Schwann Cell C5aR1 co-opts Inflammasome NLRP1 to Sustain Pain in a Mouse Model of Endometriosis

Schwann Cell C5aR1 co-opts Inflammasome NLRP1 to Sustain Pain in a Mouse Model of Endometriosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Schwann Cell C5aR1 co-opts Inflammasome NLRP1 to Sustain Pain in a Mouse Model of Endometriosis Francesco De Logu, Mustafa Titiz, Lorenzo Landini, Daniel Souza Monteiro de Araujo, and 18 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4045626/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Nov, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Over 60% of women with endometriosis experience abdominopelvic pain alongside pain in other areas of the body, such as chronic back pain, fibromyalgia and chronic fatigue, vulvodynia, and migraine. Although the imbalance of proinflammatory mediators, including the complement component C5a, has been implicated in endometriosis-associated pain symptoms, the mechanisms causing widespread pain and the role of C5a remain unclear. Female mice with endometriotic lesions displayed widespread pain and increased plasma C5a levels, similarly, observed in women with endometriosis. We hypothesized Schwann cells involvement in endometriotic pain. Silencing the C5a surface receptor (C5aR1) in mouse Schwann cells abolished C5a-induced activation of NLRP1 inflammasome and the ensuing interleukin-1β (IL-1β) release. IL-1β, from Schwann cells, recruited macrophages in sciatic/trigeminal nerve trunks. Macrophages induced oxidative stress, targeting proalgesic TRPA1, causing widespread mechanical allodynia. This pathway, initiated by C5aR1, engages Schwann cell signaling, including NLRP1/IL-1β/macrophages/oxidative stress/TRPA1, sustaining pain in an endometriosis mouse model. Health sciences/Health care/Therapeutics/Pain management Health sciences/Medical research/Experimental models of disease Health sciences/Medical research/Translational research Biological sciences/Cell biology/Mechanisms of disease Health sciences/Medical research/Preclinical research Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Endometriosis, caused by superficial peritoneal/deep infiltrating implants or ovarian cysts of endometrial-like tissue 1 , is an enigmatic and often debilitating gynecologic condition that affects 10-15% of reproductive-aged women 2 . The socioeconomic burden of the disease is high, as endometriosis affects career, everyday activities, sexual and nonsexual relationships, and quality of life{ 3 . More than 60% of women diagnosed with endometriosis suffer from abdominopelvic pain{ 4 , and often suffer from pain conditions in anatomical sites other than the pelvis/abdomen, such as chronic back pain, fibromyalgia, vulvodynia, and migraine{ 5 ;Nagai, 2015 #86}. Migraine, the most debilitating condition for women aged 20-50 years, is frequent in women with endometriosis in reproductive age 6 , 7 . A correlation between fibromyalgia and endometriosis has been proposed, given that immune mechanisms appear to contribute to chronic pain in the two conditions 8 , 9 . The complement system is a broad network of soluble and cell-surface proteins distributed throughout body fluids and tissues with no, or little, basal activity 10 , 11 . The complement system acts not just as a first line of defense against pathogens, as it functionally connects innate and adaptive immune responses via receptors expressed by myeloid and non-myeloid cells 12 , 13 . Activation of the complement system cascade involves a C3-convertase that cleaves C3, producing C3a and C3b. Two C3b molecules plus the Bb factor form the C5 convertase that cleaves C5 in C5a and C5b 12 , 13 . C5a targets a G protein coupled receptor (GPCR), C5a receptor type 1 (C5aR1), originally identified in neutrophils and monocytes/macrophages 14 . C5a contribution in post-operative 15 , 16 , inflammatory 17 , and neuropathic 18 pain has been previously proposed. Endometriosis patients undergoing surgery are classified in four stages according to the Revised Classification of Endometriosis by the American Society of Reproductive Medicine (r-ASRM 19 . Women at the early (I-II) stages have shown higher C5a serum levels compared to a control group 20 . Macrophages play a pivotal role in neurogenesis processes