Introduction

Glucocorticoids (GC) are important antiinflammatory and immunosuppresive agents in the management of inflammatory bowel disease and many other conditions. However, GC also display deleterious effects on the intestinal barrier which may limit their clinical benefit. These effects may be ascribed partly to actions on the epithelium [1]. In this regard, mice carrying a deletion of the GC receptor in intestinal epithelial cells are protected against experimental colitis [2]. It may be therefore beneficial to limit GC epithelial actions in the inflamed intestine. On the other hand, recent evidence shows that dexamethasone-treated mice aggravate acute experimental colitis due to the activation of mTOR pathway in intestinal epithelial cells [3].

Aims & Methods

Our aim is to limit harmful GC effects in intestinal epithelium in experimental colitis, with the ultimate goal of improving the management of inflammatory bowel disease with GC.
This study features two experiments. First, experimental colitis was induced by administration of 2.5% dextran sulfate sodium (DSS) for 7 days to C57BL/6J mice and treated with prednisolone 200 µg/day by either the oral or parenteral route (as prednisolone phosphate). Secondly, effects of glucocorticoid-dependent mTOR pathway activation were assessed by administering prednisolone at the same dose in combination with rapamycin 60 µg/day, a specific mTOR signal inhibitor, both by the oral route, to DSS-induced colitic C57BL/6J mice, in the same conditions as the first experiment.

Results

​Prednisolone ameliorated colitis, based on downregulated expression of inflammatory markers (S100a8, S100a9, Cxcl1, Il6) and lower colon thickening. However, oral (but not intraperitoneal) prednisolone augmented 4 kDa FITC-dextran permeability. This correlated with plasma lipopolysaccharide (LPS) levels, which were higher with oral prednisolone. In addition, colonic hemorrhage was enhanced by prednisolone treatment, particularly when given orally, as it both accelerated and enhanced blood loss. Besides, mice treated with oral prednisolone showed an increased disease activity index (DAI, based on body weight loss, feces consistency and rectal bleeding). On the other hand, GC administration suppressed Cyp11a1 expression in the colonic mucosa and also in jejunal organoids, suggesting inhibition of colonic steroidogenesis by exogenous treatment. These findings were largely reproduced using 6 µg/day of budesonide but, in this case, body weight loss was also augmented by glucocorticoid treatment. In the second experiment, rapamycin reduced DAI compared to prednisolone-only treatment, and additionally decreased bacterial adherence to colonic mucosa. This is consistent with previous findings with dexamethasone [3].

Conclusion

Our data are consistent with GC weakening of the mucosal barrier coexisting with an antiinflammatory response, which may be enhanced by oral administration. Addition of rapamycin appears to limit this detrimental effect of prednisolone, thus improving its benefit:risk ratio in this model.

References

1. Ocon, B, et al., Biochem Pharmacol, 2016. 116: p. 73-88.
2. Arredondo-Amador, M, et al., Br J Pharmacol, 2021. 178(12): p. 2482-2495.
3. Zhang, Z, et al., Cancers, 2020. 12(4), 945.

Introduction

It has emerged that the energy metabolism of colonocytes shapes the gut microbiota, and loss of epithelial hypoxia accompanied by respiration-dependent dysbiosis is observed in experimental colitis^{1, 2}. The inhibitory effect of tacrolimus on inducible nitric oxide synthase (iNOS), a vital protein in nitrate respiration, was reported in human colonic epithelial cell lines³. Our previous study reported that tacrolimus significantly alleviated colitis in mice and induced beneficial alterations of the gut microbiome⁴. However, the mechanism of how tacrolimus improves the gut microbiota in colitis remains to be elucidated.

