Fas-Independent T-Cell Apoptosis by Dendritic Cells Controls Autoimmune Arthritis in MRL/lpr Mice

Background: Although autoimmunity in MRL/lpr mice occurs due to a defect in Fas-mediated cell death of T cells, the role of Fas-independent apoptosis in pathogenesis has rarely been investigated. We have recently reported that receptor activator of nuclear factor (NF)-kB ligand (RANKL)-activated dendritic cells (DCs) play a key role in the pathogenesis of rheumatoid arthritis (RA) in MRL/lpr mice. We here attempted to establish a new therapeutic strategy with RANKL-activated DCs in RA by controlling apoptosis of peripheral T cells. Repeated transfer of RANKL-activated DCs into MRL/lpr mice was tested to determine whether this had a therapeutic effect on autoimmunity. Methods and Finding: Cellular and molecular mechanisms of Fas-independent apoptosis of T cells induced by the DCs were investigated by in vitro and in vivo analyses. We demonstrated that repeated transfers of RANKL-activated DCs into MRL/lpr mice resulted in therapeutic effects on RA lesions and lymphoproliferation due to declines of CD4+ T, B, and CD4‾CD8‾ double negative (DN) T cells. We also found that the Fas-independent T-cell apoptosis was induced by a direct interaction between tumor necrosis factor (TNF)-related apoptosis-inducing ligand-receptor 2 (TRAIL-R2) on T cells and TRAIL on Fas-deficient DCs in MRL/lpr mice. Conclusion: These results strongly suggest that a novel Fas-independent apoptosis pathway in T cells maintains peripheral tolerance and thus controls autoimmunity in MRL/lpr mice

Fas-Independent T-Cell Apoptosis by Dendritic Cells Controls Autoimmune Arthritis in MRL/<em>lpr</em> Mice

<div><h3>Background</h3><p>Although autoimmunity in MRL/<em>lpr</em> mice occurs due to a defect in Fas-mediated cell death of T cells, the role of Fas-independent apoptosis in pathogenesis has rarely been investigated. We have recently reported that receptor activator of nuclear factor (NF)-κB ligand (RANKL)-activated dendritic cells (DCs) play a key role in the pathogenesis of rheumatoid arthritis (RA) in MRL/<em>lpr</em> mice. We here attempted to establish a new therapeutic strategy with RANKL-activated DCs in RA by controlling apoptosis of peripheral T cells. Repeated transfer of RANKL-activated DCs into MRL/<em>lpr</em> mice was tested to determine whether this had a therapeutic effect on autoimmunity.</p> <h3>Methods and Finding</h3><p>Cellular and molecular mechanisms of Fas-independent apoptosis of T cells induced by the DCs were investigated by <em>in vitro</em> and <em>in vivo</em> analyses. We demonstrated that repeated transfers of RANKL-activated DCs into MRL/<em>lpr</em> mice resulted in therapeutic effects on RA lesions and lymphoproliferation due to declines of CD4⁺ T, B, and CD4⁻CD8⁻ double negative (DN) T cells. We also found that the Fas-independent T-cell apoptosis was induced by a direct interaction between tumor necrosis factor (TNF)-related apoptosis-inducing ligand-receptor 2 (TRAIL-R2) on T cells and TRAIL on Fas-deficient DCs in MRL/<em>lpr</em> mice.</p> <h3>Conclusion</h3><p>These results strongly suggest that a novel Fas-independent apoptosis pathway in T cells maintains peripheral tolerance and thus controls autoimmunity in MRL/<em>lpr</em> mice.</p> </div

T cell responses in DC-transferred MRL/<i>lpr</i> mice.

<p>(A) Proliferative responses of ILN CD4⁺ T cells from the recipients and control mice were analyzed. Purified CD4⁺ T cells were stimulated with plate-coated CD3 mAb (0–0.5 µg/ml) and CD28 mAb (10 µg/ml) for 72 hours. The proliferative response was evaluated by [³H] thymidine incorporation. Values are means ± SD (n = 4, 5, and 5 respectively per group). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048798#s2" target="_blank">Results</a> are representative of three independent experiments with similar results. (B) The culture supernatants for 24 h (anti-CD3 mAb: 0.5 µg/ml; anti-CD28 mAb: 10 µg/ml) as described above were analyzed for cytokine productions including IL-2, IFN-γ, IL-4, IL-10, and IL-17 by ELISA. Values are means ± SD (n = 4, 5, and 5 respectively per group). *p<0.05, **p<0.005.</p

T-cell apoptosis via TRAIL/TRAIL-R2.