occurring close to endometriotic lesions, suggesting their role in endometriosis-associated pain generation 21 , 22 , 23 . Activation of C5aR1 in endoneurial macrophages induces the NLRP3 inflammasome-dependent release of the pro-nociceptive mediator interleukin 1 (IL-1) β to maintain pain in a mouse model of peripheral nerve injury 24 . Recently, we showed that crosstalk between Schwann cells (SCs) and endoneurial resident monocytes/macrophages in peripheral nerves activates an oxidative stress pathway that elicits neuroinflammation and sustains chronic pain 25 , 26 , 27 , 28 . Here, we investigated the mechanism underlying mechanical allodynia produced by endometriotic lesions in the abdomen and other areas distant from the abdomen, such as the periorbital area to mimic migraine pain and the hind paw to mimic fibromyalgia, in female mice. As abdominal mechanical allodynia was associated with mechanical allodynia in the hind paw and periorbital area, we explored the cellular and molecular pathway that diffuses mechanical hypersensitivity from endometriotic lesions to the other two anatomical areas. We found that C5a increased by endometriosis, targeting the SC C5aR1, activates the NLRP1 inflammasome, which induces the release of IL-1β. The increase in IL-1β in peripheral nerves (periorbital and sciatic nerves) promotes macrophage recruitment and the release of reactive oxygen species (ROS) that target neuronal TRPA1 to signal pain. These results implicate SC C5aR1 as a new target for the treatment of endometriosis associated pain. Results The complement component C5a mediates endometriosis-induced allodynia. In C57BL/6J female (B6) mice, intraperitoneal (i.p.) injection of dissected uterus horns from donor female mice induced a time-dependent increase of endometriosis-like lesions (Supplementary Fig. 1a), which were associated with prolonged (28 days) periorbital mechanical allodynia (PMA), hind paw mechanical allodynia (HMA), and abdominal mechanical allodynia (AMA) (Fig. 1a). The simultaneous development of allodynia in anatomical areas distant from the abdomen supports the hypothesis that diffusible cellular or molecular mediators are implicated in the widespread proalgesic response. Several inflammatory cytokines/chemokines have been implicated in endometriotic pain 29 . The relative levels of forty cytokines/chemokines and acute phase proteins in plasma samples of endometriotic and sham B6 mice were investigated by a proteome profiler array at day 7 after surgery (Fig. 1b). The array revealed increased C5a levels in endometriotic mice (Fig. 1b), and the increase was confirmed by a C5a single analyte ELISA assay (Fig. 1c). A recent report showed that serum levels of C5a were higher in early stage (I-II) endometriotic patients than in control patients 20 . Here, we confirmed the higher serum levels of C5a in a group of patients with endometriosis (n=19) compared to healthy women without endometriosis (n=15) (Fig. 1d). In addition, we observed that, in endometriotic B6 mice, C5a plasma levels rose from day 1 to day 8 to then decline to basal levels at day 12-16 after surgery (Fig. 1e). As C5a have been implicated in several pain conditions 15 , 16 , 17 , 18 , we tested whether the selective allosteric C5aR1 inhibitor, DF2593A, given in two different temporal schedules would reduce PMA, HMA, and AMA in endometriotic mice. Endometriotic B6 mice treated with DF2593A (twice a day) from one day before until day 6 after the injection of the ectopic tissue, when C5a levels increase in mouse plasma, showed a significant reduction in the development of PMA, HMA, and AMA (Fig. 1f). Conversely, daily treatment with DF2593A, from day 12 to day 16, failed to reduce PMA, HMA, and AMA (Fig. 1g). Overall, the data indicate that, although transient (from day 2 to day 10 after surgery), the increase in C5a plasma levels was sufficient to initiate and sustain PMA, HMA, and AMA. Neuronal macrophages mediate endometriosis-induced mechanical allodynia Immune cells, including macrophages and nerve fibers, interact to promote pain symptoms associated with endometriosis 30 , 31 . Our previous findings proposed that macrophages infiltrating peripheral nerve trunks interact with SCs to sustain mechanical allodynia in different