P2Y2R deficiency attenuates experimental autoimmune uveitis development

Abstract

We aimed to study the role of the nucleotide receptor P2Y2R in the development of experimental autoimmune uveitis (EAU). EAU was induced in P2Y2+/+ and P2Y2-/- mice by immunization with IRBP peptide or by adoptive transfer of in vitro restimulated semi-purified IRBP-specific enriched T lymphocytes from spleens and lymph nodes isolated from native C57Bl/6 or P2Y2+/+ and P2Y2-/- immunized mice. Clinical and histological scores were used to grade disease severity. Splenocytes and lymph node cell phenotypes were analyzed using flow cytometry. Semi-purified lymphocytes and MACS-purified CD4+ T lymphocytes from P2Y2+/+ and P2Y2-/- immunized mice were tested for proliferation and cytokine secretion. Our data show that clinical and histological scores were significantly decreased in IRBP-immunized P2Y2-/- mice as in P2Y2-/- mice adoptively transfered with enriched T lymphocytes from C57Bl/6 IRBP-immunized mice. In parallel, naïve C57Bl/6 mice adoptively transferred with T lymphocytes from P2Y2-/- IRBP-immunized mice also showed significantly less disease. No differences in term of spleen and lymph node cell recruitment or phenotype appeared between P2Y2-/- and P2Y2+/+ immunized mice. However, once restimulated in vitro with IRBP, P2Y2-/- T cells proliferate less and secrete less cytokines than the P2Y2+/+ one. We further found that antigen-presenting cells of P2Y2-/- immunized mice were responsible for this proliferation defect. Together our data show that P2Y2-/- mice are less susceptible to mount an autoimmune response against IRBP. Those results are in accordance with the danger model, which makes a link between autoreactive lymphocyte activation, cell migration and the release of danger signals such as extracellular nucleotides.

Fig 1. P2Y₂ ^-/- mice are partially protected against uveitis development.

EAU was induced in P2Y₂ ^+/+ (n = 15 ->12) and P2Y₂ ^-/- (n = 15 ->10) mice by s.c. immunization with IRBP 1–20 peptide emulsified in CFA and i.p. injection of PTX. A blind fundoscopy was performed at days 7, 14 and 21 to detect vitritis, optic neuropathy, vasculitis and retinitis. Data represent the median of EAU clinical scores established for all mice at each time point, in the two groups. Three independent experiments were done, starting with 5 animals in each group. *p < 0,05.

Fig 2. P2Y₂ ^-/- mice are partially protected against uveitis development induced by the adoptive transfer of IRBP-specific enriched TL—Efferent phase.

P2Y₂ ^+/+ (n = 9) and P2Y₂ ^-/- (n = 11) mice have received an adoptive transfer of semi-purified IRBP-specific enriched TL from C57Bl/6 immunized mice. Data are from two independent experiments. (A) A blind fundoscopy was performed at days 7, 14 and 21 and an EAU clinical score was established for each eye of every animal in the two groups. Data represent the median of clinical scores established for all mice at each time point. (B) At day 21, all the mice were sacrificed, the eyes enucleated and processed for histology. Hematoxylin-eosine coloration was realized in order to perform a double blind histological grading. Each symbol represents the established EAU histological score for one mouse. Lines represent median histological scores. *P < 0,05; **P < 0,01; ***p < 0,001.

Fig 3. In vivo decreased capacity of IRBP-specific enriched TL from P2Y₂ ^-/- mice to induce uveitis—Afferent phase.