Aims & Methods

In this study, we aim to explore the effects of tacrolimus on colonocyte metabolism in colitis. Male C57BL/6J mice aged 8 to 10 weeks were employed. Solutions of 2.5% dextran sulfate sodium (DSS) were prepared in the drinking water to induce colitis for 7 consecutive days. Tacrolimus (Tacro) at a dose of 10 mg/kg was administered via gavage, while aminoguanidine hydrochloride (AG), the selective iNOS inhibitor, was given intraperitoneally at a dose of 100 mg/kg per day. The body weight was observed daily. For hypoxia detection, mice were treated with 60 mg/kg of pimonidazole HCl intraperitoneally 1 hour before euthanasia. Colon lengths were measured, and the colon samples were fixed in 4% paraformaldehyde for subsequent hematoxylin and eosin staining and hypoxia staining. Total RNA and protein were extracted from the isolated colonocytes for the measurement of iNOS expression. For the in vitro study, the human colorectal adenocarcinoma cells (Caco-2) were incubated with 10 ug/mL tacrolimus or 0.1% DMSO for 4 hours, then the oxygen consumption rates were measured on the Seahorse XFe24 Analyzers according to the manufacturer’s protocols.

Results

Hypoxia in the colonic epithelium was significantly disrupted in DSS-induced colitis mice, which was partially restored by tacrolimus treatment. The iNOS expression on both mRNA and protein levels was significantly upregulated by DSS and downregulated after tacrolimus administration, while the addition of AG enhanced the effects of tacrolimus. Compared with the individual tacrolimus intervention, the combined treatment of tacrolimus and AG achieved a better remission in weight loss, colonic shortening, and colonic pathological changes including the disruption of crypt-villus structure, goblet cell loss and immune cell infiltration. In vitro measurement of mitochondrial oxidative phosphorylation showed that tacrolimus significantly enhanced oxygen consumption, and promoted ATP production and spare respiratory capacity in Caco-2 cells.

Conclusion

Our results indicated that tacrolimus improved the oxidative metabolism of colonocytes in experimental colitis, which could be an important contributor to the alterations in the gut microbes induced by tacrolimus. The observed effect of tacrolimus in colonocytes may be associated with the inhibition of iNOS.

References

1. Litvak Y, Byndloss MX, Bäumler AJ. Colonocyte metabolism shapes the gut microbiota. Science 2018;362.
2. Cevallos SA, Lee JY, Velazquez EM, et al. 5-Aminosalicylic Acid Ameliorates Colitis and Checks Dysbiotic Escherichia coli Expansion by Activating PPAR-γ Signaling in the Intestinal Epithelium. mBio 2021;12.
3. Hämäläinen M, Lahti A, Moilanen E. Calcineurin inhibitors, cyclosporin A and tacrolimus inhibit expression of inducible nitric oxide synthase in colon epithelial and macrophage cell lines. Eur J Pharmacol 2002;448:239-44.
4. Lv W, Zhang D, He T, et al. Combination of Lactobacillus plantarum improves the effects of tacrolimus on colitis in a mouse model. 2023;13.

Introduction

Inflammatory bowel diseases are chronic disabling conditions with a complex and multifactorial aetiology, which is still not completely understood. OCTN1, an organic cation transporter could have a role in modulating the inflammatory response, whose genetic polymorphisms were associated to increased risk of inflammatory bowel diseases. Up to now, limited information exists on its role in predicting/modulating response to therapies.

Aims & Methods

The aim of this study was to evaluate the role of OCTN1 in modifying gut microbiota and immune composition to Infliximab therapy in murine colitis. Dextran sodium sulphate model of colitis on C57BL/6 mice and ocnt1 gene knockout mice was used to assess clinical efficacy of infliximab administered intravenously. Stool, colon and mesenteric lymph node samples were collected to assess differences in gut microbiota composition, histology for Rachmilewitz score and T cell populations respectively.

Results

Octn1^-/-influences microbiota profile and is associated with a worse dysbiosis in colitis mice. Infliximab treatment attenuates colitis-associated dysbiosis with increase of bacterial richness and evenness in both strains. In comparison with wild type, octn1^-/- mice have a higher baseline percentage of Treg, Tmemory, Th2 and Th17.

