TRAIL regulates T cell activation and suppresses inflammation in autoimmune diseases

In recent years, it has become evident that the immune tolerance and homeostasis of T cells is highly dynamic and tightly regulated by internal stimuli, including signals from T cell receptor (TCR) and immune checkpoint receptors such as CTLA-4 1. Autoimmune diseases begin with the breakdown of immune tolerance and result in the development of tissue inflammation. The manifestations of autoimmune inflammation are driven by innate immunity and autoreactive pathogenic T and B cells. T cells play a central role in the regulation and initiation of autoimmune responses.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)¹ is a type II transmembrane protein in the TNF superfamily that shares high homology with the Fas ligand.^2–4 TRAIL induces cell apoptosis by binding to its death receptor, resulting in the activation of apoptosis signaling through the caspase cascade;^5,6 however, most normal primary cells are resistant to TRAIL-induced apoptosis.⁷ Although the actual biological role of TRAIL remains obscure, recent accumulated evidence has focused on the emerging role of TRAIL in T cell homeostasis and in regulating immune responses, and evidence in several autoimmune animal models suggests that TRAIL has an immunoregulatory role in autoimmune diseases.^8–14 In autoimmune arthritis, TRAIL deficiency¹⁵ or TRAIL blockade¹² increased joint inflammation and enhanced disease activity, and TRAIL administration suppressed joint inflammation and inhibited synovial lymphocyte infiltration in autoimmune arthritis models.⁸ To determine whether the anti-inflammatory effects of TRAIL are due to the induction of apoptosis, we further evaluated TRAIL-induced anti-inflammatory effects in mice with collagen antibody-induced arthritis (CAIA) in the presence and absence of the pan-caspase inhibitor z-VAD-fmk. The results demonstrated that the pan-caspase inhibitor z-VAD-fmk was not able to reverse the TRAIL-mediated suppression of joint inflammation. In addition, very little cell apoptosis was detected in the synovial tissue of mice treated with TRAIL (Fig. 1a). Moreover, TRAIL treatment did not induce apoptosis in most of the primary immune cells within joints, including T cells, macrophages and synovial fibroblasts (Fig. 1b), which indicated that the suppression of inflammation was not due to the induction of cell apoptosis in the joint tissues by TRAIL. In contrast, TRAIL directly suppressed the activation of T cells and inhibited joint inflammation, suggesting that TRAIL mediates an apoptosis-independent anti-inflammatory pathway⁸. To further explore the possible mechanisms by which TRAIL inhibits joint inflammation in inflammatory arthritis, we examined the effects of TRAIL on T-cell activation. The results demonstrated that the proliferation of primary T cells stimulated with anti-CD3/CD28 Abs was significantly inhibited by TRAIL, while these effects were abolished in the presence of soluble TRAIL receptor (sTRAIL-R) (Fig. 1c). The TRAIL-induced inhibitory effects on the T cells were not due to the proapoptotic effects of TRAIL but instead involved the suppression of the downstream TCR signaling pathway (Fig. 1d). All these results indicated that instead of inducing apoptosis, TRAIL directly inhibited T-cell activation, thus suppressing T-cell proliferation and cytokine production.⁸ Similar results were also observed in mice with experimental autoimmune encephalomyelitis (EAE). TRAIL inhibited T cell reactivity to neuroantigens and suppressed autoimmune encephalomyelitis in murine EAE, and these effects were dependent on TRAIL-R.¹⁶ Moreover, TRAIL directly inhibited the activation of T cells and suppressed neuroinflammation in adoptive transfer-induced EAE.¹⁶ Taken together, our results show a novel mechanism through which the TRAIL/TRAIL-R interaction regulates T cell activation and suppresses T cell activation via an apoptosis-independent pathway in autoimmune inflammation.

Fig. 1.

TRAIL suppressed TCR signaling and T cell activation in an apoptosis-independent manner. a C57BL/6 mice were immunized with a cocktail of five monoclonal antibodies to type II collagen on day 0 followed by LPS on day 3 (collagen antibody-induced arthritis, CAIA). Beginning day 3 postimmunization, mice were treated with vehicle, z-VAD-fmk (20 μg/mouse, i.p.), TRAIL (50 μg/mouse, i.p.) plus vehicle, or TRAIL plus z-VAD-fmk daily. Clinical scores (upper panel) of groups were measured at the indicated time points (n = 10 in each group). *p < 0.05, **p < 0.01 as determined by Student’s t test, compared to vehicle (CAIA) group; †p < 0.05, ††p < 0.01 as determined by Student’s t test, compared to z-VAD-fmk (CAIA) group. Immunohistochemistry staining for cleaved caspase-3 in the ankle joints in each group was carried out on day 14 (lower panel). The arrow indicates cleaved caspase-3⁺ cells. b T cells, macrophages, and synovial fibroblasts from the joints of C57BL/6 mice with CAIA induction and the Jurkat cell line were treated with TRAIL as indicated for 24 h followed by Annexin V and 7-AAD staining. The percentages of apoptotic cells, including Annexin V⁺ 7-AAD^- (early apoptotic) and Annexin V⁺ 7-AAD⁺ (late apoptotic) cells, are shown. c A total of 10⁵ splenic CD4 T cells from C57BL/6 mice were labeled with CFSE and treated with medium, anti-CD3 Ab (1 μg/ml) and anti-CD28 Ab (1 μg/ml) in the presence or absence of TRAIL (10 μg/ml) and soluble TRAIL-R (10 μg/ml) for 5 days. Representative figures of the CFSE staining (upper panel) and quantified data (lower panel) are shown. **p < 0.01 as determined by Student’s t test. d A total of 2 × 10⁶ splenic CD4 T cells from C57BL/6 mice were stimulated at the indicated time points with medium, anti-CD3 (1 µg/ml) and anti-CD28 (1 µg/ml) Abs, TRAIL (10 µg/ml), or a combination of anti-CD3/anti-CD28 Abs and TRAIL. Lysates of each group were prepared and immunoblotted with the indicated antibodies

