Non-apoptotic Fas (CD95) Signaling on T Cells Regulates the Resolution of Th2-Mediated Inflammation

Abstract

Fas (CD95/APO-1) and its ligand (FasL/CD95L) promote the resolution of type 2 lung inflammation and eosinophilia. We previously found that Fas-deficiency on T cells, but not eosinophils, delayed resolution of inflammation. However, Fas can signal both cell death and have a positive signaling function that can actually activate cells. In this study, we investigated whether Fas-induced death or Fas-activated signaling pathways promote resolution of allergic lung inflammation. By increasing T cell survival through two Fas-independent pathways, using Bim-deficient T cells or Bcl-x_L overexpressing T cells, no differences in resolution of Th2-mediated inflammation was observed. Furthermore, Th2 cells were inherently resistant to Fas-mediated apoptosis and preferentially signaled through non-apoptotic pathways following FasL treatment. Utilizing Fas-mutant mice deficient in apoptotic but sufficient for non-apoptotic Fas signaling pathways, we demonstrate that non-apoptotic Fas signaling in T cells drives resolution of Th2-mediated airway inflammation. Our findings reveal a previously unknown role for non-apoptotic Fas signaling on Th2 cells in the induction of resolution of type 2 inflammation.

Keywords: Apoptosis; Asthma; Eosinophilia; Fas-FasL; Th2 cells; allergy.

Figure 1

Increasing T cell survival through Bim-deficiency is not sufficient to induced delayed resolution of inflammation. (A) Protocol for adoptive T cell transfer, sensitization, and challenge. Bim^−/− or WT T cells were transferred into Rag^−/− mice which were then sensitized and challenged. Mice were sacrificed on day 4, day 14, or day 28 after challenge and assayed for (B) T cell and (C) eosinophil infiltration into the lungs. (D) H&E staining of histologic sections from the lungs of sensitized and challenged mice. (E) T cells from the lungs at day 14 after challenge were restimulated with media or SEA and assayed for Th2-cytokine secretion. Data are representative of three independent experiments for each time point. For each independent experiment, n ≥ 5 mice per group were used (^*p ≤ 0.05, ^**p ≤ 0.01, ^***p ≤ 0.001).

Figure 2

Bcl-x_L transgenic T cells promote T cell survival but do not affect eosinophilia in the lungs following sensitization and challenge. Bcl-x_L transgenic T cells were transferred into Rag^−/− mice then sensitized and challenged. Mice were sacrificed on day 4, day 14, or day 28 after challenge and assayed for (A) T cell and (B) eosinophil infiltration into the lungs. (C) H&E staining of histologic lung sections of sensitized and challenged mice at day 14. Data are representative of three independent experiments for each time point with n ≥ 5 mice used per group for each experiment (^*p ≤ 0.05).

Figure 3

Th2 cells preferentially signal through non-apoptotic Fas pathways following FasL treatment. In vitro skewed Th1 and Th2 cells from WT, LPRcg/wt, and LPR mice were treated with LzCD95L and assayed for induction of apoptosis 4 h later by propidium iodide staining. (A) Representative PI staining from WT Th1 and Th2 cells showing apoptotic cells in sub-G1, and relative survival rate of T cells from different mice following LzCD95L treatment. (B) WT Th1 and Th2 cells were treated with LzCD95L and assayed for cytoplasmic levels of p65 by western blot. (C) WT Th1 and Th2 cells were treated with LzCD95L and assayed for nuclear NFκB binding activity by EMSA. Densitometry measurements (right) are displayed as mean pixel intensities in arbitrary units. Data are representative data from three or more independent experiments. In panel (A) apoptosis assays included 5 replicated for each independent experiment (^**p ≤ 0.01, ^***p ≤ 0.001).

Figure 4

Non-apoptotic Fas signaling in T cells is sufficient for the normal resolution of Th2-mediated inflammation. T cells from the indicated strains were adoptively transferred into Rag^−/− mice which were then sensitized and challenged then assayed for numbers of (A) CD4 T cells and (B) eosinophils in the lungs at days 4, 14, and 28 after the final challenge. (C) H&E stains of lung sections of sensitized and challenged mice at day 14. Total lung cells were co-cultured with irradiated splenocytes with SEA antigen (D) or anti-CD3 antibody (E) for T cell cytokine production. (A–C) Data are representative of three independent experiments for each time point with n ≥ 5 mice per group for each experiment. (D,E) data are representative of two independent experiments with n- of 3 or 4 mice per group (^*p ≤ 0.05, ^**p ≤ 0.01, ^***p ≤ 0.001).