Immunosuppressive Treatment Alters Gut Microbiota and Modified Gut Microbiota Affects Immune Status

Introduction: Immunosuppressive therapies in transplantation could change the host-microbial interactions, especially gut microbiota in transplant recipients, which is an important component in systemic immunity.[1][2][3] We evaluated the relationship between immunosuppression and gut microbiota, to investigate the immune function by fecal microbiota transplantation in mice models. Materials and Methods: Mice models were established using C57BL6 mice, male, 6–8 weeks. Tacrolimus has been used to establish immunosuppressive model in mice. Tacrolimus group(T group)(n = 8) were given 10 mg/kg tacrolimus by gavage each day; fecal microbiota transplantation group(F group)(n = 8) took fresh feces from T group (No tacrolimus left tested by ELISA); control group(C group)(n = 5) transplant fresh feces from blank mice.[5][6] On 0 to 28 days, flow cytometry has been used to characterize peripheral blood lymphocytes expressing CD3, CD4, CD8, CD25, Foxp3, IL-2.[3][4] On 3, 7, 14 day, gut microbiota was analyzed by 16S rRNA gene sequencing. Skin transplantation acute rejection model is established by Babl/C mice to C57BL6 mice. Results: Phylogenetic classification of 16S rDNA frequencies in the fecal from samples collected from control group, tacrolimus group on 14 days, or FMT group on 14 days. Erysipelotrichales at the order level shows the most significant differences. (P < 0.001, ANOVA test) (Figure 1). Comparing with T group, F group shows the similar tendency on gut microbiota after 7 days, and blood tacrolimus level is below detection limit (0.074 ng/ml, T group 5-7 ng/ml p < 0.01). The percentage of Treg cell was significantly increased in both groups, T group has higher percentage and earlier increase than F group. Both group has lower IL-2 expression in many type of T cell (CD3+, CD4+, CD8+, Treg cell)(Figure 2). Allograft skin in mice showed striking differences after day 5. Survival analysis showed that FMT and Mixture group prolonged the survival time of allograft skin, which is significantly different to Saline group(p < 0.01) (Figure 3).FIGURE 1: Tacrolimus treatment alters the composition of the intestinal microbiota. (A) Phylogenetic classification of 16S rDNA frequencies in the fecal from samples collected from control mice, mice treated with tacrolimus for 14 days, or mice treatedwith fecal microbiota transplantation for 14 days. (B) Color code for the most predominant bacterial taxa found in the murine fecal. (C) Heatmap shows the frequencies of 16s rDNA clusters classifien at the order level in the fecal of three group mice. (D) Erysipelotrichales is the most significant differences at the order level.FIGURE 2: Peripheral blood lymphocytes by Flow Cytometry. (A) Tacrolimus has no eefect on the proportion of CD3+ cells, while FMT shows increasing in expression of CD3. (B) CD4+ cells shows no significant change (C) Oral tacrolimus significantly increased the expression of FoxP3 lymphocytes, bacterial flora accurately reproduces the increase FoxP3 as oral tacrolimus treatment. (D) (E) (F) Transplant feces from tacrolimus-treatedmice also has a significant effect on IL-2 expression in lymphocytes, as the same effect by oral tacrolimus treatment.FIGURE 3: Skin transplataion in mice treated with a combination of lowdose tacrolimus (0.1mg/kg) and FMT (n=8). (A) Representative photographs of allograft skin in mice showing striking differences beginning after day 5. At day 10, mice treate with FMT and mixture treatment has showed significantly difference which has no significant signs of tissue necrosis. At day 15, graft skin in saline and tacrolimus group has been completely lost, while FMT and mixture group is simply damaged. (B) Survival analysis showed that FMT and Mixture group prolonged the survival time of allograft skin, which is significantly different to Saline group (p<0.01).Conclusion: Based on these findings, the gut microbiota could be changed by systemic immune status, such as immunosuppressive therapies, and modifications of gut microbiota is considered to affect the immune status. A specific composition of gut microbiota may be able to induce immunosuppressive state or strengthen the effect of immunosuppressive agents. References: 1. Kamada, N., Seo, S.U., Chen, G.Y. & Nunez, G. Role of the gut microbiota in immunity and inflammatory disease. Nature reviews. Immunology 13, 321-335 (2013). 2. Jérôme Tourret, et al. Immunosuppressive treatment alters secretion of ileal antimicrobial peptides and gut microbiota, and favors subsequent colonization by uropathogenic Escherichia coli. Transplantation Publish Ahead of Print. 3. Xiaofei Xu, et al. Effects of cyclophosphamide on immune system and gut microbiota in mice. Microbiological Research 171, 97–106 (2015). 4. Gong, N., et al. Immunoregulatory effects of sirolimus vs. tacrolimus treatment in kidney allograft recipients. Cellular immunology 297, 87-93 (2015). 5. Ellekilde, M., et al. Transfer of gut microbiota from lean and obese mice to antibiotic-treated mice. Scientific Reports 4(2014). 6. Peng, J., et al. Long term effect of gut microbiota transfer on diabetes development. Journal of autoimmunity 53, 85-94 (2014). 7. Baroja-Mazo, A., Revilla-Nuin, B., Ramirez, P. & Pons, J.A. Immunosuppressive potency of mechanistic target of rapamycin inhibitors in solid-organ transplantation. World journal of transplantation 6, 183-192 (2016). 8. Hartel, C., et al. Sensitivity of whole-blood T lymphocytes in individual patients to tacrolimus (FK 506): impact of interleukin-2 mRNA expression as surrogate measure of immunosuppressive effect. Clinical chemistry 50, 141-151 (2004).