Infection with the intracellular bacterium, Listeria monocytogenes, overrides established tolerance in a mouse cardiac allograft model

Abstract

Infections and TLR signals at the time of transplantation have been shown to prevent the induction of tolerance, but their effect on allografts after tolerance has been established is unclear. We here report that infection with Listeria monocytogenes precipitated the loss of tolerance and the MyD88- and T cell-dependent rejection of accepted cardiac allografts in mice. This loss of tolerance was associated with increases in the numbers of graft-infiltrating macrophages and dendritic cells, as well as CD4(+)FoxP3(-) and CD8(+) T cells. Rejection was also associated with increased numbers of graft-infiltrating alloreactive as well as Listeria-reactive IFNgamma-producing T cells. Rejection of the established grafts required both IL-6 and IFNss, cytokines produced during acute Listeria infection. However, IL-6 and IFNss alone, even when present at higher concentrations than during Listeria infection, were insufficient to break tolerance, while the combination of IL-6 and IFNss was sufficient to break tolerance. These and in vitro observations that IL-6 but not IFNss enhanced T cell proliferation while IFNss but not IL-6 enhanced IFNgamma production support a hypothesis that these cytokines play nonredundant roles. In conclusion, these studies demonstrate that the proinflammatory effects of infections can induce the loss of tolerance and acute rejection of accepted allografts.

Figure 1. Listeria infection breaks anti-CD154/DST-mediated cardiac allograft tolerance

(A) B6 recipients of B/c heart grafts were untreated (Rej; n=5) or were treated with anti-CD154/DST to induce long-term graft acceptance (Tolerant). On day 60 post-transplantation, tolerant recipients were infected i.p. with 1.5 × 10⁶ Listeria (Tolerant+LM; n=41); this control cohort was cumulative over the project period. Controls were recipients with syngeneic cardiac grafts infected with Listeria on day 60 post-transplantation (n=5). p=0.0118 for Tolerant vs Tolerant+LM. (B) Tolerant MyD88^−/− or wild type B6 mice were infected with LM on day 60 post-transplantation (n=6). p=0.006 for Tolerant MyD88^−/−+LM vs Tolerant WT+LM. (C) Two days before Listeria infection, tolerant recipients were treated with GK1.5 (0.2mg/mouse) or YTS-169 (1mg/mouse) mAbs to deplete CD4⁺ or CD8⁺ T cells (n=5/group). (D) No increase in allo-IgG titers following the abrogation of tolerance by Listeria infection (Tolerant+D60-LM), but significantly increased allo-IgG titers were detected in anti-CD154/DST-treated recipients infected with Listeria on the day of transplantation (Tolerant+D0-LM), or in untreated recipients (No Rx). p=0.0108 for Tolerant+D0-LM vs Tolerant+D60-LM on day 7; p=0.0153 for Tolerant+D0-LM vs Tolerant+D60-LM on day14; p=0.0012 for No Rx vs Tolerant+D60-LM on day21; p=0.0004 for No Rx vs Tolerant+D60-LM on day28. All results represent the mean and standard deviation of at least 3 independent experiments.

Figure 2. Analyses of intra-graft immune responses

(A) Histology and immunohistochemistry of rejecting cardiac allografts harvested on day 7 post-Listeria infection (representative of 3 allografts/group). (B & C) Graft infiltrating cells were isolated and analyzed by flow cytometry. Total CD11b⁺CD11c⁻ (macrophages), dendritic cells (CD11c⁺) and dendritic cell subsets (pDC: B220⁺CD11b⁻CD11c⁺; lDC:CD8⁺CD11b⁻CD11c⁺; mDC: CD11b⁺CD11c⁺) as well as T cell subsets (CD4⁺, CD8⁺, FoxP3⁺, and FoxP3⁻) were calculated for Tolerant and Tolerant+LM groups. ***p<0.001; **p<0.01; *p<0.05.

Figure 3. Specificity of intra-graft immune responses

(A) The frequency of IFNγ-producing T cells isolated from the allograft on day 7 post-Listeria infection. The results represent the mean and standard deviation of 3 independent experiments. (B) 10×10⁶ B/c and 5×10⁶ B6.Ly5.1 spleen cells were injected into naïve B6 mice or on day 7-post Listeria (1.5×10⁶) infection or 7 days after B/c skin transplantation plus 30×10⁶ B/c splenocyte (i.p.). Six hours after cell transfer, spleen cells were harvested, and the total B/c cell and B6.Ly5.1 cells recovered enumerated. **p<0.01; *p<0.05.

Figure 4. Type I IFN signaling is necessary but not sufficient for abrogating tolerance following Listeria infection

(A) IFNαR1^−/− mice received B/c cardiac grafts and were untreated (Rej; n=4) or treated with anti-CD154/DST followed by Listeria infection on day 60 post-transplantation (Tolerant+LM; n=41). The control cohort is the same as in Fig 1. p=0.0005 for Tolerant IFNαR1^−/−+LM vs Tolerant WT+LM. (B) Wild type B6 mice were infected with Listeria or with mouse IFNβ-expressing plasmid and serum IFNβ levels were determined by ELISA. (C) Graft survival after IFNβ (n=5) or control plasmid (n=4) injection (p>0.05).

Figure 5. IL-6 is necessary but not sufficient for abrogating tolerance following Listeria infection

(A) IL-6^−/− mice transplanted with B/c cardiac grafts and were untreated (Rej; n=3), or were treated with anti-CD154/DST followed by Listeria infection on day 60 post-transplantation (Tolerant+LM; n=5). The control cohort is the same as in Fig 1. p=0.0118 for Tolerant IL-6^−/−+LM vs Tolerant WT+LM. (B) Wild type B6 mice were infected with Listeria or with mouse IL-6-expressing plasmid, and serum IL-6 levels determined (n=3/group). (C) Graft survival after IL-6 (n=4) or control plasmid (n=4) injection (p>0.05).

Figure 6

IFNβ and IL-6 are sufficient for abrogating tolerance. Tolerant B6 receipinets were injected with mouse IL-6- followed by IFNβ-expressing plasmids 5 days later (n=3/group). Serum IFNβ (A) and IL-6 (B) were quantified and (C) graft survival determined, following IL-6+IFNβ (n=8) or control plasmid (n=4) injections (p=0.0019).

Figure 7. Listeria infection of tolerant recipients induces a transient enhancement of alloreactive T cell responses and abrogation of tolerance

(A) Sorted CD4⁺ T cells (CD25⁺GFP⁺ and CD25⁻GFP⁻) from naïve FoxP3^gfp knock-in mice were stimulated at the indicated ratios with immobilized anti-CD3 and IL-2 and irradiated T cell-depleted syngeneic splenocytes. In some cultures, rIL-6 (500 ng/ml) or rIFNβ (500 U/ml) was added, and cell proliferation was determined by [³H]-thymidine incorporation. (B) Spleen cells from naïve or tolerant B6 recipients were stimulated with irradiated syngeneic (B6), allogeneic (B/c) or third-party (C3H) splenocytes in the presence of IL-6 (1000 ng/ml) or mouse IFNβ (1000 U/ml). (C) Whole splenocytes, CD4⁺ or CD8⁺ cells from the spleens of Tolerant+LM recipients, or (D) The frequency of IFNγ-producing T cells from Tolerant+LM or Tolerant recipients, stimulated in rIL-6 (500ng/ml) or/and rIFNβ (1000U/ml), was determined with an IFNγ ELISPOT assay. All results represent the mean and standard deviation of 3 independent experiments. ***p<0.001; **p<0.01; *p<0.05.