The Effect of Tacrolimus on Gut Microbiota in a Mice Model and In Vitro Cellular Responses

Abstract

Tacrolimus (FK506) is a cornerstone calcineurin inhibitor used in solid organ transplantation and has been more and more implicated in gut microbiota dysregulation, but its mechanism of action has not been well comprehended. This paper examined the dose-dependent cytotoxic and pro-inflammatory effects of tacrolimus, using a BALB/c murine model to study the gut microbial dysbiosis caused by tacrolimus. The male BALB/c mice (n=30) were given control, low dose (1mg/kg/day) tacrolimus and high dose (5mg/kg/day) tacrolimus over 28 days and gut microbial composition was determined by 16S rRNA gene sequencing. Simultaneously, to determine cell viability (MTT assay), inflammatory cytokines (IL-6 and TNF-a) and cell cycle dynamics (flow cytometry), Vero cells were subjected to tacrolimus (0.1-50 mg/mL) over the course of 24-48 hours. Tacrolimus at high doses led to severe microbial dysbiosis, characterized by decreased diversity and severe compositional changes, such as loss of beneficial short-chain fatty acid-producing genera (Lactobacillus, Bifidobacterium, Faecalibacterium) and increase in Proteobacteria. Tacrolimus induced dose- and time-dependent cytotoxicity in vitro, increased IL-6 and TNF-alpha levels significantly and G1 phase arrest with greater apoptosis at higher concentrations. Taken together, these results indicate that tacrolimus encourages the state of gut microbiota imbalance and inflammatory cellular reactions, and immunosuppressive treatment and microbiome perturbation are closely connected. These findings also highlight the possibility of using microbiota-specific interventions, such as probiotic supplementation and short-chain fatty acid regulation, to alleviate the adverse effects of tacrolimus use in transplant patients.