Conclusion

Our data support murine model to study OCTN1 genetic asset in inflammatory bowel diseases. This could be the first step to consider this membrane transporter as a biomarker in inflammatory conditions and as a predictor of response to therapies.

Introduction

A deeper understanding of the pathogenesis of UC has led to the emergence of new therapeutic strategies. One of them consists of targeted exposure with biological drugs to certain molecules that regulate the interaction between leukocytes and endothelial cells in the inflammatory zone (mainly leukocyte integrins and endothelial cell adhesion molecules of the immunoglobulin superfamily).

Aims & Methods

Purpose of the study: To study the content of adhesion molecules ICAM-1, ICAM-2 and L-selectin in the peripheral blood of patients with ulcerative colitis (UC) and the effect of vedolizumab (Intivio) on their levels.
Materials and methods: We examined 12 patients with moderate and severe forms of UC and the ineffectiveness of previous standard therapy. The severity of the UC attack was calculated according to the Mayo index. Antivio® 300 mg was administered to patients intravenously according to the attached instructions. The content of adhesion molecules in the peripheral blood was studied before and 6 weeks after the first injection of vedilizumab. The control group consisted of 15 healthy volunteers. The content of ICAM-1, ICAM-2 and L-selectin in blood serum was determined by multilayer ELISA analysis (Bender med.Systems, Austria). Statistical processing of the obtained results was carried out using SPSS 13.0 software.

Results

It was found that the initial levels of ICAM-1 (461.9±28.4 ng/ml), ICAM-2 (323.3±18.4 ng/ml) and sL-selectin (1650±12.25 U/ml) ) in the peripheral blood of patients with UC were increased (P<0.05 with control), the Mayo index was 9 points. After four infusions of Vedolizumab (week 14), the levels of soluble molecules decreased in 4 patients, with a predominant decrease in sL-selectin (201±9.32 ng/ml, 197±8.54 pg/ml and 1008±15.3 U/ml respectively). The Mayo index was 5 points. In 8 patients, by the 6th week of treatment, the levels of the studied adhesive molecules did not change, although the Mayo index decreased by 3 points.

Conclusion

The initial level of ICAM-1, ICAM-2 and sL-selectin in the peripheral blood of patients with severe forms of UC is increased. The dynamics of the synthesis of adhesive molecules by immunocompetent cells under the influence of the fourth administration of Vedolizumab may predict the possibility of continuing the administration of the drug.

Introduction

Inflammatory bowel disease (IBD) is a complex chronic and relapsing inflammatory disorder of the gastrointestinal tract with unclear pathogenesis. Accumulating evidence suggests food emulsifiers as a risk factor for the development of IBD. It is indicated that food emulsifiers carboxymethylcellulose (CMC) can alter gut microbiota and promote intestinal inflammation in IBD under normal diet. Since high-fat diet is the main life scenario for CMC use, little is known about the effect of CMC on colitis model in different high-fat diet.

Aims & Methods

C57/BL6 mice (3-5 weeks old) were randomly divided into three high-fat diet (HFD) groups, which only differed by fat composition (40 %), derived from corn oil (HCO, n-6 polyunsaturated fatty acids), olive oil (HOO, monounsaturated fatty acids) and milk fat (HMF, saturated fatty acids), and exposed to CMC (1.0% w/v) in drinking water for 4 weeks before the establishment of dextran sulfate sodium (DSS) induced acute colitis mice model. Colon inflammation severity was evaluated using survival rate, HE staining, colon length, body weight change and DAI score. Ki67 and TUNEL assays were performed to detect proliferation and apoptosis.

