Rocio Lopez-Posadas

Differential effects of α4β7 and GPR15 on homing of effector and regulatory T cells from patients with UC to the inflamed gut in vivo

Objective Gut homing of lymphocytes via adhesion molecules has recently emerged as new target for therapy in IBDs. We aimed to analyse the in vivo homing of effector (Teff) and regulatory (Treg) T cells to the inflamed gut via α4β7 and G protein receptor GPR15. Design We assessed the expression of homing receptors on T cells in peripheral blood and inflamed mucosa. We studied the migration pattern and homing of Teff and Treg cells to the inflamed gut using intravital confocal microscopy and FACS in a humanised mouse model in dextran sodium sulfate-treated NSG (NOD.Cg-Prkdcscid-Il2rgtm1Wjl/SzJ) mice. Results Expression of GPR15 and α4β7 was significantly increased on Treg rather than Teff cells in peripheral blood of patients with UC as compared with Crohn’s disease and controls. In vivo analysis in a humanised mouse model showed augmented gut homing of UC Treg cells as compared with controls. Moreover, suppression of UC (but not control) Teff and Treg cell homing was noted upon treatment with the α4β7 antibody vedolizumab. In contrast, siRNA blockade of GPR15 had only effects on homing of Teff cells but did not affect Treg homing in UC. Clinical vedolizumab treatment was associated with marked expansion of UC Treg cells in peripheral blood. Conclusions α4β7 rather than GPR15 is crucial for increased colonic homing of UC Treg cells in vivo, while both receptors control UC Teff cell homing. Vedolizumab treatment impairs homing of UC Treg cells leading to their accumulation in peripheral blood with subsequent suppression of systemic Teff cell expansion.

Blockade of αEβ7 integrin suppresses accumulation of CD8+ and Th9 lymphocytes from patients with IBD in the inflamed gut in vivo

Objective Therapeutically targeting lymphocyte adhesion is of increasing relevance in IBD. Yet, central aspects of the action of antiadhesion compounds are incompletely understood. We investigated the role of αEβ7 and α4β7 integrins and their blockade by vedolizumab and etrolizumab for trafficking of IBD T lymphocytes in an in vivo model of homing to and retention in the inflamed gut. Design We explored integrin expression in patients with IBD by flow cytometry and immunohistochemistry, while regulation of integrins was studied in T cell cultures. The functional relevance of integrins was assessed by adhesion assays and a recently established humanised mouse model in dextran sodium sulfate-treated immunodeficient mice. Results High expression of αEβ7 was noted on CD8+ and CD4+ Th9 cells, while α4β7 was expressed on CD8+, Th2 and Th17 cells. T cell receptor stimulation and transforming growth factor β were key inducers of αEβ7 on human T cells, while butyric acid suppressed αEβ7. In comparison to α4β7 blockade via vedolizumab, blockade of β7 via etrolizumab surrogate antibody superiorly reduced colonic numbers of CD8+ and Th9 cells in vivo after 3 hours, while no difference was noted after 0.5 hours. AEβ7 expression was higher on CD8+ T cells from patients with IBD under vedolizumab therapy. Conclusions AEβ7 is of key relevance for gut trafficking of IBD CD8+ T cells and CD4+ Th9 cells in vivo and mainly retention might account for this effect. These findings indicate that blockade of αEβ7 in addition to α4β7 may be particularly effective in intestinal disorders with expansion of CD8+ and Th9 cells such as IBD.

The α4β1 Homing Pathway Is Essential for Ileal Homing of Crohn's Disease Effector T Cells In Vivo.

Background: The precise mechanisms controlling homing of T effector (Teff) cells to the inflamed gut in Crohns disease (CD) are still unclear, and clinical outcome data from patients with inflammatory bowel disease treated with the anti-&agr;4&bgr;7 integrin antibody vedolizumab suggest differences between ulcerative colitis and CD. Methods: Expression of homing molecules was studied with flow cytometry and immunohistochemistry. Their functional role was investigated in in vitro adhesion assays and in a humanized mouse model of T cell homing to the inflamed gut in vivo. Results: Despite in vitro blockade of CD Teff adhesion to mucosal vascular addressin cell adhesion molecule-1 (MadCAM-1) and in contrast to previous observations in ulcerative colitis, anti-&agr;4&bgr;7 treatment did not result in reduced Teff cell homing to the colon in vivo. However, the integrin &agr;4&bgr;1 was expressed in higher levels on Teffs from patients with CD compared with controls, while its expression in the peripheral blood declined, and its expression in the intestine increased during the course of clinical vedolizumab treatment. Consistently, adhesion of CD Teffs to vascular cell adhesion molecule-1 (VCAM-1) was blocked by inhibition of &agr;4 and &agr;4&bgr;1 in vitro. Moreover, in vivo homing of CD Teffs to the ileum was reduced by inhibition of &agr;4 and &agr;4&bgr;1 integrins, but not &agr;4&bgr;7 integrins. Conclusions: Our findings suggest that Teff cell homing to the ileum through the axis &agr;4&bgr;1–VCAM-1 is an essential and nonredundant pathway in CD in vivo, possibly affecting efficacy of clinical treatment with antiadhesion compounds.

