In a recent study published in Cellular & Molecular Immunology, researchers explored the therapeutic impact of Thiostrepton (TST) on experimental colitis by examining its effects on colon inflammation and determining its targets through various biochemical approaches. They analyzed its influence on gut microbiota, with a focus on its potential as a treatment for inflammatory bowel disease (IBD).
BackgroundÂ
IBD is a chronic, relapsing condition significantly impacting life quality and necessitating innovative treatments due to the inadequacy of current therapies to maintain long-term remission. TST, a multi-functional antibiotic, presents a promising solution, demonstrating anti-inflammatory, antimicrobial, and anticancer properties, potentially modulating immunological and microbial factors in IBD. The inconsistent outcomes of antibiotics in IBD treatments underscore the importance of further investigations to validate and refine TST’s therapeutic efficacy, addressing the urgent need for advanced, effective solutions in managing IBD’s global burden.
About the study
In the present study at the Third Military Medical University, varied mouse strains underwent multiple stringent experiments, adhering to ethical guidelines. The experiments focused on analyzing the impacts and interactions of drug interventions and diseases, utilizing elaborate methods like multiplex cytokine assays and histopathology assessments on mouse colons. Additionally, meticulous in vitro techniques involving quantitative polymerase chain reaction (PCR) and precise cell culture methodologies were employed to study the human embryonic kidney (HEK) 293 T cells.
The study also included an ex vivo ubiquitination assay on specific cells, incorporating varied biochemical methods, solutions, and analyzes to evaluate the ubiquitination of Retinoic acid-related orphan receptor gamma t (RORγt). This was supplemented by sophisticated short-chain fatty acid quantification and gut microbiota depletion assays on designated mouse models to comprehend the consequences of diverse treatments.
A process was employed to generate the Itch-knockout cell line using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–Cas9 technology, involving the design of three specific single guide ribonucleic acid (sgRNAs) and the construction of sgRNA expression plasmids. The targeted sequence was transfected into HEK 293T cells and was followed by rigorous isolation and confirmation of correct clones through immunoblotting and Sanger sequencing.
The study extended to preparing protein structures of RORγt and Itch and executing detailed molecular docking studies. These were used to understand the binding dynamics between the proteins, employing advanced tools and software to establish the most favorable binding structures. Lastly, Statistical analysis was meticulously conducted with GraphPad Prism 9 to validate the results, reinforcing the precision and credibility of the entire study.Â
Study resultsÂ
In the study, the therapeutic and prophylactic efficacy of TST on Dextran sulfate sodium (DSS)-induced colitis in mice was investigated, revealing significant alleviation of disease phenotypes, evident through attenuated weight loss, reduced disease activity index (DAI) scores, and mitigated colon shortening and histological severity. The assessment of TST’s influence on T-cell mediated colitis confirmed a limitation in the development of T-cell transfer colitis. Further examination into inflammatory cytokine production revealed that TST diminished the levels of several proinflammatory cytokines, suggesting an impairment in inflammatory response in DSS-induced colitis. The frequency of interleukin- 17A (IL-17A)-producing cells in colons of DSS-exposed mice was notably reduced following TST treatment, affecting mostly Gamma delta T (γδ T) cells in the inflamed colons.Â
This was validated by the observed corresponding reductions in RORγt-expressing populations post-TST treatment. It was also observed that the protective effect of TST is independent of the gut microbiota, exhibiting immunomodulatory effects directly on T cells. Additionally, TST selectively inhibits T helper 17 (Th17) differentiation in vitro, without impacting the differentiation of other T cells.
Experiments on Rorc mice suggested that the protective efficacy of TST is reliant on the presence of RORγt, marking IL-17A-producing cells expressing RORγt as selective targets of TST in DSS-induced colitis. Thus, TST emerges as a prospective immunosuppressant for treating autoimmune diseases like IBD.
To understand the mechanism through which TST reduces RORγt expression, the EL4 cell line was studied due to its consistent expression of RORγt and production of IL-17A and IL-17F. TST displayed antiproliferative activity, inhibiting the growth of EL4 cells, with a significant decrease in protein levels of Forkhead Box M1 (FOXM1) and RORγt. However, TST primarily modulates RORγt expression posttranslationally, not affecting its transcriptional activity. Experiments showed the half-life of RORγt, but not FOXM1 was significantly reduced after TST treatment.
The research highlighted TST’s induction of ubiquitination of RORγt in a dose-dependent manner, with more exogenous RORγt co-immunoprecipitation with Itch, essential for TST-induced degradation of RORγt. The knockout of Itch nullified the TST-induced ubiquitination of RORγt. Molecular docking studies suggested that TST may function as a molecular glue degrader, facilitating the assembly of RORγt and Itch.
Additionally, the study explored the impact of TST on gut microbiota, revealing that TST shapes the gut microbiota, offering protection against DSS-induced colitis by altering microbial diversity and composition, including the normalization of the Firmicutes/Bacteroidetes ratio, which is usually imbalanced in dysbiosis.
Specific bacterial genera producing short-chain fatty acids (SCFAs), crucial for maintaining intestinal homeostasis, were more abundant in TST-treated groups, indicating TST’s potential therapeutic role in intestinal inflammatory disorders. The protective effect of TST was found to be partially dependent on the gut microbiota, impacting the ability of microbiota to control intestinal inflammation.
