P2RX7 Deletion in T Cells Promotes Autoimmune Arthritis by Unleashing the Tfh Cell Response

Abstract

Rheumatoid arthritis (RA) is an autoimmune disease that affects ~1% of the world's population. B cells and autoantibodies play an important role in the pathogenesis of RA. The P2RX7 receptor is an ATP-gated cation channel and its activation results in the release of pro-inflammatory molecules. Thus, antagonists of P2RX7 have been considered to have potential as novel anti-inflammatory therapies. Although originally identified for its role in innate immunity, P2RX7 has recently been found to negatively control Peyer's patches (PP) T follicular helper cells (Tfh), which specialize in helping B cells, under homeostatic conditions. We have previously demonstrated that PP Tfh cells are required for the augmentation of autoimmune arthritis mediated by gut commensal segmented filamentous bacteria (SFB). Thus, we hypothesized that P2RX7 is required to control autoimmune disease by keeping the Tfh cell response in check. To test our hypothesis, we analyzed the impact of P2RX7 deficiency in vivo using both the original K/BxN autoimmune arthritis model and T cell transfers in the K/BxN system. We also examined the impact of P2RX7 ablation on autoimmune development in the presence of the gut microbiota SFB. Our data illustrate that contrary to exerting an anti-inflammatory effect, P2RX7 deficiency actually enhances autoimmune arthritis. Interestingly, SFB colonization can negate the difference in disease severity between WT and P2RX7-deficient mice. We further demonstrated that P2RX7 ablation in the absence of SFB caused reduced apoptotic Tfh cells and enhanced the Tfh response, leading to an increase in autoantibody production. It has been shown that activation of TIGIT, a well-known T cell exhaustion marker, up-regulates anti-apoptotic molecules and promotes T cell survival. We demonstrated that the reduced apoptotic phenotype of P2rx7^-/- Tfh cells is associated with their increased expression of TIGIT. This suggested that while P2RX7 was regulating the Tfh population by promoting cell death, TIGIT may have been opposing P2RX7 by inhibiting cell death. Together, these results demonstrated that systemic administration of general P2RX7 antagonists may have detrimental effects in autoimmune therapies, especially in Tfh cell-dependent autoimmune diseases, and cell-specific targeting of P2RX7 should be considered in order to achieve efficacy for P2RX7-related therapy.

Keywords: P2RX7; TIGIT; apoptosis; autoimmune; microbiota.

Figure 1

P2RX7 deficiency leads to enhanced disease development in the SFB(–) K/BxN model. (A) Ankle thickness was followed over time for K/BxN and P2rx7^−/−.K/BxN mice between ~3.5 and 6 weeks of age. Data are shown as change in ankle thickness compared to first measurement. N = 9–14/group, 6 assays combined. (B) Anti-GPI auto-Ab titers in serum obtained from the end point of each experiment were measured by ELISA. N = 4–8/group, 6 assays combined. Error bars represent SEM. ^*p < 0.05.

Figure 2

P2RX7 deletion induces the Tfh cell response in PPs but not spleen of the SFB(–) K/BxN mice. (A) Splenocytes and PP cells from 5 to 6 week old K/BxN and P2rx7^−/−.K/BxN mice were stained with Abs against CD4, CD19, PD-1, and CXCR5. Values in the representative plots indicate the percentage of Tfh cells in total CD4⁺ T cells. Compiled graphs of the number of Tfh cells are also shown as mean + SEM. N = 17–18/group, 8 independent assays combined. (B) Tfh cells from spleen and PPs of K/BxN and P2rx7^−/−.K/BxN mice were analyzed for Annexin V binding among Live/Dead Yellow⁻ cells. Representative plots and compiled graphs are shown. Ratios calculated by dividing the average of the K/BxN (WT) group by the average of the P2rx7^−/−.K/BxN (KO) group. N = 6–7/group, 4 independent assays combined. (C) P2RX7 expression was detected by flow cytometry on non-Tfh and Tfh cells from spleen and PPs of K/BxN and P2rx7^−/−.K/BxN mice. Representative plots and compiled graphs are shown. N = 8–9/group, 3 independent assays combined. (D) Bcl-6 expression was detected by flow cytometry on non-Tfh cells from spleen and PPs of K/BxN and P2rx7^−/−.K/BxN mice. Representative plots and compiled graphs are shown. N = 13–17/group, 6 independent assays combined. Error bars represent means + SEM. ^*p < 0.05, ^**p < 0.01, ^****p < 0.0001.