mouse models of pain 25 , 26 , 27 , 28 . To understand whether macrophage accumulation plays a role in the present model of endometriosis, sciatic and trigeminal nerve trunks were examined. Due to their limited size, the nerve fibers of the endometriotic lesions were not included in these studies. In endometriotic B6 mice, the number of F4/80 + macrophages inside sciatic and trigeminal nerve trunks increased in a time-dependent manner starting from day 3 after surgery (Fig. 2a). To explore the role of macrophages in endometriosis-induced mechanical allodynia, macrophage Fas-induced apoptosis (MaFIA) mice, injected with the apoptosis inducer, AP20187, which abates the number of macrophages (GFP + /F4/80 + cells) 32 , were used. Ectopic tissue inoculation in the peritoneum of MaFIA mice induced time-dependent PMA, HMA, and AMA, as observed in B6 mice (Fig. 2b). To better understand the action of C5a on Schwann cells and macrophages, macrophages were depleted in MaFIA mice by AP20187 administration following three different time schedules: i) before the increase in C5a levels (from day -2 to day 2); ii) during the C5a increase (from day 2 to day 6); and iii) when C5a declined to baseline (from day 21 to day 25). Macrophage depletion obtained with the two later (ii and iii) (Fig. 2b,c and Supplementary Fig. 1b,c respectively), but not with the early (i) (Supplementary Fig. 1d,e) treatment schedule reduced PMA, HMA, and AMA. These findings are consistent with the transient (~48 hours) monocyte/macrophage depletion obtained in MAFiA mice 33 . Depletion of monocytes/macrophages in MaFIA mice after AP20187 treatment was evaluated as the number of F4/80 + cells in both sciatic and trigeminal nerves (Fig. 2c, Supplementary Fig. 1c and Supplementary Fig. 1e). The C5aR1 inhibitor, DF2593A, given to endometriotic mice during the increase of C5a in plasma (from day 2 to day 6), reduced macrophages in sciatic and trigeminal nerve tissues (Supplementary Fig. 1f). In contrast, treatment when C5a serum levels declined (from day 12 to 16) failed to reduce the macrophage accumulation (Supplementary Fig. 1g). Collectively, our data suggest that C5a release, most likely from ectopic endometrial lesions during the first seven days after surgery, increases macrophages in nerve trunks. However, when C5a serum levels decline, increased macrophages promote PMA, HMA, and AMA in a C5a-independent manner. Schwann cell C5aR1 mediates macrophage recruitment and endometriosis-induced mechanical allodynia. Given that, in several mouse models of pain, SCs play a crucial role in sustaining mechanical allodynia, we investigated whether these glial cells contributed to PMA, HMA, and AMA in the present model of endometriosis. Immunofluorescent staining analysis of mouse sciatic and trigeminal nerve tissues revealed that C5aR1 staining was present in 92.2% ± 1.1 and 93.3% ± 0.44 of nucleated cells, respectively (Supplementary Fig. 2a). SCs, which represent 45.1% of the nucleated cells in mouse peripheral nerve tissues 34 , are identified by SOX10 expression. Immunofluorescent staining analysis in mouse and human sciatic and mouse trigeminal nerves revealed that all SOX10+ cells co-express C5aR1 (Fig. 3a,b and Supplementary Fig. 2b). C5aR1 mRNA and protein expression were confirmed in cultured mouse and human SCs by qRT-PCR and western immunoblot assays (Fig. 3c,d). To investigate the role of Schwann cell C5aR1 in mechanical allodynia, we used a Plp Cre driver that functions as a lineage tracer to express a short hairpin RNA (shRNA) for selective silencing of C5aR1 in SCs by the injection of a Cre-dependent adeno associated viral vector (AAV). A virus packaged with the AAVrh10 serotype for efficient infection of SCs was used with a loxP flanked shRNA to express shRNA in SCs (AAV- C5aR1 ) (Fig. 3e). The efficacy of the gene silencing was evaluated in sciatic and trigeminal nerves by immunofluorescent staining (Fig. 3f). Systemic (intravenous, i.v.) administration of AAV- C5aR1 in Plp Cre+ but not in Control mice attenuated endometriosis-induced PMA, HMA, and AMA (Fig. 3g) and macrophage increase in sciatic and trigeminal nerves (Fig. 3h). To further investigate the ability of C5a to stimulate cultured mouse and human SCs to recruit macrophages, we used