Semi-purified IRBP-specific enriched TL were recovered from five P2Y₂ ^+/+ and P2Y₂ ^-/- immunized mice, pooled and adoptively transferred in two groups of naïve C57Bl/6 mice after a 48h in vitro restimulation with IRBP. Data are from three independent experiments where 16 mice in all received P2Y₂ ^+/+ TL and 15 mice P2Y₂ ^-/- TL. (A) A blind fundoscopy was performed at days 7, 14 and 21 and a clinical score was established for each eye of every animal in the two groups. Data represent the median of clinical scores established for all mice at each time point. (B) At day 21, all the mice were sacrificed, the eyes enucleated and processed for histology. Hematoxylin-eosine coloration was realized in order to performed a double blind histological grading. Each symbol represents the established histological score for one mouse. Lines represent median histological scores. * p < 0,05; ***p < 0,001.

Fig 4. No difference in cell number or in cell phenotype between P2Y₂ ^+/+ and P2Y₂ ^-/- IRBP-immunized mice.

Twelve days after s.c. immunization with IRBP 1–20 peptide emulsified in CFA and combined with an i.p. injection of PTX, P2Y₂ ^+/+ and P2Y₂ ^-/- mice were sacrificed and their spleen and draining lymph nodes collected and dissociated. (A) Total cell number count of mixture of splenic T lymphocytes semi-purified by passage on nylon wool fiber columns and LN cells. Mean +/- SEM. (B) Spleens and LN were independently characterized by flow cytometry for cellular phenotype by using FITC- or PE- anti-mouse antibodies directed against CD3, CD11b, CD11c and MHCII surface molecules. TL: CD3+; DC: CD11c+/CD11b+ or MHCII+; APC tot: DC + CD11c-/CD11b+ or MHCII+. Mean +/- SEM. Data are from at least 10 different mice in each group.

Fig 5. P2Y₂ ^-/- TL proliferate less and secrete less cytokines in response to in vitro IRBP restimulation.

Twelve days after s.c. immunization with IRBP 1–20 peptide, P2Y₂ ^+/+ and P2Y₂ ^-/- mice were sacrificed and their spleen and draining lymph nodes collected and dissociated. (A) Splenic TL were semi-purified by passage on nylon wool fiber columns and pooled with LN cells before being in vitro restimulated in medium alone (CTRL) or supplemented with IRBP 1–20 peptide. TL proliferation was tested after 72h by (³H)- incorporation. Data are from a representative experiment of 10 with 4 mice per group. (B) Cytokine secretion was quantified, by specific ELISA, in culture supernatants after 48h of IRBP restimulation. Data are from a representative experiment of 5 or 6 or 3 respectively, with 4 mice per group. ns: not significant; **p < 0,01; ***p < 0,001.

Fig 6. P2Y₂ ^-/- reduced TL proliferation in response to in vitro IRBP restimulation is linked to a defect in P2Y₂ ^-/- APC activation capacities.

Twelve days after s.c. immunization with IRBP 1–20 peptide, P2Y₂ ^+/+ and P2Y₂ ^-/- mice were sacrificed and their spleen and draining lymph nodes collected and dissociated. (A) Splenic TL were semi-purified by passage on nylon wool fiber columns and pooled with LN cells before being in vitro restimulated in medium alone (CTRL) or supplemented with Dynabeads Mouse T-activator CD3/CD28. TL proliferation was tested after 72h by (³H)-thymidine incorporation. Data are from a representative experiment of 6 with 4 mice per group. ns: not significant (B) Spleens from P2Y₂ ^+/+ and P2Y₂ ^-/- IRBP-immunized were independently processed and analyzed by qPCR for β-Actin, CD3 and Foxp3 mRNA expression. Data are from 5 different mice in each group and are expressed as relative expression of Foxp3 versus CD3. ns: not significant (C) CD4+ TL were isolated from LN of P2Y₂ ^+/+ and P2Y₂ ^-/- mice by magnetic separation and co-cultured with total irradiated (30 Gy) splenocytes (APC) in autologous and cross-culture conditions, in presence of IRBP 1–20 peptide. TL proliferation was tested after 72h by (³H)-thymidine incorporation. Data, expressed in cpm, are from a representative experiment of 3 with 4 mice per group. ns: not significant; **p < 0,01; ***p < 0,001.