Results

Compared with the CMC+DSS group, the HMF+CMC+DSS group markedly increased survival rate (P=0.013), colon length (P=0.027) and body weight (day4, day5, day6 and day7, P<0.05, respectively), and relieved DAI score (day4 and day6, P<0.05, respectively) and histological damage (P<0.0001). The HOO+CMC+DSS group seemly had no significant difference from the CMC+DSS group. In contrast, the HCO+CMC+DSS group developed more aggressive colitis with increased DAI score (day5, P<0.01) and decreased body weight (day1, day2, day5 and day6, P<0.05, respectively). In terms of maintaining homeostasis of the intestinal barrier, the HMF+CMC+DSS group showed the highest Ki67 (P=0.012, P=0.0002 P=0.042, respectively) and TUNEL positive cells (P=0.324, P<0.0001, P<0.0001, respectively) compared with the CMC+DSS, HOO+CMC+DSS or HCO+CMC+DSS group. Between CMC+DSS and HOO+CMC+DSS or HCO+CMC+DSS group, there was no difference in Ki67 positive cells, while TUNEL positive cells significantly decreased (P=0.002, P=0.008, respectively).

Conclusion

The function of CMC on DSS colitis varies in different high-fat. Unexpectedly, CMC protects against the development of high-milk-fat related colitis by maintaining homeostasis of proliferation and apoptosis.

References

The function of CMC on DSS colitis varies in different high-fat. Unexpectedly, CMC protects against the development of high-milk-fat related colitis by maintaining homeostasis of proliferation and apoptosis.

Disclosure

none

Introduction

Inflammatory bowel disease (IBD), a chronic inflammatory disease of the gastrointestinal tract, is caused by various factors, including genetic factors and environmental factors, as well as dysregulated immune responses. LMT503, a novel organic small-molecule compound, has the potential to suppress pro-inflammatory cells and induce anti-inflammatory cells by modulating their cell metabolisms. We evaluated the anti-inflammatory effects of LMT503 in a murine adoptive T cell transfer-induced colitis model.

Aims & Methods

This study was conducted to evaluate the anti-inflammatory effect of LMT503 in the murine chronic colitis model. Naïve CD4⁺CD25^-CD45RB^high CD62L^high T cells that were isolated from wild-type mice by flow cytometry based-method and transferred to Rag1 knock-out mice by intraperitoneal injection to induce murine adoptive T cell transfer-induced colitis. LMT503 was orally administrated (50mg/kg, 100mg/kg) for 2 weeks starting at 5 weeks after naïve T cell transfer. The disease activity index (DAI) was checked daily, and histopathological score, macroscopic injury score, and colon length were evaluated upon sacrifice. Macrophages and T cells from the spleen were analyzed by flow cytometry. Gene expression in colon tissue was measured by qRT-PCR and cytokine profiles in plasma was measured by cytometric bead array. Myeloperoxidase (MPO) activity was measured using the MPO activity kit.

Results

Oral administration of LMT503 ameliorated adoptive T cell transfer-induced colitis in mice. The DAI, histopathological score, and macroscopic injury score were significantly decreased after the administration of LMT503 in a dose-dependent manner. The LMT503-treated group showed significantly increased mucin production in the colon. Th1, Th17 cells, and M1 macrophages in the spleen were suppressed, and M2 macrophages were induced by the administration of LMT503. MPO activity and gene expressions of pro-inflammatory cytokines, such as IL-6, IL-1β, and TNF-α, in colon tissue were suppressed by the administration of LMT503. And the level of inflammatory cytokines, such as IL-2, TNF-α, and IFN-γ, in plasma were significantly suppressed by the administration of LMT503. Especially, LMT503 treatment significantly increased the gene expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Ppargc1a), a master regulator of mitochondrial biogenesis, suggesting that LMT503 modulates cellular metabolism towards an anti-inflammatory metabolism.

Conclusion

Our data suggest that the anti-inflammatory effect of LMT503 ameliorates murine adoptive T cell transfer-induced colitis by modulating cellular metabolism and immune cell modulation. Therefore, LMT503 may be a novel therapeutic drug that opens new avenues for the treatment of IBD.

