la IL-10 en la actividad del epitelio intestinal favoreciendo el aislamiento de la lamina propria, facilitando la respuesta a los esteroides en pacientes con colitis ulcerosa activa. Los objetivos de esta Tesis doctoral fueron: a) comprobar in vitro en monocapas de células epiteliales Caco-2, si la presencia de IL-10 puede favorecer la respuesta a los glucocorticoides actuando sobre la función barrera; y b) averiguar si estos cambios in vitro también son percibidos en la mucosa cólica de pacientes con colitis ulcerosa activa con diferente respuesta al tratamiento con glucocorticoides. Los resultados en las monocapas de células Caco-2 demostraron que la presencia de IL-10, conjuntamente con los glucocorticoides, influye sobre el epitelio intestinal reforzando las uniones intercelulares a través de un mecanismo que implica a la fosforilación de p38 MAPK. En estos ensayos comprobamos que la presencia conjunta de IL-10 y glucocorticoides incrementaba los núcleos positivos para p38 MAPK fosforilada en las células epiteliales. En este sentido, la presencia nuclear de la p38 MAPK fosforilada también fue más elevada en las biopsias intestinales previas al tratamiento de pacientes respondedores que en las de los no respondedores, lo que podría suponer mayor capacidad de aislamiento de la lamina propria en los pacientes sensibles al tratamiento. El análisis efectuado por microscopia electrónica de transmisión en células Caco-2 reveló que la principal área de desacoplamiento celular se producía a nivel de desmosomas, fenómeno que conseguía revertir el tratamiento con IL-10 y glucocorticoides. Por otra parte, en pacientes con colitis ulcerosa activa se observó un patrón diferencial en la localización y expresión de los receptores α de IL-10 y glucocorticoides según su respuesta al tratamiento esteroidal. En definitiva, la acción sinérgica de la IL-10 y los glucocorticoides parece favorecer la recuperación del epitelio intestinal lesionado en la colitis ulcerosa activa, fortaleciendo las uniones paracelulares. En esta acción biológica es clave el papel de la p38 MAPK, cuya actividad esta modulada principalmente por la IL-10 que favorece el aislamiento de la lamina propria intestinal frente a los antígenos luminales. De esta manera, la actividad inflamatoria mediada por las células colitogénicas puede verse disminuida, lo que facilitaría la respuesta a los glucocorticoides induciendo su apoptosis. Los cambios moleculares que afectan a la respuesta frente a los glucocorticoides pueden ser detectados en el epitelio intestinal de pacientes con colitis ulcerosa, sugiriendo su potencial papel como dianas en el tratamiento con esteroides.Inflammatory bowel disease (IBD) consists in two main clinical forms: ulcerative colitis and Crohn's disease. IBD is a complex and chronic disease with an increasing incidence in our country. Glucocorticoids are the first-line therapy in active ulcerative colitis, however, several studies have shown that up to 60% of patients do not respond adequately to steroid treatment. So the glucocorticoid treatment failure is an important complication in the treatment of IBD, with hight impact on patient evolution. Previous studies from our group have associated the presence of interleukin (IL)-10 in intestinal biopsies of patients with active Crohn's disease with a favourable response to glucocorticoid treatment. Moreover, the Il-10 deficiency in animal models has been associated with an increased intestinal permeability. Likewise,, IL-10 is capable of reducing TNF-α endoplasmic reticulum stress in intestinal epithelial cells. Our group and other groups suggest an important role of intestinal epithelium in ulcerative colitis. Taken together these last premises , it is been suggested that IL-10 plays a key role in intestinal epithelial promoting the lamina propria sealing and enabling steroid response in patients with active ulcerative colitis. The objectives of this thesis were: a) test in vitro, in Caco-2 epithelial monolayers cells, if the presence of IL-10 facilitates the glucocorticoid response acting on the barrier function, and b) determine whether these changes in vitro also are observed in the colonic mucosa of active ulcerative colitis patients with different response to glucocorticoid treatment. The results in Caco-2 monolayers cells showed that IL-10 in combination with glucocorticoids, influence the strengthening intestinal epithelium junctions through a mechanism involving the p38 MAPK phosphorylation. In these studies we found that the presence of IL-10 and glucocorticoids, increase positive nuclei p38 MAPK phosphorylation in epithelial cells. In this sense, the nuclear presence of phosphorylated p38 MAPK was higher in intestinal biopsies prior treatment in responders compared to non-responders patients, fact that may involve a higher sealing capacity of lamina propria in patients who were sensitive to treatment. Analysis by transmission electron microscopy in Caco-2 cells revealed that the main area of cell detachment occurred at desmosomes level, which was reversed getting IL-10 and glucocorticoids treatment. Moreover, in patients with active ulcerative colitis a differential pattern in the location and expression of IL-10 and glucocorticoid alpha receptor was observed depending of their steroid treatment response. In short, the synergistic action of IL-10 and glucocorticoids seems to favor the recovery of injured intestinal epithelium in active ulcerative colitis, strengthening the paracellular junctions. In this biological action p38 MAPK has a key role. Its activity is modulated mainly by IL-10 and favors the intestinal lamina propria sealing against luminal antigens. Thus the inflammatory cells activity may be diminished, thereby facilitating the apoptotic glucocorticoid response. Molecular changes that affect the glucocorticoid response can be detected in the intestinal epithelium of ulcerative colitis patients, suggesting their role as potential targets in the steroid treatment