the Boyden chamber assay. Human and mouse SCs stimulated with C5a promoted macrophage migration through the microporous membrane that was inhibited in the presence of DF2593A (Fig. 3i). Previous data reported C5aR1 mRNA expression in mouse DRG neurons where C5a, by activating and sensitizing cutaneous nociceptors, amplified the capsaicin response 35 . To identify the role for neuronal C5aR1 in endometriotic pain, we used a Adv Cre driver to silence C5aR1 selectively in sensory neurons and a virus packaged with the AAV2/9n serotype for efficient infection of nociceptors. Selective silencing of C5aR1 in sensory neurons was confirmed by immunofluorescent staining in lumbar (L4-L6) dorsal root ganglion (DRG) tissues (Supplementary Fig. 2c). Intrathecal administration of AAV- C5aR1 in Adv Cre+ did not attenuate endometriosis-induced PMA, HMA, and AMA and macrophage increases in nerve (sciatic and trigeminal) trunks (Supplementary Fig. 2d,e). These results indicate that, in endometriotic mice, C5a targeting of the SC C5aR1 is necessary and sufficient to induce neuroinflammation and mechanical allodynia. Schwann cell NLRP1 drives endometriosis-induced macrophage increases and mechanical allodynia. C5aR1 is known to activate the NLR family pyrin domain containing 3 (NLRP3) inflammasome, thus promoting the expression of the interleukin-1β precursor (pro-IL-1β) and secretion of IL-1β through the cleavage by caspase-1 in different immune cells 36 . Here, we report that human SC C5aR1 encoded a signaling pathway that released IL-1β, a response attenuated by the C5aR1 inhibitor, DF2593A (Fig. 4a). IL-1β plasma levels from endometriotic mice were more elevated from day 10 until day 28 after surgery as compared to sham mice (Fig. 4b). Increased IL-1β plasma levels were also detected in women with endometriosis as compared to healthy women without endometriosis (Fig. 4c). To assess caspase-1 activation in cultured human SCs, cells were exposed to C5a that increased a bioluminescent signal due to caspase-1 activation. The increase was reduced by DF2593A and by the selective caspase-1 inhibitor, Ac-YVAD-CHO (Supplementary Fig. 3a). Confirmation of caspase-1 activation was obtained by genetically encoded fluorescent sensor (SCAT1) based on fluorescence resonance energy transfer (FRET) 37 . SCAT1 contains a consensus peptide preferentially cleaved by caspase-1 that reduces the FRET signal 37 . C5a decreased SCAT1 Venus/ECFP (V/C) ratio, a response that was prevented by DF2593A and Ac-YVAD-CHO in human SCs (Fig. 4d). Ac-YVAD-CHO inhibited the release of IL-1β from human SCs after C5a stimulation, supporting the role of C5aR1 in promoting inflammasome activation and IL-1β release (Supplementary Fig. 3b). Endometriotic mice with AAV-mediated selective silencing of C5aR1 ( Plp- AAV- C5aR1 mice) showed reduced levels of IL-1β in sciatic and trigeminal nerve tissues compared to Control mice (Supplementary Fig. 3c). To test the role of SC NLRP3 in endometriotic mechanical allodynia, we injected Plp Cre+ mice with an AAV- Nlrp3 for selective silencing of NLRP3 in SCs ( Plp- AAV- NLRP3 mice). However, PMA, HMA, AMA, neuroinflammation and IL-1β levels in sciatic and trigeminal nerve tissue in endometriotic Plp- AAV- NLRP3 mice were unaffected. (Supplementary Fig. 3d,e, f). The efficacy of the gene silencing was evaluated in sciatic and trigeminal nerve tissue by immunofluorescent staining (Supplementary Fig. 3g) The inflammasome family currently includes three subtypes, NLRP1, NLRP3, and NLRC4, which recognize specific classes of pathogens or risk signals 38 . The qRT-PCR analysis of mRNA expression in both human and mouse SCs revealed mainly the presence of NLRP1 and NLRP3 transcripts (Supplementary Fig. 3h). Given the very low level of NLRC4 expression, we tested the role of SC NLRP1 in endometriotic pain by infecting Plp Cre+ mice with an AAV- Nlrp1 for selective silencing of NLRP1 in SCs ( Plp- AAV- NLRP1) . Plp- AAV- NLRP1 mice showed reduced PMA, HMA, and AMA IL-1β levels and neuroinflammation in sciatic and trigeminal nerve tissue compared to Control mice (Fig. 4e-g). The efficacy of NLRP1gene silencing was evaluated in sciatic and periorbital nerve tissue by immunofluorescent staining (Supplementary Fig. 3i) Previous studies