References

1. Erben U, Loddenkemper C, Doerfel K, et al. A guide to histomorphological evaluation of intestinal inflammation in mouse models. Int J Clin Exp Pathol. 2014;7(8):4557-4576.
2. Ostanin DV, Bao J, Koboziev I, et al. T cell transfer model of chronic colitis: concepts, considerations, and tricks of the trade. Am J Physiol Gastrointest Liver Physiol. 2009;296(2):G135-G146.
3. Ghoreschi K, Jesson MI, Li X, et al. Modulation of innate and adaptive immune responses by tofacitinib (CP-690,550). J Immunol. 2011;186(7):4234-4243.
4. Burke JR, Cheng L, Gillooly KM, et al. Autoimmune pathways in mice and humans are blocked by pharmacological stabilization of the TYK2 pseudokinase domain. Sci Transl Med. 2019;11(502):eaaw1736.
5. Kiesler P, Fuss IJ, Strober W. Experimental Models of Inflammatory Bowel Diseases. Cell Mol Gastroenterol Hepatol. 2015;1(2):154-170.

Introduction

Inflammatory Bowel Diseases (IBD) are chronic inflammatory disorders characterised by alterations of acute relapses and remission. The past decades have brought substantial advances in the pharmacological management of IBD by the introduction of immunosuppressive agents, biologics and lately small molecules. Although, the sustained efficacy of the currently available anti-inflammatory therapies is still far from optimal. Therefore, the development of novel therapies represents a major unmet clinical need in this field. Recently the enrichment of the coagulation pathway genes and among these, plasminogen activator inhibitor 1 (PAI-1) was described in IBD, whereas PAI-1 emerged as a link between the epithelium and inflammation. Moreover, the genetic deletion of PAI-1 impaired the severity of experimental colitis in mice.

Aims & Methods

Based on these, our aim was to develop and test novel PAI-1 inhibitors for the treatment of IBD. Novel, peptide-based PAI-1 inhibitors were designed and screened in silico, then the selected ones were synthetized using the ixPeptide® platform. The inhibitory potential of the peptides was determined by an in vitro PAI-1 activity assay. Cytotoxicity was monitored in vitro in Hela cells with the CellTiter Glo assay. Based on the in vitro results LDN-071 was selected for further analysis. Experimental colitis was induced in mice with 3% DSS and after the induction of colitis 10 mg/bwkg LDN-071 was administrated per os for four consecutive days. The changes of body weight, spleen weight and colon length were measured. Haematoxylin eosin (HE) and Periodic acid-Schiff (PAS) staining were performed for histology. ELISA and qRT-PCR measurements were applied to analyse the mucosal cytokine expressions. In vivo toxicity of LDN-071 was determined in mice.

Results

LDN-071 significantly inhibited PAI-1 in vitro without decreasing the cell viability in a concentration range of 1µM-1mM. The administration of 10 mg/bwkg LDN-071 in the DSS-treated mice prevented the body weight loss, the development of bloody diarrhoea, the decrease of colon length and increase of the spleen weight, which were displayed by the DSS+vehicle-treated animals. The histological analysis of the colon revealed that DSS treatment induced a severe transmural inflammation, ulceration, loss of crypt structures and goblet cell depletion. Importantly, all of these parameters were significantly impaired by the administration of 10 mg/bwkg LDN-071. Additionally, mucosal gene and protein expressions of TNF-α, IL-1β and IL-6 were significantly impaired by PAI-1 inhibition. Importantly, no signs of toxicity were LDN-071 observed in mice exposed to 100 mg/bwkg LDN-071 for 7 days.

Conclusion

Our results showed that LDN-071, a novel peptide-based inhibitor of PAI-1. It significantly reduces the severity of experimental colitis in mice. We propose that the inhibition of PAI-1 could be a potential novel therapeutic strategy in IBD.

Disclosure

LDN-071 PAI-1 inhibitor is owned by Ladon Therapeutics Ltd.