Schwann cell C5aR1 co-opts inflammasome NLRP1 to sustain pain in a mouse model of endometriosis

Abstract

Over 60% of women with endometriosis experience abdominopelvic pain and broader pain manifestations, including chronic back pain, fibromyalgia, chronic fatigue, vulvodynia, and migraine. Although the imbalance of proinflammatory mediators, including the complement component C5a, is associated with endometriosis-related pain, the mechanisms causing widespread pain and the C5a role remain unclear. Female mice and women with endometriosis exhibit increased plasma C5a levels and pain. We hypothesize the Schwann cells involvement in endometriotic pain. Here, we show that silencing the C5a receptor (C5aR1) in Schwann cells blocks the C5a-induced activation of the NLRP1 inflammasome and subsequent release of interleukin-1β (IL-1β). Macrophages, recruited to sciatic/trigeminal nerves by IL-1β from Schwann cells, increase oxidative stress, which activates the proalgesic TRPA1 pathway, resulting in widespread pain. These findings reveal a pathway involving Schwann cell C5aR1, NLRP1/IL-1β activation, macrophage recruitment, oxidative stress, and TRPA1 engagement, contributing to pain in a mouse model of endometriosis.

Fig. 1. Endometriosis-associated mechanical allodynia is mediated by the complement system C5a.

a Time-dependent periorbital (PMA), hind paw (HMA) and abdominal (AMA) mechanical allodynia in endometriotic (endo) or Sham C57BL/6 J female (B6) mice. Proteome profiler array (b) and single analyte C5a assay (c) in plasma samples from endo and Sham B6 mice. d C5 assay in plasma samples from endo patients (n = 19) or healthy subjects (n = 15) (control). e Time-dependent C5a levels in plasma samples from endo and Sham B6 mice. f and g Timeline of the DF2593A (DF25) treatment schedule and time-dependent PMA, HMA, and AMA in endo B6 mice after intragastric (i.g.) administration of DF25 (1 mg/kg) or vehicle (Veh) and Sham mice. (n = 8 mice per group). Data are mean ± s.e.m. a, e, f, g 2-way ANOVA, Bonferroni correction; c, d 2- tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Sham ^##P < 0.01, ^####P < 0.0001 vs. Endo+Veh DF25. Source data are provided as a Source Data file.

Fig. 2. Neuroinflammation mediates endometriosis-associated mechanical allodynia.

a Representative images and cumulative data of the time-dependent increase of F4/80⁺ cells in sciatic and trigeminal nerve in endometriotic (endo) or Sham C57BL/6 J female (B6) mice. (n = 4 independent experiments). b Time-dependent C5a levels in sciatic and trigeminal nerve homogenates from endo and Sham B6 mice. (n = 6 independent experiments). c Timeline of the AP20187 (AP) treatment schedule and time-dependent periorbital (PMA), hind paw (HMA) and abdominal (AMA) mechanical allodynia in endo and Sham MaFIA mice after intraperitoneal (i.p.) injection of AP or vehicle (Veh). (n = 8 mice per group). d Representative images and cumulative data of F4/80⁺ cells in sciatic and trigeminal nerve of endo B6 mice after AP or Veh and in Sham mice (treatment schedule as in b). (n = 4 independent experiments). (Scale bar, 50 μm, dashed lines, perineurium). Data are mean ± s.e.m. a, b, d 1-way ANOVA, c, 2-way ANOVA, Bonferroni correction; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Sham, ^####P < 0.0001 vs. Endo-MaFIA+ Veh AP. Source data are provided as a Source Data file.

Fig. 3. Schwann cell C5aR1 mediates endometriosis-associated mechanical allodynia and neuroinflammation.