Rho-A prenylation and signaling link epithelial homeostasis to intestinal inflammation

Although defects in intestinal barrier function are a key pathogenic factor in patients with inflammatory bowel diseases (IBDs), the molecular pathways driving disease-specific alterations of intestinal epithelial cells (IECs) are largely unknown. Here, we addressed this issue by characterizing the transcriptome of IECs from IBD patients using a genome-wide approach. We observed disease-specific alterations in IECs with markedly impaired Rho-A signaling in active IBD patients. Localization of epithelial Rho-A was shifted to the cytosol in IBDs, and inflammation was associated with suppressed Rho-A activation due to reduced expression of the Rho-A prenylation enzyme geranylgeranyltransferase-I (GGTase-I). Functionally, we found that mice with conditional loss of Rhoa or the gene encoding GGTase-I, Pggt1b, in IECs exhibit spontaneous chronic intestinal inflammation with accumulation of granulocytes and CD4+ T cells. This phenotype was associated with cytoskeleton rearrangement and aberrant cell shedding, ultimately leading to loss of epithelial integrity and subsequent inflammation. These findings uncover deficient prenylation of Rho-A as a key player in the pathogenesis of IBDs. As therapeutic triggering of Rho-A signaling suppressed intestinal inflammation in mice with GGTase-I-deficient IECs, our findings suggest new avenues for treatment of epithelial injury and mucosal inflammation in IBD patients.

Immunomodulatory properties of the protein fraction from Phorphyra columbina.

The phycobiliproteins from Rhodophyta , R-phycoerythrin (R-PE) and C-phycocyanin (C-PC), have been shown to exert immunomodulatory effects. This study evaluated the effects of a Phorphyra columbina protein fraction (PF) and R-PE and C-PC on rat primary splenocytes, macrophages, and T-lymphocytes in vitro. PF featured various protein species, including R-PE and C-PC. PF showed mitogenic effects on rat splenocytes and was nontoxic to cells except at 1 g L(-1) protein. IL-10 secretion was enhanced by PF in rat splenocytes, macrophages, and especially T-lymphocytes, whereas it was markedly diminished by R-PE and C-PC. The production of pro-inflammatory cytokines by macrophages was inhibited. The effect of PF on IL-10 was evoked by JNK/p38 MAPK and NF-κB-dependent pathways in macrophages and T-lymphocytes. It was concluded that PF has immunomodulatory effects on macrophages and lymphocytes that appear to be predominantly anti-inflammatory via up-regulated IL-10 production and cannot be accounted for by R-PE and C-PC.

Designer Thiopurine-analogues for Optimised Immunosuppression in Inflammatory Bowel Diseases

BACKGROUND AND AIMS The clinical use of azathioprine and 6-mercaptopurine is limited by their delayed onset of action and potential side effects such as myelosuppression and hepatotoxicity. As these drugs specifically target the Vav1/Rac1 signalling pathway in T lamina propria lymphocytes via their metabolite 6-thio-GTP, we studied expression and optimised suppression of this pathway in inflammatory bowel diseases [IBD]. METHODS Rac1 and Vav1 expressions were analysed in mucosal immune cells in IBD patients. Targeted molecular modelling of the 6-thio-GTP molecule was performed to optimise Rac1 blockade; 44 modified designer thiopurine-analogues were tested for apoptosis induction, potential toxicity, and immunosuppression. Activation of the Vav1/Rac1 pathway in lymphocytes was studied in IBD patients and in lamina propria immune cells in the presence or absence of thiopurine-analogues. RESULTS Several thiopurine-analogues induced significantly higher T cell apoptosis than 6-mercaptopurine. We identified a compound, denoted B-0N, based on its capacity to mediate earlier and stronger induction of T cell apoptosis than 6-mercaptopurine. B-0N-treatment resulted in accelerated inhibition of Rac1 activity in primary peripheral blood T cells as well as in intestinal lamina propria immune cells. Compared with 6-thio-GTP and 6-mercaptopurine, B-0N-treatment was associated with decreased myelo- and hepatotoxicity. CONCLUSIONS The Vav1/Rac1 pathway is activated in mucosal immune cells in IBD. The designer thiopurine-analogue B-0N induces immunosuppression more potently than 6-mercaptopurine.