indicated a crucial role of IL-1β in sustaining inflammation through macrophage recruitment 39 . To support the role of IL-1β in increasing macrophage number in nerve trunks, mouse and human SCs and macrophages were exposed to a neutralizing monoclonal antibody against IL-1β (anti-IL-1β mAb) and C5a in a Boyden chamber assay. The anti-IL-1β mAb reduced macrophage migration evoked by C5a (Supplementary Fig. 4a). The anti-IL-1β mAb treatment also reduced PMA, HMA, and AMA and macrophage numbers in sciatic and trigeminal nerves in endometriotic mice (Supplementary Fig. 4b,c). To identify the SC role in releasing IL-1β following C5a stimulation in endometriotic mice, we selectively silenced IL-1β in SCs by infecting Plp Cre+ mice with an AAV- IL-1β (Plp- AAV- IL-1β mice) . Plp- AAV- IL-1β mice showed reduced PMA, HMA, and AMA (Fig. 4h), IL-1β levels (Fig. 4i), and neuroinflammation in sciatic and trigeminal nerve tissue (Fig. 4j) compared to Control mice. The efficiency of gene silencing was evaluated in sciatic and trigeminal nerve tissue by immunofluorescent staining (Supplementary Fig. 4d). Overall, these data support the view that C5a from endometriotic lesions targets SC C5aR1, which activates inflammasome NLRP1 to release IL-1β, which in turn increases macrophages within peripheral nerve trunks to sustain mechanical allodynia. Neuronal TRPA1 mediates endometriosis-induced mechanical allodynia. We previously reported that targeting the transient receptor ankyrin 1 (TRPA1) in primary sensory neurons by oxidative stress signals mechanical allodynia in several mouse models of pain 25 , 26 , 27 , 28 . Here, endometriotic Adv Cre mice with selective deletion of TRPA1 in sensory neurons ( Adv-TRPA1) showed attenuation of PMA, HMA, and AMA compared to Control mice (Fig. 5a). Conversely, IL-1β levels, as well as neuroinflammation in sciatic and trigeminal nerve tissue, were unaffected (Fig. 5b,c). These data show that neuronal TRPA1 is the final common pathway that, from endometriotic lesions and via SCs, signals mechanical allodynia. Discussion Here, we provide evidence that, in the present mouse model of endometriosis, peritoneal lesions, by increasing serum levels of C5a, not only exert local effects in mesenteric afferent neurons, but also spread a macrophage-dependent proalgesic mechanism throughout the body, reaching distant anatomical regions, including the periorbital or hind paw areas. We reveal that C5a, in both proximal and remote areas, such as the abdomen and periorbital area/hind paw, respectively, targets the SC C5aR1, where it encodes a proalgesic mechanism that results in neuroinflammation and the ensuing widespread mechanical allodynia. Another unexpected finding of the present study highlights the role of the inflammasome NLRP1, instead of the classical NLRP3, normally activated by C5a, as we found that SC C5aR1 via NLRP1 enhances the caspase-1 processing of pro-IL-1β. The resulting IL-1β release increases the macrophage number that, via oxidative stress and neuronal TRPA1 activation, promotes and sustains mechanical allodynia. C5a has been implicated in various pain conditions, including post-operative 16 , inflammatory 17 , and neuropathic pain 24 . To date, although little is known about the association between the complement system components and endometriotic pain, fluctuation of C5a plasma levels during the different disease stages of endometriosis has been documented in female patients 20 , 40 . Time course analysis of C5a plasma levels in the present mouse model of endometriosis showed similar fluctuations in female mice with endometriotic lesions, which were identified as the initial stimulus responsible for the abdominal and diffuse proalgesic condition. A considerable proportion of women with endometriosis-associated pain reported a neuropathic-like pain component 41 . Recently, it has been observed that neuropathic-like pain caused by peripheral nerve injury in mice was modulated by activation of neuron associated C5aR1+ macrophages 24 . In our study, results obtained with the C5aR1 inhibitor and transgenic MaFIA mice confirm that increases in plasma C5a and nerve trunk macrophages are essential for the development of endometriosis-associated pain-like responses. We also found that