Representative images of SOX10 and C5aR1 (#orb393229, rabbit polyclonal, Biorbyt Ltd, 1:100) co-expression in sciatic (a) and trigeminal (b) nerve tissue from C57BL/6 J female (B6) mice, (n = 4 independent experiments) (Scale bar, 50 μm). (c) C5aR1 mRNA relative expression in primary mouse and human Schwann cells (n = 3 independent experiments). d Schematic representation of AAV-(loxP-shRNA)-C5aR1 vector pre- and post-Cre switch. e Representative images and cumulative data (Rcoloc) of SOX10 and C5aR1 (#orb393229, rabbit polyclonal, Biorbyt Ltd, 1:100) co-expression in sciatic and trigeminal nerve tissue in Plp-AAV-C5aR1 and Control mice (n = 4 independent experiments) (Scale bar, 50 μm). f Time-dependent periorbital (PMA), hind paw (HMA), and abdominal (AMA) mechanical allodynia in endometriotic (endo) or Sham Plp-AAV-C5aR1 and Control mice. (n = 8 mice per group). g Representative images and cumulative data of F4/80⁺ cells in sciatic and trigeminal nerve tissue in endo or Sham Plp-AAV-C5aR1 and Control mice. (n = 4 independent experiments) (Scale bar, 50 μm, dashed lines, perineurium). h Representative images and cumulative data of transmigrated macrophages after stimulation of human and mouse Schwann cells with C5a or vehicle (Veh) and in the presence of DF2593A (DF25) or Veh (n = 6 independent experiments). Data are mean ± s.e.m. c, Student’s t-test, f, 2-way, g, h 1-way ANOVA, Bonferroni correction; ****P < 0.0001 vs. Sham/Control, ^####P < 0.0001 vs. Endo/Control. Source data are provided as a Source Data file.

Fig. 4. Schwann cell C5aR1 activation induces NLRP1-dependent release of IL-1β.

a IL-1β assay in human Schwann cell conditioned medium after stimulation with C5a or vehicle (Veh) and in the presence of DF2593A (DF25) or Veh (n = 6 independent experiments). b Time-dependent C5a levels in plasma samples from endometriotic (endo) or Sham C57BL/6 J female (B6) mice. (n = 8 independent experiments). c IL-1β assay in plasma samples from endo patients (n = 16) or healthy subjects (n = 15) (control). d Typical traces and cumulative data of SCAT1 V/C ratio changes in human Schwann cells stimulated with C5a or Veh and in the presence of DF25or Ac-YVAD (AUC, area under the curve) (cell numbers: C5a = 20, Veh=16, DF25 = 16 and Ac-YVAD = 20; n = 3 independent experiments) e Time-dependent periorbital (PMA), hind paw (HMA) and abdominal (AMA) mechanical allodynia in endo or Sham Plp-AAV-Nlrp1 and Control mice (n = 8 mice per group). f IL-1β assay in sciatic and trigeminal nerve tissue homogenates in endo or Sham Plp-AAV-Nlrp1 and Control mice (n = 6 independent experiments). g Representative images and cumulative data of F4/80⁺ cells in sciatic and trigeminal nerves in endo or Sham Plp-AAV-Nlrp1 and Control mice (n = 4 independent experiments). h Time-dependent PMA, HMA, and AMA in endo or Sham Plp-AAV-IL-1β and Control mice (n = 8 mice per group). i Il-1β assay in sciatic and trigeminal nerve tissue homogenates in endo or Sham Plp-AAV-Il-1β and Control mice. j Representative images and cumulative data of F4/80⁺ cells in sciatic and periorbital nerve in endo or Sham Plp-AAV- Il-1β and Control mice (Scale bar, 50 μm, dashed lines, perineurium) (n = 8 mice per group). Data are mean ± s.e.m. a, c, d, f, g 1-way ANOVA, b, e, h, 2-way ANOVA, Bonferroni correction; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Sham, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001 vs. Endo/Control. Source data are provided as a Source Data file.

Fig. 5. Schwann cell TRPA1 channel mediates endometriosis-associated mechanical allodynia and neuroinflammation.

a Time-dependent periorbital (PMA), hind paw (HMA) and abdominal (AMA) mechanical allodynia in endometriotic (endo) or Sham Plp-Trpa1 and Control mice (n = 8 mice per group). Representative images and cumulative data of (b) F4/80⁺ cells and (c) 4-HNE staining in sciatic and trigeminal nerve in endo or Sham Plp-Trpa1 and Control mice (Scale bar, 50 μm, dashed lines, perineurium) (n = 4 and n = 5 independent experiments). d Time-dependent PMA, HMA and AMA in endometriotic (endo) or Sham Adv-Trpa1 and Control mice (n = 8 mice per group). Representative images and cumulative data of (e) F4/80⁺ cells and (f) 4-HNE staining in sciatic and trigeminal nerve in endo or Sham Adv-Trpa1 and Control mice (Scale bar, 50 μm, dashed lines, perineurium) (n = 4 independent experiments). Data are mean ± s.e.m. a,d 2-way ANOVA, b,c,e,f 1-way ANOVA, Bonferroni correction; *P < 0.05, ***P < 0.001, ****P < 0.0001 vs. Sham, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001 vs. Endo/Control. Source data are provided as a Source Data file.