Fig 2: The role of neutrophil P2X7 receptor expression on S. pneumoniae corneal infection.(a–c). S. pneumoniae infected corneas of C57BL/6 and P2X7-/- mice. Total neutrophils and macrophages quantified by flow cytometry (a), and bacterial CFU (b) 24 h post infection. (c). Western blot analysis of pro- and cleaved caspase-1 and IL-1ß from 24 h infected corneas. (d–f). CD18-/- mice were infected with S. pneumoniae and 3 h later injected intravenously with donor bone marrow neutrophils from C57BL/6 or P2X7-/- mice. Total neutrophils in infected corneas of recipient CD18-/- mice given C57BL/6 or P2X7-/- neutrophils (d); CFU in corneas of infected C57BL/6 and CD18-/- mice (e); and in infected CD18-/- mice given donor bone marrow neutrophils from C57BL/6 or P2X7-/- mice (f). These experiments were repeated twice with similar results. 24 h post infection mice were sacrificed and and bacterial quantified. Each data point represents one cornea and histograms are mean±s.d. of at least three corneas per group. A P value =0.05 was considered significant using a Student's t-test.

Fig 3: Cerebral Synaptosomal Purinergic Receptors were Downregulated in the MIA Model and Restored to Normal by Antipurinergic Therapy.(A) Western Analysis of Metabotropic P2Y2 and Ionotropic P2X7 receptors. Each lane contains the synaptosomes from 2–3 males isolated at 16-weeks of age (n = 4–5 per group). (B) P2Y2 receptor expression was decreased by over 50% by gestational poly(IC) exposure and normalized by suramin treatment (Sal-Sal = 100+/-7.3%; Sal-Sur = 62+/-4.6%; PIC-Sal = 48+/-4.7%; PIC-Sur = 84+/-4.7%; one-way ANOVA F(3,12) = 18.1; p<0.0001; n = 4–5 males per group). (C) P2X7 receptor expression was decreased over 50% by gestational poly(IC) exposure and normalized by suramin treatment (Sal-Sal = 100+/-2.2%; Sal-Sur = 39+/-12%; PIC-Sal = 47+/-0.5%; PIC-Sur = 81+/-1.5%; one-way ANOVA F(3,12) = 23.2; p<0.0001; n = 4–5 males per group). Post-synaptic density 95 (PSD95) protein was used as a loading control. Values are expressed as mean +/- SEM.

Fig 4: Activated Müller cells cause microglia activation via ATP/P2X7R pathway. a Representative immunoblots showing the changes in TSPO and P2X7R protein expression in cultured microglia which were co-cultured with normal or pre-activated Müller cells in the absence or presence of the P2X7R blocker BBG. Müller cells were pre-activated by incubating with DHPG for 72 h. BBG (10 µM) was added to culture medium of microglia 2 h before the activated Müller cells were added to the Transwell system. b, c A comparison of average relative densitometric quantifications of the immunoreactive bands obtained following different kinds of treatment is shown in the bar charts (b for TSPO; c for P2X7R). d Representative western blotting results showing the changes in protein levels of TSPO and P2X7R in cultured microglia which were co-cultured with normal or pre-activated Müller cells in the absence or presence of the Cx43 blocker Gap62. Gap26 (200 µM) was added to culture medium of Müller cells. e, f A comparison of average relative densitometric quantifications of the immunoreactive bands obtained following different kinds of treatment is shown in the bar charts (e for TSPO; f for P2X7R). All the data are normalized to empty transwell group. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. empty transwell group using ordinary one-way ANOVA with Dunnett’s multiple comparisons test (b, c, e, f); #P < 0.05 and ###P < 0.001 using unpaired two-tailed Student’s t test against the activated Müller cells group (b, c, e, f). All in vitro experiments: n = 3 biological replicates × 3 technical replicates

Fig 5: The mixed rod and cone ERG is altered across a range of light intensities in the P2X7-KO mouse.Mice were dark adapted overnight and mixed (rod and cone) ERG responses were recorded in response to full field flashes from intensity -4.2 to 2.1 log cd.s/m2. (A–B) Representative mixed ERG waveforms for (A) WT and (B) P2X7-KO mice are presented. (C) There was no change in the amplitude of the rod photoreceptor derived component, a-wave, as a function of intensity. (D) The amplitude of the post-photoreceptor response, the b-wave was larger in P2X7-KO mice than in WT mice across almost all intensities tested. * indicates p<0.05, a significant difference between WT and P2X7-KO.