endometriotic lesions promote mechanical allodynia diffused to various tissues by orchestrating a series of temporally distinct and strictly coordinated phases. The role of C5a was confined to its initial sharp increase. Although C5aR1 expression has not been reported in SCs located at neuromuscular junctions 42 , we found that both cultured primary human and mouse SCs and SOX10+ cells in mouse and human peripheral nerve fibers expressed C5aR1. The present data obtained by C5aR1 selective silencing highlighted the presence and the role of SC C5aR1 in endometriosis-associated mechanical allodynia and neuroinflammation. Recent studies suggest that the C5a-C5aR1/C5aR2 interaction regulates the NLRP3 inflammasome in several immune system cells, including macrophages 43 , 44 and T cells 45 . Furthermore, NLRP3 has been implicated in the pathobiology of chronic neuropathic and inflammatory pain 46 , 47 , 48 . Inflammation is one of the key features of endometriosis, and the inhibition of NLRP3 inflammasome has been associated with reduction of IL-1β secretion from endometrial stromal cells 49 , 50 and mast cells 51 . However, the present observation that selective silencing in SCs of the NRLP3 subtype in endometriotic mice was ineffective, whereas silencing of the NRLP1 SC subtype reduced IL-1β release and mechanical allodynia, highlights the NRLP1 involvement in the proalgesic pathway promoted by endometriosis. The time course of the various soluble and cellular mediators implicated in endometriosis-induced mechanical allodynia is complex and under a strict sequential order; it encompasses the contribution of macrophages and DRG afferents but recognizes the central role of SCs. The most parsimonious hypothesis indicates as the originator of endometriosis associated pain the transient increase in C5a plasma levels, which targets the SC C5aR1. C5aR1 encodes an intracellular signaling pathway that consists in the NRLP1-dependent caspase-1 activation and the ensuing cleavage of pro IL-1β and IL-1β release. IL-1β increases macrophages within the nerve trunks that, via their oxidative stress burst, target the neuronal TRPA1. The role of C5a as a soluble and diffusible pain mediator explains the epidemiological observation that other painful conditions, like migraine or fibromyalgia, coexist with endometriosis 6 , 7 , 9 , 52 . The expression and the ability to promote mechanical allodynia and neuroinflammation of the C5a receptor, C5aR1, in SCs explains how endometriosis features chronic pain as a major symptom of the disease. One limitation of our study is that we documented all the cellular and molecular steps of the endometriotic inflammatory and proalgesic pathways in sciatic and trigeminal nerve trunks, implicated in fibromyalgia and migraine pain, respectively, but in contrast, due to the limited size of mesenteric nerve fibers, we could not assess the steps underlying mechanical allodynia in the abdomen, the primary site of endometriotic pain. However, functional experiments in mice with selective silencing in SCs suggest that abdominal mechanical allodynia is produced by the same cellular and molecular mechanisms identified in sciatic and trigeminal nerves. Development of therapies targeted to the cellular and molecular mediators of the C5a-C5aR1/NLRP1/IL-1β pathway in SCs may be a future option for the treatment of pain in endometriosis patients. Methods Experimental model and content details Animals Female mice were used throughout (25–30 g, 5–8 weeks old). The following strains of mice were used: C57BL/6 J (Charles River, RRID: IMSR_JAX:000664), B6.Cg-Tg(Plp1-CreERT)3Pop/J mice ( Plp-Cre ERT , RRID: IMSR_JAX:005975 Jackson Laboratory) expressing a tamoxifen-inducible Cre in Schwann cells (Plp1, proteolipid protein myelin 1). Both positive and negative mice for Cre ERT ( Plp-Cre ERT + or Plp-Cre ERT - (control) respectively) were treated with intraperitoneal (i.p.) 4-hydroxytamoxifen (4-OHT, 1 mg/100 μL in corn oil once a day consecutively for 3 days) before the infection with AAV for selective silencing of the different genes in Schwann cells. To selectively delete Trpa1 in primary sensory neurons, homozygous 129S-Trpa1 tm2Kykw/J (floxed Trpa1, Trpa1 fl/fl , RRID:IMSR_JAX: 008649 Jackson Laboratory) mice were crossed