Microglial pannexin-1 channel activation is a spinal determinant of joint pain

Abstract

Chronic joint pain such as mechanical allodynia is the most debilitating symptom of arthritis, yet effective therapies are lacking. We identify the pannexin-1 (Panx1) channel as a therapeutic target for alleviating mechanical allodynia, a cardinal sign of arthritis. In rats, joint pain caused by intra-articular injection of monosodium iodoacetate (MIA) was associated with spinal adenosine 5'-triphosphate (ATP) release and a microglia-specific up-regulation of P2X7 receptors (P2X7Rs). Blockade of P2X7R or ablation of spinal microglia prevented and reversed mechanical allodynia. P2X7Rs drive Panx1 channel activation, and in rats with mechanical allodynia, Panx1 function was increased in spinal microglia. Specifically, microglial Panx1-mediated release of the proinflammatory cytokine interleukin-1β (IL-1β) induced mechanical allodynia in the MIA-injected hindlimb. Intrathecal administration of the Panx1-blocking peptide ¹⁰panx suppressed the aberrant discharge of spinal laminae I-II neurons evoked by innocuous mechanical hindpaw stimulation in arthritic rats. Furthermore, mice with a microglia-specific genetic deletion of Panx1 were protected from developing mechanical allodynia. Treatment with probenecid, a clinically used broad-spectrum Panx1 blocker, resulted in a striking attenuation of MIA-induced mechanical allodynia and normalized responses in the dynamic weight-bearing test, without affecting acute nociception. Probenecid reversal of mechanical allodynia was also observed in rats 13 weeks after anterior cruciate ligament transection, a model of posttraumatic osteoarthritis. Thus, Panx1-targeted therapy is a new mechanistic approach for alleviating joint pain.

Fig. 1. Spinal microglia critically contribute to MIA-induced joint pain.

(A) Representative images (scale bar, 100 μm) and (B) quantification of CD11b immunofluorescence in the L3-L5 spinal dorsal horn from MIA (2 mg) and saline CTR rats 7 days after injection (CTR/Ipsi, n = 27; MIA/Ipsi, n = 21; MIA/Contra, n = 21). (C) Schematic depicting drug administration paradigm in rats injected with intra-articular (i.a.) MIA (2 mg) or saline (CTR) and intrathecal (i.t.) Mac1-saporin (Sap; 15 μg) or saporin (15 μg). (D) Mechanical paw withdrawal threshold (PWT) (CTR/Mac1-Sap, n = 4; MIA/Mac1-Sap, n = 6; MIA/Sap, n = 4). (E) ATP levels in rat CSF and (F and G) flow cytometric analysis of P2X7R expression in spinal cord cell populations 7 days after injection of MIA (n = 9) or saline (CTR, n = 9). (F) Representative dot plot from CTR and MIA rats depicting gating parameters for CD11b⁻ (black) and Cd11b⁺ (blue) populations. (G) Histogram of P2X7R mean fluorescence intensity of CD11b⁻ and CD11b⁺ populations (MIA, n = 3; CTR, n = 3). NS, not significant. Effect of intrathecal A740003 on mechanical threshold following (H) continuous delivery [CTR/A740003, n = 4; MIA/A740003 10 μM, n = 5; MIA/saline (SA), n = 5] and (I) single injection intrathecally on day 7 (arrow) (MIA/A740003 30 μM, n = 5; MIA/SA, n = 6). *P < 0.05; ****P < 0.0001, one-way analysis of variance (ANOVA) (B and G), two-way repeated-measures ANOVA (D, H, and I) followed by Sidak post hoc test, and unpaired t test (E).

Fig. 2. Microglial Panx1 channel activation is crucial for MIA-induced mechanical allodynia.

(A) Representative traces (scale bar, 50 μm) and (B) quantification of BzATP (300 μM)–evoked YO-PRO-1 dye uptake in spinal microglia isolated from adult rats 7 days after intra-articular saline (CTR, n = 24 cells) or MIA (n = 18 cells). (C) Effect of ¹⁰panx (10 μM) and ^scrpanx (10 μM) control peptide on dye uptake (CTR/^scrpanx, n = 14 cells; CTR/¹⁰panx, n = 31 cells; MIA/^scrpanx, n = 20 cells; MIA/¹⁰panx, n = 24 cells). (D and E) Effect of intrathecal ¹⁰panx (0.5 μg/hour) or ^scrpanx (0.5 μg/hour) on mechanical threshold following (D) continuous (CTR/¹⁰panx, n = 4; MIA/¹⁰panx, n = 6; MIA/^scrpanx, n = 6) or (E) acute delivery on day 7 (arrow) (20 μg) (MIA/¹⁰panx, n = 6; MIA/^scrpanx, n = 5). (F) Mechanical threshold in vehicle (VEH) and tamoxifen (TMX) Cx3cr1-Cre^ERT2::Panx1^flx/flx–treated mice (CTR/VEH, n = 4; MIA/VEH, n = 4; MIA/TMX, n = 5; CTR/TMX, n = 5). *P < 0.05; **P < 0.001; ****P < 0.0001, unpaired t test (B), one-way ANOVA (C), and two-way repeated-measures ANOVA (D to F) followed by Sidak post hoc test.

Fig. 3. Panx1-mediated IL-1β release contributes to joint pain.