In addition, to investigate the immunoregulatory role and potential therapeutic application of TRAIL in inflammatory bowel disease (IBD), we further demonstrated that TRAIL effectively suppressed gut inflammation and reduced colitis severity in a DSS-induced colitis animal model.¹⁷ To confirm that TRAIL directly inhibits colitogenic T cells in the development of autoimmune colitis, in this study, we used an adoptive transfer colitis model to evaluate the ability of TRAIL-treated T cells to promote the development of gut inflammation and colitis. The results demonstrated that TRAIL directly inhibited colitogenic T cells and the development of mucosal inflammation and colitis in the adoptive transfer-induced colitis model.¹⁷ The results implicate a novel immunoregulatory role of TRAIL in gut inflammation and mucosal immunity. All these studies further demonstrated that TRAIL effectively suppresses autoimmune inflammation via an apoptosis-independent pathway.^8,16

TRAIL has long been regarded as an apoptosis-inducing ligand; however, in addition to inducing apoptosis, recent emerging evidence has demonstrated that TRAIL can modulate inflammation in autoimmune diseases. Although the mechanisms by which TRAIL inhibits autoimmune inflammation remain to be elucidated, the anti-inflammatory effect was previously thought to be due to its proapoptotic activity, namely, inducing inflammatory cell death.^10,18,19 However, recent reports and our studies clearly demonstrated that TRAIL does not promote inflammatory cell apoptosis in several autoimmune animal models, and the anti-inflammatory effect of TRAIL occurs via an apoptosis-independent pathway.^{8,12,13,16,17} Instead, TRAIL directly suppresses the activation of T cells and inhibits autoimmune inflammation. These results also imply the potential therapeutic potential of TRAIL in treating human autoimmune diseases.

T cell activation is a critical step for the development of inflammation in autoimmune diseases. Our studies have demonstrated that TRAIL inhibits T cell activation and suppressed autoimmune inflammation in an apoptosis-independent manner in several autoimmune disease animal models.^8,16,17 In support of these findings, TRAIL-treated T cells significantly suppressed the development of encephalitis and gut inflammation, indicating that TRAIL inhibited effector T cell functions in the pathogenesis of autoimmune diseases. On the other hand, it has been well addressed that regulatory T cells (Tregs) are critical in the regulation of immune responses in many autoimmune diseases. It has been demonstrated that both Th1 and Th17 effector T-cell responses contribute to colonic inflammation.^20–22 In contrast, the adoptive transfer of Tregs could protect recipient mice from colitis and mucosal inflammation.²³ Recent results in mouse models suggest that T cell plasticity, in particular, the Th17-Treg axis, plays an important role in regulating immune responses in the intestines.²⁴ These findings suggest that TRAIL may have a role in regulating proinflammatory T cells and Tregs.

Our results provide in vivo evidence that TRAIL suppresses the activation of autoreactive T cells and prevents the development of autoimmune disease through an apoptosis-independent pathway via TRAIL-R signaling. To further investigate the molecular mechanism of TRAIL in the inhibition of T cell activation. A large number of genes in CD4⁺ T cells regulated by TRAIL were analyzed. Most of these genes were involved in the TCR signaling pathway, cell cycle, cytokine-cytokine receptor interactions, extracellular matrix (ECM) receptor interactions, and metabolic pathways. Among these pathways, most TCR signaling pathways were downregulated by TRAIL, indicating a regulatory role of TRAIL in TCR signaling.¹⁶ To confirm TRAIL-mediated regulation of T cell activation and TCR signaling, we isolated CD4 T cells and stimulated them with anti-CD3/anti-CD28 Abs in the presence or absence of TRAIL. The results demonstrated that when CD4 T cells were activated in the presence of TRAIL, the phosphorylation of proximal TCR-associated signaling molecules, including ZAP70, LAT, and PLCγ1, was profoundly inhibited. In addition, the TRAIL-mediated suppression of phosphorylated TCR-associated signaling molecules was abolished in TRAIL-R deficiency.¹⁶ Furthermore, to confirm whether TRAIL could directly inhibit the activation of T cells, we demonstrated that upon stimulation with anti-CD3/anti-CD28 Abs, TRAIL-treated T cells inhibited the phosphorylation of proximal TCR signaling-associated molecules, including Lck, ZAP70, PLCγ1, and LAT, and repressed the lipid raft recruitment of these signaling molecules.¹⁶ Taken together, these results clearly indicate that the TRAIL/TRAIL-R interaction directly inhibits the phosphorylation of TCR-associated proximal tyrosine kinases and subsequent lipid raft recruitment, resulting in the suppression of signaling downstream of TCR and T cell activation. Moreover, these results provide a novel mechanism through which TRAIL-R signaling suppresses T cell activation, a signaling pathway distinct from traditional death receptor signaling. We conclude that TRAIL is a critical regulator of T cell activation in autoimmune inflammation. Our study implies that TRAIL-R can serve as a novel immune checkpoint in T cell responses and sheds light on future therapeutic applications for autoimmune diseases.

Competing interests

The authors declare no competing interests.