with hemizygous Advillin-Cre mice (Adv-Cre). Mice positive or negative for Cre and homozygous for floxed Trpa1 ( Adv-Trpa1 and control respectively) were used. The successful Cre-driven deletion of TRPA1 mRNA was confirmed using reverse transcription quantitative real-time PCR (RT-qPCR). Some Adv-Cre + or Adv-Cre - (control) were used for the infection with AAV for selective gene silencing in primary sensory neurons. To evaluate the involvement of macrophages, transgenic Macrophage Fas-Induced Apoptosis (MaFIA) mice (C57BL/6-Tg(Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6)2Bck/J, stock No: 005070, RRID:IMSR_JAX:005070, Jackson Laboratories) were used. These transgenic mice express a mutant human FK506 binding protein 1A, 12kDa (FKBP12)-Fas inducible suicide/apoptotic system, driven by the mouse Csf1r promoter conjugated with a green fluorescent protein (GFP), which preferentially binds the B/B dimerizing agent (B/B-HmD, AP20187). Treatment of mice with AP20187 induces the dimerization of the suicide protein to activate the cytoplasmic FKBP12-Fas fragments, leading to the apoptosis of transgene‐expressing cells and consequent macrophage depletion 32 . The group size of n=8 mice for behavioral experiments was determined by sample size estimation using G Power [v3.1 53 ] to detect the size effect in a post-hoc test with type 1 and 2 error rates of 5% and 20%, respectively. Allocation concealment of mice into the vehicle(s) or treatment groups was performed using a randomization procedure (http://www.randomizer.org/). The assessors were blinded to the identity of the animals (genetic background) or allocation to treatment groups. None of the animals were excluded from the study. Mice were housed in a temperature- and humidity-controlled vivarium (12 h dark/light cycle, free access to food and water, 5 animals per cage). At least 1 h before behavioral experiments, mice were acclimatized to the experimental room and behavior was evaluated between 9:00 am and 5:00 pm. Animals were anesthetized with a mixture of ketamine and xylazine (90 mg/kg and 3 mg/kg, respectively, i.p.) and euthanized with inhaled CO 2 plus 10-50% O 2 . All behavioral experiments followed Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and were in accordance with European Union (EU) guidelines for animal care procedures and the Italian legislation (DLgs 26/2014) application of the EU Directive 2010/63/EU. The study was approved by the National Committee for the Protection of Animals used for Scientific Purposes of the Italian Ministry of Health (research permits # 452/2020-PR). Human samples The use of blood plasma samples collected from human individuals diagnosed with endometriosis and healthy control was approved by the Local Ethics Committee of the Florence University Hospital (Area Vasta Toscana Centro, CEAVC) (EndoTRP study, 15211.oss and subsequent amendments), according to the Helsinki Declaration and good clinical practice guidelines, and all patients gave their informed consent. Participants did not receive any form of compensation. The blood plasma samples derived from nineteen endometriosis diagnosed patients [female, median age 30 years (range 22-43)] and fifteen healthy individuals [female, median age 27 years (23-42)]. Considering the exploratory nature of the blood sampling analyses, the sample size was not determined based on statistical considerations but enrolling all consecutive outpatients and healthy controls. Cell lines Mouse Schwann cells (MSC). MSC were isolated from sciatic nerves of C57BL/6J mice 54 . Briefly, the epineurium was removed, and nerve explants were cut into 1 mm segments and enzyme-dissociated in Hank's Balanced Salt Solution (HBSS, 2 hr, 37 °C) added with collagenase (0.05%) and hyaluronidase (0.1%). Cells were collected by centrifugation (150xg, 10 min, room temperature RT) and the pellet was resuspended and cultured in Dulbecco's Modified Eagle Medium (DMEM) containing fetal calf serum (10%), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 mg/ml), neuregulin (10 nM) and forskolin (2 μM). Cytosine arabinoside (Ara-C, 10 mM) was added three days later, to remove fibroblasts. The culture medium was replaced every 3 days. Cells were cultured at 37 °C in 5% CO 2 and 95% O 2 for 15 days