<p>(A) Real-time RT-PCR for a wide array of apoptosis-related genes was performed using mRNA samples of MRL/<i>lpr</i> CD4⁺ T cells repeatedly stimulated with MRL/<i>lpr</i> and MRL+/+ BMDCs. Gene expression of CD4⁺ T cells repeatedly stimulated with MRL/<i>lpr</i> BMDCs was compared with those stimulated with MRL+/+ BMDCs (controls). Genes with increased and decreased expression are shown as fold of control. The experiments were repeated twice with similar results. (B) The mRNA expression of TRAF3, TRAIL-R2, caspase 8, and Bcl-2 was confirmed by quantitative real-time PCR analysis. Relative expression to β-actin level is shown as means ± SD from triplicate samples. The experiments were repeated three times with similar results. (C) Up-regulation of TRAIL expression on MRL/<i>lpr</i> BMDCs by RANKL was detected by flow cytometry. Staining of DC with FITC-labeled isotype control Ab is shown as a dotted line. The experiments were repeated three times with similar results. (D) <i>lpr</i> CD4+ T cells were repeatedly co-cultured with activated <i>lpr</i> DCs in the presence of anti-TRAIL mAb. Data are shown as means ± SD of triplicate samples. *p<0.05, **p<0.005.</p

Fas-independent T-cell apoptosis in DC-transferred MRL/<i>lpr</i> mice.

<p>(A) Experimental protocol of T cell apoptosis by repeated co-culture with DCs. Total T cells from MRL/<i>lpr</i> mice (5×10⁴) were repeatedly (three times) co-cultured with BMDCs (2.5×10⁵) from MRL+/+ or MRL/<i>lpr</i> mice for 8 hours without interval. BMDCs were stimulated with RANKL and CII for 24 hours before the co-culturing. (B) After the third co-culture, apoptosis of CD4⁺ and CD8⁺ T cells expressing annexin-V was detected by flow cytometry. Staining of T cell with FITC-labeled isotype control Ab is shown as a dotted line. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048798#s2" target="_blank">Results</a> are shown as representative of three independent experiments with similar results. (C) MFI of annexin-V on CD4⁺ T cells was calculated, and the data are shown as the means ± SD of triplicate samples. (D) Induction of T-cell apoptosis by repeated co-culturing with activated DCs. Purified CD4⁺, CD8⁺, DNT, and B220⁺ cells (5×10⁴) were repeatedly co-cultured with BMDCs (5 and 25×10⁴). Annexin-V⁺ cells are shown as the means ± SD of triplicate samples. The experiments were repeated three times with similar results. *p<0.05. (E) BMDCs were stimulated with RANKL and, OVA or BSA (10 µg/ml) for 24 hours before the co-culturing. Purified CD4⁺ cells (5×10⁴) were repeatedly co-cultured with BMDCs (5 and 25×10⁴). Annexin-V⁺ cells are shown as the means ± SD of triplicate samples. The experiments were repeated three times with similar results. (F) Purified CD4⁺ cells (5×10⁴) from ILNs in MRL+/+ mice were repeatedly co-cultured with <i>lpr</i> BMDCs (25×10⁴). Annexin-V⁺ cells are shown as the means ± SD of triplicate samples. The experiments were repeated three times with similar results.</p

Therapeutic effect of repeated transfers of DCs on autoimmune arthritis.

<p>(A) Experimental protocol is shown. BMDCs from female MRL+/+ and MRL/<i>lpr</i> mice were stimulated with RANKL and CII, and then female MRL/<i>lpr</i> mice received a total of 3 injections of the BMDCs every other day distributed over 6 day period. At 16 weeks after transfer (20 weeks of age), the recipient MRL/<i>lpr</i> mice were analyzed. (B) Histology of joint from recipient mice. Histological photos with HE staining are shown as representative of the recipient mice at 16 weeks after transfers. Arrow; bone erosion or synovial proliferation, star; mononuclear inflammatory infiltrate, fibrosis, or panus. Scale bar: 100 µm (n = 7, 10 and 12 per group respectively). (C) The histological score of the recipient mice was evaluated at 16 weeks after repeated transfers. Data are shown as means ± SD. (n = 7, 10 and 12 per group respectively). (D) Rheumatoid factor (RF) (IgM and IgG) antibody was measured by ELISA. Values are shown as means ± SD (n = 7, 10 and 12 per group respectively). OD = optical density. (E) RA lesions of control, a single DC transferred (1× DC), and multiple DC transferred (3× DC) MRL/<i>lpr</i> mice were compared. Histological photos with HE staining are shown as representative of the recipient mice at 12 weeks after transfers. Scale bar: 100 µm (n = 5 per group respectively). (F) The histological score of the recipient mice was evaluated at 12 weeks after repeated transfers. Data are shown as means ± SD (n = 5 per group respectively). *p<0.05.</p

Reduced lymphoproliferation of MRL/<i>lpr</i> mice following repeated transfers of DCs.

<p>(A) Spleen and ILNs from the recipient mice are shown. Photos are representative of the recipient mice (16 weeks of age) at 12 weeks after the transfer. Values are shown as means ± SD (n = 5, 7 and 7 per group respectively). (B) The total cell number in the spleen, and ILNs is shown. Scale bar: 5 mm. (C) T cell numbers in the spleen and ILNs of the recipient mice. Flow cytometry was performed using spleen and ILN cells. The number of CD4⁺ and CD8⁺ T cells is shown. (D) B cell (B220⁺ Thy1.2⁻) number in the spleen and ILNs of the recipient mice. (E) CD4⁻ CD8⁻ CD3⁺ DNT cell number in the spleen and ILNs of the recipient mice. Values are shown as means ± SD (n = 5, 7, and 7 respectively per group). *p<0.05, **p<0.005.</p