(A) IL-1β release from BV2 microglia-like cell cultures and effect of ¹⁰panx (10 μM) or ^scrpanx (10 μM) peptide (ECS, n = 10; BzATP, n = 10; ^scrpanx, n = 8; ¹⁰panx, n = 8). (B) IL-1β levels in rat CSF 7 days after intra-articular MIA (n = 6) or saline (CTR, n = 4) injection. (C) IL-1β mRNA levels of spinal microglia-specific transcripts isolated from RiboTag mice (Cx3cr1-Cre^ERT::Rpl22^HA) (MIA, n = 9; CTR, n = 8). (D) IL-1β levels in vehicle or tamoxifen Cx3cr1-Cre^ERT2::Panx1^flx/flx mice 7 days after intra-articular MIA or saline injection (VEH/CTR, n = 6; VEH/MIA, n = 7; TMX/CTR, n = 7; TMX/MIA, n = 8). (E) Mechanical threshold following a single intrathecal injection on day 7 (arrow) of IL-1β (100 pg) or IL-1ra, an IL-1β receptor antagonist (50 ng), in vehicle or tamoxifen-treated Cx3cr1-Cre^ERT2::Panx1^flx/flx mice (VEH/IL-1ra, n = 8; TMX/IL-1β, n = 8; TMX IL-1ra/IL-1β, n = 7). *P < 0.05; ***P < 0.001, one-way ANOVA (A and D), unpaired t test (B and C), and two-way repeated-measures ANOVA (E) followed by Bonferroni or Sidak post hoc tests.

Fig. 4. Blocking Panx1 suppresses neuronal excitability in spinal L_I-L_II dorsal horn neurons.

(A and B) Quantification and representative images of cFos immunofluorescence in the L3-L5 spinal dorsal horn from vehicle- and tamoxifen-treated Cx3cr1-Cre^ERT2::Panx1^flx/flx mice after MIA or saline CTR intra-articular injection. Scale bar, 50 μm. (C and D) Rat in vivo spinal cord recordings from spinal L_I-L_II neurons 7 days after intra-articular MIA or saline injection. (C) Spike frequency evoked by brush (dynamic mechanical allodynia) at 30 and 60 min after ¹⁰panx or ^scrpanx peptides (CTR/¹⁰panx, n = 37 cell recordings; MIA/^scrpanx, n = 22 cell recordings; MIA/¹⁰panx, n = 29 cell recordings). BL, baseline. (D) Stimulus-response curve showing mean response at each von Frey (static mechanical allodynia) stimulation force (MIA/^scrpanx, n = 20 cell recordings; MIA/¹⁰panx, n = 27 cell recordings; CTR/¹⁰panx, n = 20 cell recordings). *P < 0.05; **P < 0.01, one-way ANOVA (A and C) and two-way repeated-measures ANOVA (D) followed by Bonferroni or Sidak post hoc tests.

Fig. 5. Treatment with probenecid, a broad-spectrum Panx1 blocker, alleviates MIA-induced pain behaviors.

(A) Mechanical paw withdrawal threshold assessed by von Frey filaments using the SUDO method in rats injected twice daily intraperitoneally with probenecid (PRB) or saline [CTR/SA, n = 6; MIA/SA, n = 6; MIA/PRB (100 mg/kg), n = 6]. Baseline threshold in gram force: 14.5 g. (B) IL-1β levels in rat CSF 7 days after intra-articular MIA/CTR and daily intraperitoneal injection of saline or probenecid (CTR/SA, n = 6; MIA/SA, n = 6; MIA/PRB 100 mg/kg, n = 6). (C) Mechanical paw withdrawal threshold assessed by von Frey filament test on day 7 after MIA-induced joint injury (saline, n = 6; probenecid 50 mg/kg, n = 6; probenecid 100 mg/kg, n = 4). (D) IL-1β levels in rat CSF 7 days after intra-articular MIA/CTR and acute intraperitoneal injection of saline or probenecid (CTR/SA, n = 4; MIA/SA, n = 8; MIA/PRB 100 mg/kg, n = 8). (E and F) Dynamic weight bearing assessed on day 7 after MIA-induced joint injury (CTR/PRB, n = 8; MIA/PRB, n = 7; MIA/SA, n = 8). Effect of probenecid (50 mg/kg) on (E) weight bearing (day 7; P = 0.0036, two-way ANOVA) and (F) paw surface area (day 7; P = 0.05, two-way ANOVA). Arrow represents single intraperitoneal injection of probenecid. *P < 0.05; **P < 0.01, two-way repeated-measures ANOVA (A, C, E, and F) and one-way ANOVA (B and D) followed by Sidak or Bonferroni post hoc tests.

Fig. 6. Probenecid alleviates ACLx-induced joint pain.

(A) Illustration depicting an ACLx. (B) Mechanical paw withdrawal threshold measured in vehicle- and tamoxifen-treated Cx3cr1-Cre^ERT2::Panx1^flx/flx mice up to 13 weeks after ACLx surgery (Naive, n = 4; VEH/ACLx, n = 6; TMX/ACLx, n = 6). Mechanical paw withdrawal threshold following single injection of probenecid (100 mg/kg intraperitoneally; arrow) 13 weeks after surgery. Mechanical paw withdrawal threshold in mice was assessed by von Frey filaments using the SUDO method. Baseline threshold in gram force: 1.1 g. *P < 0.05, two-way repeated-measures ANOVA (B) followed by Sidak post hoc test.