Interleukin-10 enhances the intestinal epithelial barrier in the presence of corticosteroids through p38 MAPK activity in Caco-2 monolayers : a possible mechanism for steroid responsiveness in ulcerative colitis

Altres ajuts: 2012 Spanish Gastroenterological Association i CIBER G0034Glucocorticosteroids are the first line therapy for moderate-severe flare-ups of ulcerative colitis. Despite that, up to 60% of patients do not respond adequately to steroid treatment. Previously, we reported that low IL-10 mRNA levels in intestine are associated with a poor response to glucocorticoids in active Crohn's disease. Here, we test whether IL-10 can favour the response to glucocorticoids by improving the TNFα-induced intestinal barrier damage (assessed by transepithelial electrical resistance) in Caco-2 monolayers, and their possible implications on glucocorticoid responsiveness in active ulcerative colitis. We show that the association of IL-10 and glucocorticoids improves the integrity of TNFα-treated Caco-2 cells and that p38 MAPK plays a key role. In vitro, IL-10 facilitates the nuclear translocation of p38 MAPK-phosphorylated thereby modulating glucocorticoids-receptor-α, IL-10-receptor-α and desmoglein-2 expression. In glucocorticoids-refractory patients, p38 MAPK phosphorylation and membrane desmoglein-2 expression are reduced in colonic epithelial cells. These results suggest that p38 MAPK-mediated synergism between IL-10 and glucocorticoids improves desmosome straightness contributing to the recovery of intestinal epithelium and reducing luminal antigens contact with lamina propria in ulcerative colitis. This study highlights the link between the intestinal epithelium in glucocorticoids-response in ulcerative colitis

IL-10 and GC preserve p38 MAPK-dependent desmosome integrity and strength in Caco-2 monolayers.

<p>Representative transmission electron microscopy images of the paracellular junctions in Caco-2 cell monolayers treated with GC (Panel A), GC plus IL-10 (Panel B) or GC, IL-10 and SB203580 (Panel C) D: desmosomes; TJ: tight junctions. Magnification varies from 25,000 x to 100,000 x.</p

Interleukin-10 enhances the intestinal epithelial barrier in the presence of corticosteroids through p38 MAPK activity in Caco-2 monolayers : a possible mechanism for steroid responsiveness in ulcerative colitis

