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. 2016 Jul 1;126(7):2736-44.
doi: 10.1172/JCI85295. Epub 2016 Jun 20.

"VSports" The composition of the microbiota modulates allograft rejection

Yuk Man Lei, Luqiu Chen, Ying Wang, Andrew T Stefka, Luciana L Molinero, Betty Theriault, Keston Aquino-Michaels, Ayelet S Sivan, Cathryn R Nagler, Thomas F Gajewski, Anita S Chong, Caroline Bartman, Maria-Luisa Alegre

VSports手机版 - The composition of the microbiota modulates allograft rejection

Yuk Man Lei (VSports在线直播) et al. J Clin Invest. .

Abstract

Transplantation is the only cure for end-stage organ failure, but without immunosuppression, T cells rapidly reject allografts VSports手机版. While genetic disparities between donor and recipient are major determinants of the kinetics of transplant rejection, little is known about the contribution of environmental factors. Because colonized organs have worse transplant outcome than sterile organs, we tested the influence of host and donor microbiota on skin transplant rejection. Compared with untreated conventional mice, pretreatment of donors and recipients with broad-spectrum antibiotics (Abx) or use of germ-free (GF) donors and recipients resulted in prolonged survival of minor antigen-mismatched skin grafts. Increased graft survival correlated with reduced type I IFN signaling in antigen-presenting cells (APCs) and decreased priming of alloreactive T cells. Colonization of GF mice with fecal material from untreated conventional mice, but not from Abx-pretreated mice, enhanced the ability of APCs to prime alloreactive T cells and accelerated graft rejection, suggesting that alloimmunity is modulated by the composition of microbiota rather than the quantity of bacteria. Abx pretreatment of conventional mice also delayed rejection of major antigen-mismatched skin and MHC class II-mismatched cardiac allografts. This study demonstrates that Abx pretreatment prolongs graft survival, suggesting that targeting microbial constituents is a potential therapeutic strategy for enhancing graft acceptance. .

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Figures

Figure 1
Figure 1. Abx pretreatment results in prolonged skin graft survival and reduced bacterial diversity.
(A) B6 females untreated or pretreated for 10 days with Abx received a skin graft from B6 males untreated or pretreated for 10 days with Abx. SPF → SPF, n = 12; SPF (Abx) → SPF, n = 5; SPF → SPF (Abx), n = 5; Abx → Abx, n = 19; syngeneic, n = 5. log-rank test. (B) Bacterial load by qPCR in fecal samples of GF and SPF B6 females at the indicated time points after initiation of Abx treatment. n = 3 (1 SPF and 1 GF samples shown for reference). LOD, level of detection. (C–E) Bacterial DNA was isolated from gut and skin of female SPF controls and age-matched mice on day 10 of Abx treatment and was analyzed by high-throughput sequencing. Data are displayed as richness (C), diversity (D), and PCA (E). Each line (C) and dot (B and E) represents an individual mouse. PC1, principal component 1; Exp, experiment. Bars (D) represent the mean ± SEM of 4 mice per group. (C–E) Results are representative of 4 experiments with n = 3–4. *P < 0.05; ***P < 0.001, Student’s t test.
Figure 2
Figure 2. GF mice display prolonged skin graft survival.
(A) Schematics of transplantation in the gnotobiotic facility. (B) B6 female SPF, GF, and GF mice gavaged with PBS-diluted fecal material from SPF mice (GF-SPF.F) or Abx-pretreated SPF mice (GF-Abx.F) 5 to 7 days prior, were transplanted with male skin grafts from the indicated donors. SPF → SPF, n = 8; GF → GF, n = 8; GF → SPF, n = 11; GF-SPF.F → GF-SPF.F, n = 10; GF-Abx.F → GF-Abx.F, n = 7. log-rank test. (C) Representative cecum appearance at day 7 after gavage. (D) qPCR of 16S rRNA gene to assess bacterial load in SPF, GF, and GF-Abx.F mice before and after gavage. n = 3–5. One-way ANOVA. (E) Normalized abundance of B. coccoides and Lactobacillus spp. as determined by qPCR in feces of SPF, 10-day Abx-treated SPF mice, and GF-Abx.F mice after fecal gavage. n = 3–5. One-way ANOVA. Data are representative of 2 experiments (D–F) or combined from 2 to 3 experiments (B). (B and E) Data represent the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Figure 3
Figure 3. Abx pretreatment and GF status result in reduced allogeneic T cell priming.
(A–C) Graft-infiltrating cells were isolated from control and Abx-pretreated SPF mice on day 10 after transplantation for flow cytometric analysis to determine the percentage of TCR-β+ (A) or CD4+ (B) cells among CD45+ cells and the percentage of IFN-γ–producing cells (C) among PMA/ionomycin-stimulated CD4+ cells. (D and E) Congenic Marilyn T cells were labeled with CFSE and transferred (106 cells/mouse) into SPF female recipients 1 day prior to transplantation with male skin grafts; donors and recipients were both untreated or both Abx pretreated. Mice were sacrificed 4 days after transplantation, and cells were isolated from the graft dLNs for analysis of CFSE dilution. Representative plots (D) and quantitation (E) of divided Marilyn T cells. (F and G) APCs from skin dLN cells were isolated from control or 10-day Abx-pretreated SPF B6 males or females and cultured with CFSE-labeled T cells from naive Marilyn females. (F) Quantitation of CFSE dilution in Marilyn-gated T cells on day 4 of culture. (G) Percentage of IFN-γ+ cells among Marilyn T cells after a 3-day culture and following restimulation with PMA/ionomycin for 4 hours. (A–G) n = 3–5 mice per group. Experiments were repeated 3 to 4 times. No txp, no transplant. Student’s t test. (H) Marilyn T cell–CFSE dilution after culture as in F with male APCs from different groups. APCs were harvested 7 days after gavage of GF mice. Quantitation of divided Marilyn T cells (I) and IFN-γ production after restimulation (J). n = 2–8. One-way ANOVA. Data represent the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 4
Figure 4. Abx pretreatment results in downregulation of the type I IFN pathway.
(A and B) DCs from skin dLN of control and 10-day Abx-pretreated SPF mice were sorted, and cDNA was analyzed for gene expression profiling. (A) Examples of genes differentially expressed (n = 3 per group). (B) DAVID analysis of enriched pathways in DCs from control relative to Abx-treated groups. (C) Female B6 control and Ifnar–/– SPF mice were transplanted with skin grafts from males of the host genotype. Results are combined from 2 experiments. WT, n = 12; Ifnar–/–, n = 8. log-rank test. **P < 0.01.
Figure 5
Figure 5. Abx pretreatment delays rejection of major antigen–mismatched skin and MHC class II–mismatched cardiac allografts.
(A) Graft survival of BALB/c skin transplanted onto B6 recipients. Untreated, n = 5; Abx pretreatment, n = 9. log-rank test. (B) Clinical graft functional score of Bm12 heart transplanted into B6 recipients. n = 6 per group. Two-way ANOVA. Data represent the mean ± SEM. ****P < 0.0001.
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