Figure 3

P2rx7^−/− T cells enhance autoimmunity in the SFB(–) K/BxN T cell transfer model. (A–E) CD4⁺ T cells were isolated from either WT K/BxN or P2rx7^−/−.K/BxN donors and transferred to TCRα^−/−.BxN recipients. (A) Schematic for T cell transfer. (B) Arthritis development was followed by ankle measurements starting at the day of transfer (day 0). Values are shown as averages of the change in ankle thickness for each individual compared to its first measurement. N = 20–24/group, 5 assays combined. (C) Auto-Ab titer as determined by ELISA against GPI. N = 20–24/group, 5 assays combined. (D) Representative plots and compiled graphs of Tfh and non-Tfh cell numbers in spleen and PPs. N = 15–18/group, 4 assays combined. (E) Representative plots of Tfh and non-Tfh cells stained for Annexin V and Live/Dead Yellow and compiled graphs of Live/Dead Yellow⁻ Annexin V⁺ Tfh or non-Tfh cells as a percentage of total Tfh or non-Tfh cells in spleen and PPs. WT = K/BxN, KO = P2rx7^−/−.K/BxN. Ratios determined by dividing the average of the WT group by the average of KO group. N = 8–11/group, 3 assays combined. ^*p < 0.05 ^**p < 0.01 ^****p < 0.0001.

Figure 4

Treg numbers do not differ between SFB(–) K/BxN and P2rx7^−/−.K/BxN mice. (A) Representative plots and compiled graphs of Treg numbers in spleen and PPs of K/BxN and P2rx7^−/−.K/BxN mice. N = 5–11/group, 3 assays combined. (B) P2RX7 surface expression was detected by flow cytometry on non-Tfh, Tfh, and Treg populations from spleen and PPs of K/BxN mice. N = 7/group. Error bars represent SEM. ^***p < 0.001 ^****p < 0.0001.

Figure 5

SFB colonization negates the difference in disease severity between WT and P2rx7^−/− mice in the original K/BxN model and K/BxN T cell transfer model. (A–G) K/BxN mice (A–C) or TCRα^−/−. BxN mice (D–G) were gavaged with SFB 1–2 days after weaning. (A) Ankle thickness was followed over time for K/BxN and P2rx7^−/−.K/BxN mice between ~3.5 and 6 weeks of age. Data are shown as change in ankle thickness compared to first measurement. Error bars represent SEM. N = 7–17/group, multiple litters combined. (B) Anti-GPI auto-Ab titers in serum obtained from mice 5–7 weeks old were measured by ELISA. N = 8–11/group, 5 assays combined. (C) SFB level 10 days after gavage was determined by quantitative PCR on 16S rDNA, normalized to a standardized positive control. N = 5–12/group, 5 assays combined. (D–G) SFB(+) TCRα^−/−.BxN recipients were given either K/BxN or P2rx7^−/−.K/BxN CD4⁺ cells retro-orbitally at 6 weeks of age. (D) Ankle thickness was followed beginning at day of transfer for TCRα^−/−.BxN mice transferred with either K/BxN or P2rx7^−/−.K/BxN CD4⁺ cells. Data are shown as change in ankle thickness compared to first measurement. Error bars represent SEM. N = 12/group, 3 assays combined. (E) Anti-GPI auto-Ab titers in serum obtained from the end point of each experiment were measured by ELISA. N = 12/group, 3 assays combined. (F) SFB level determined by quantitative PCR on 16S rDNA, normalized to a standardized positive control. Feces were collected at end point of each experiment (14 days after T cell transfer which was equivalent to 5 weeks after SFB gavage). N = 23–24/group, 5 assays combined. (G) Representative plots and compiled graphs of spleen and PP Tfh numbers. N = 11/group, 3 assays combined. ^**p < 0.01.

Figure 6

TIGIT is increased on P2rx7^−/− Tfh cells in the SFB(–) K/BxN transfer model. (A,B) Representative histograms and compiled graphs of TIGIT expression on (A) splenic or (B) PP Tfh cells from WT or P2rx7^−/−.K/BxN CD4⁺ cells transferred into TCRα^−/−.BxN mice. Compiled graphs show TIGIT⁺ Tfh as a percentage of total Tfh cells. N = 7–9/group, 2 assays combined. ^**p < 0.01 ^****p < 0.0001.

Figure 7

TIGIT expression on Tfh cells is associated with decreased apoptosis in the SFB(–) K/BxN transfer model. (A,B) WT or P2rx7^−/−.K/BxN CD4⁺ cells were transferred into TCRα^−/−. BxN recipients. Shown are representative plots and compiled graphs of Live/Dead Yellow⁻ Annexin V⁺ staining as a percentage of either TIGIT⁺ or TIGIT⁻ Tfh cells. N = 11–14/group, 3 assays combined. ^*p < 0.05 ^**p < 0.01 ^****p < 0.0001.

Figure 8

Model of interactions between P2RX7 and TIGIT, and their impact on cell death. (A) P2RX7 promotes apoptosis in PP Tfh cells to regulate Tfh numbers. P2RX7 may also regulate TIGIT expression on Tfh cells. TIGIT, conversely, may inhibit apoptosis to promote Tfh cell survival. (B) In the absence of P2RX7, apoptosis is disrupted, allowing the expansion of the PP Tfh population. In addition, the absence of P2RX7 leads to greater TIGIT expression, which may further down-regulate apoptosis.