Altres ajuts: 2012 Spanish Gastroenterological Association i CIBER G0034Glucocorticosteroids are the first line therapy for moderate-severe flare-ups of ulcerative colitis. Despite that, up to 60% of patients do not respond adequately to steroid treatment. Previously, we reported that low IL-10 mRNA levels in intestine are associated with a poor response to glucocorticoids in active Crohn's disease. Here, we test whether IL-10 can favour the response to glucocorticoids by improving the TNFα-induced intestinal barrier damage (assessed by transepithelial electrical resistance) in Caco-2 monolayers, and their possible implications on glucocorticoid responsiveness in active ulcerative colitis. We show that the association of IL-10 and glucocorticoids improves the integrity of TNFα-treated Caco-2 cells and that p38 MAPK plays a key role. In vitro, IL-10 facilitates the nuclear translocation of p38 MAPK-phosphorylated thereby modulating glucocorticoids-receptor-α, IL-10-receptor-α and desmoglein-2 expression. In glucocorticoids-refractory patients, p38 MAPK phosphorylation and membrane desmoglein-2 expression are reduced in colonic epithelial cells. These results suggest that p38 MAPK-mediated synergism between IL-10 and glucocorticoids improves desmosome straightness contributing to the recovery of intestinal epithelium and reducing luminal antigens contact with lamina propria in ulcerative colitis. This study highlights the link between the intestinal epithelium in glucocorticoids-response in ulcerative colitis

IL-10 reverts the GC-mediated decrease in phosphorylated-p38 MAPK activity.

<p>Figure shows (median (IQR, limits)) the ratio for total p38 MAPK (Panel A) and phosphorylated p38 MAPK (Panel B) versus control group in TNF-treated Caco-2 cell monolayers. An example of Western blot bands obtained for these proteins and α-tubulin (as housekeeping protein) is provided in Panel C. *p≤ 0.026 <i>vs</i>. all other groups, ^#p = 0.028 <i>vs</i>. TNF_IL10 and TNF_GC_IL10;.</p

IL-10 and GC restore control TEER values in Caco-2 cell monolayers.

<p> Figure represents median values (IQR, limits) of percent TEER changes in Caco-2 cell monolayers after 48h of incubation with different stimuli. *p ≤ 0.02 <i>vs</i> control and TNF_GC_IL10.</p

Steroid-sensitive UC patients show a greater DSG2 staining.

<p>Immunofluorescence staining score (mean±SEM) of DSG2 (Panel A) in colonic biopsies of steroid-sensitive and steroid-refractory UC patients, before and after treatment. Representative DSG2 staining intensity of steroid-sensitive (Panel B) and steroid-refractory (Panel C) patients are also provided. All images are 630 x. *p≤ 0.004 <i>vs</i> steroid-refractory patients, ^#p = 0.023 <i>vs</i> after GC treatment.</p

SB203580 reverses the synergistic action of IL-10 and GC on TEER in Caco-2 cell monolayers.

<p>Values are median (IQR, limits) of percent TEER changes in Caco-2 cell monolayers after 48h of incubation with different stimuli. *p≤ 0.042 <i>vs</i> control.</p

p38 MAPK phosphorylation is reduced in colonic epithelial cells from GC-refractory UC patients before treatment.

<p>Panel A shows the immunostaining score (mean±SEM) of phosphorylated p38 MAPK in colonic biopsies of steroid-responsive and steroid-refractory active UC patients, before and after GC treatment. *p = 0.028 <i>vs</i> steroid-sensitive patients before treatment. Panel B provides representative images from steroid-refractory UC biopsies (score value 0–1), and from steroid-sensitive UC biopsies (score value 2–3). Scale bar: 20 μm.</p

Response Variability to Drug Testing in Two Models of Chemically Induced Colitis

The lack of knowledge regarding the pathogenesis of IBD is a challenge for the development of more effective and safer therapies. Although in vivo preclinical approaches are critical for drug testing, none of the existing models accurately reproduce human IBD. Factors that influence the intra-individual response to drugs have barely been described. With this in mind, our aim was to compare the anti-inflammatory efficacy of a new molecule (MTADV) to that of corticosteroids in TNBS and DSS-induced colitis mice of both sexes in order to clarify further the response mechanism involved and the variability between sexes. The drugs were administered preventively and therapeutically, and real-time bioluminescence was performed for the in vivo time-course colitis monitoring. Morphometric data were also collected, and colonic cytokines and acute plasma phase proteins were analyzed by qRT-PCR and ELISA, respectively—bioluminescence images correlated with inflammatory markers. In the TNBS model, dexamethasone worked better in females, while MTADV improved inflammation in males. In DSS-colitis, both therapies worked similarly. Based on the molecular profiles, interaction networks were constructed to pinpoint the drivers of therapeutic response that were highly dependent on the sex. In conclusion, our results suggest the importance of considering sex in IBD preclinical drug screening