Genomic analysis of sulfasalazine effect in experimental colitis is consistent primarily with the modulation of NF-κB but not PPAR-γ signaling

Objective Sulfasalazine (SAZ) is a widely used drug in inflammatory bowel disease patients but its mechanism of action is incompletely understood. The objective of our study was to further characterize SAZ mechanism by studying its effect on the colonic transcriptome in a suitable preclinical model of inflammatory bowel disease. Methods The trinitrobenzenesulfonic acid model of colitis in rats was used. The effect of SAZ on mRNA expression was assessed with Affymetrix Rat Expression 230 2.0 arrays used in triplicate (sextuplicate for controls), validated in separate samples with quantitative reverse-time PCR analysis. Some nongenomic experiments were also carried out. Results SAZ (500 mg/kg) had a marked anti-inflammatory effect as expected, which was correlated with a dramatic impact on colonic gene expression. In addition to immune/inflammatory genes, SAZ responsive genes were involved in distinct metabolic and signaling pathways. The effect of sulfasalazine was generally of normalization of colitis-modulated genes to control levels, with very few exceptions. Postgenomic validation showed an excellent correlation with microarray data and seemed to be slightly more sensitive. SAZ generally modulated the expression of nuclear factor-κB-driven genes. SAZ was also shown to inhibit IκB-&agr; phosphorylation in rat primary splenocytes and in HT29 and IEC18 cells. In contrast, SAZ had only a modest effect on peroxisome proliferator-activated receptor (PPAR)-&ggr;-regulated genes and it was confirmed to induce PPAR-&ggr; in enterocytes but not splenocytes. Conclusion Mechanistically, our data are consistent primarily with nuclear factor-κB inhibition, and there is little evidence of a prominent role of activation of PPAR-&ggr; receptors or antioxidative actions.

Interplay of GTPases and Cytoskeleton in Cellular Barrier Defects during Gut Inflammation

An essential role of the intestine is to build and maintain a barrier preventing the luminal gut microbiota from invading the host. This involves two coordinated physical and immunological barriers formed by single layers of intestinal epithelial and endothelial cells, which avoid the activation of local immune responses or the systemic dissemination of microbial agents, and preserve tissue homeostasis. Accordingly, alterations of epithelial and endothelial barrier functions have been associated with gut inflammation, for example during inflammatory bowel disease (IBD). The discriminative control of nutriment uptake and sealing toward potentially pathological microorganisms requires a profound regulation of para- and transcellular permeability. On the subcellular level, the cytoskeleton exerts key regulatory functions in the maintenance of cellular barriers. Increased epithelial/endothelial permeability occurs primarily as a result of a reorganization of cytoskeletal–junctional complexes. Pro-inflammatory mediators such as cytokines can induce cytoskeletal rearrangements, causing inflammation-dependent defects in gut barrier function. In this context, small GTPases of the Rho family and large GTPases from the Dynamin superfamily appear as major cellular switches regulating the interaction between intercellular junctions and actomyosin complexes, and in turn cytoskeleton plasticity. Strikingly, some of these proteins, such as RhoA or guanylate-binding protein-1 (GBP-1) have been associated with gut inflammation and IBD. In this review, we will summarize the role of small and large GTPases for cytoskeleton plasticity and epithelial/endothelial barrier in the context of gut inflammation.

Molecular pathways driving disease-specific alterations of intestinal epithelial cells

Due to the fact that chronic inflammation as well as tumorigenesis in the gut is crucially impacted by the fate of intestinal epithelial cells, our article provides a comprehensive overview of the composition, function, regulation and homeostasis of the gut epithelium. In particular, we focus on those aspects which were found to be altered in the context of inflammatory bowel diseases or colorectal cancer and also discuss potential molecular targets for a disease-specific therapeutic intervention.

The Bisphosphonate Pamidronate is an Intestinal Antiinflammatory Agent in Rat and Mouse Experimental Colitis.

Background:Statins have antiinflammatory effects at the cardiovascular level because of inhibition of prenylation, which also probably underlies their therapeutic effects in preclinical models of inflammatory bowel disease. Another inhibitor of prenylation, namely alendronate, reduces colitis in rodents. In this study, we aim to explore the therapeutic potential of second-generation, nitrogen-containing bisphosphonates in 3 preclinical models of colitis. Methods:The trinitrobenzenesulfonic acid and dextran sulfate sodium models of rat colitis and the adoptive lymphocyte transfer model of colitis in mice were used. Pamidronate, alendronate, and ibandronate were tested. Treatments were administered in equimolar doses through the oral or intraperitoneal route. The effect of pamidronate on prenylation and cytokine release was assessed in vivo and in vitro. Results:Pretreatment with pamidronate, but not with ibandronate or alendronate, improves chemically induced trinitrobenzenesulfonic acid and dextran sulfate sodium colitis in rats. Moreover, this beneficial effect is extended to lymphocyte transfer colitis. Pamidronate has no effect on intestinal epithelial cells in vitro in terms of cytokine/chemokine release, but enhances IFN-&ggr;, IL-6, and IL-10 production by T cells in coculture. Pamidronate also exerts a direct immunomodulatory effect on T cells, favoring Th1 differentiation and impairing Th17 polarization. Conclusions:Pamidronate presents antiinflammatory and immunomodulatory properties in 3 different models of experimental colitis in rodents. This effect requires oral administration and may involve T cells in the gut mucosa, although the exact mechanism is unclear.