before experiments. Human Schwann cells (HSCs). HSCs (#P10351; Innoprot) were grown in Schwann cell medium (#P60123, Innoprot) at 37 ◦ C with 5 % CO 2 and 95 % O 2 and discarded and replaced after 12 passages 55 . The murine macrophage cell lineage, RAW 264.7 (#TIB-71™; ATCC, RRID: CVCL_0493), was cultured and maintained in DMEM supplemented with FBS (10%), penicillin (100 U/mL), streptomycin (100 mg/mL) and L-glutamine (2 mM) at 37 ◦C in 5% CO 2 and 95% O 2 . The human monocytic cell line, U937 (#CRL-1593.2™; ATCC, RRID:CVCL_0007) was maintained at 37 ◦C, 5% CO 2 and 95% O 2 in Roswell Park Memorial Institute (RPMI-1640) medium supplemented with heat-inactivated FBS (10 %), L-glutamine (2 mM), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; 10 mM), and sodium pyruvate (1 mM). The differentiation of U937 cells (1 × 10 5 cells/mL) towards macrophage-like lineage was promoted by the exposure to 200 ng/mL of phorbol 12-myristate 13- acetate (PMA) for 2 days before experiments 56 . AAVpro293T cell line. AAVpro 293T cells (#632273, Takara, RRID:CVCL_B0XW), were maintained in DMEM high glucose supplemented with 10% heat inactivated FBS, 4 mM L-glutamine, 1 mM penicillin/streptomycin and 1 mM sodium pyruvate at 37 °C in 5% CO 2 and 95% O 2 . The day before transfection, cells were plated in DMEM supplemented with 2% FBS. Induction of endometriosis Endometriosis was induced in mice as previously reported 57 . Briefly, donor mice, received a subcutaneous injection of estradiol benzoate (3 μg/100 μL) to stimulate the endometrium growth. Seven days later, the uteri of the donor mice were dissected and divided longitudinally. Uterine horns from each donor mouse were minced in tissue fragment smaller than 1 mm in Hank’s balanced salt solution (HBSS). Each dissociated uterine horn was then injected intraperitoneally (50 mg/500 μL i.p.) in recipient mice. One donor mouse was used for every 2 endometriosis mice. Sham mice received an intraperitoneal injection of 500 μL of HBSS. Behavioral experiments were performed during a 28-day period. After 28 days from the injection of the endometrium suspension, mice were sacrificed, and tissues collected. Lesion implantation was also quantified in a time-course manner by counting the number of lesions in endometriotic mice at day 7, 14, 21, and 28. Treatment protocols If not otherwise indicated, all reagents were obtained from Merck Life Science SRL. Mice received systemic administration of C5a complement antagonist (DF2593A, 1 mg/kg, intragastric, i.g.) or vehicle (0.5% carboxymethyl cellulose, CMC) twice a day, by two different time schedules: from one day before the endometriotic tissue injection to day 6 or from day 12 to day 16. Anti-IL1β (#BE02463, clone B122, Bio X Cell, RRID:AB_2687727) monoclonal antibody (mAb) or IgG isotype control were administered (50 µg/200 μL, i.p.) at day 8 and day 14 after endometriosis/sham induction. MaFIA mice were treated with B/B homodimerizer (AP20187, 2 mg/kg, i.p., once a day) or vehicle (10% PEG-400, 1.7% tween 80 in 0.9% NaCl) by three different time schedules: from day -2 to day 2; from day 2 to day 6, and from day 21 to day 25. Plp-Cre + and control mice were infected with an intravenous (1 mL/kg, 1x10 12 v/g, i.v.) injection of different AAVs. Adv-Cre + and control mice were infected with an intrathecal (1x10 12 v/g, i.th., 5 μL) injection of AAV. Animals were used 3 weeks after AAVs infection. Sciatic and trigeminal nerve tissues were harvested for evaluating AAVs infection. Behavioral assays Abdominal Mechanical allodynia (AMA). The mechanical pain sensitivity of the abdominal region was evaluated with von Frey filaments by methods described previously 58 . Mice were habituated in small compartments on a perforated grid for 1 h before the test. Subsequently von Frey filaments were applied to the abdomen (between diaphragm and genitals). The test was performed by a trained blinded observer. The individual filaments were tested in an ascending order covering 0.008, 0.04, 0.16, and 0.4 g forces. Each force was applied 10 times to the abdominal surface. The maximal duration of each force application was 2 s, and the inter-stimulus interval was 2–3 min. Following each challenge, the withdrawal response was quantified either as 1 (withdrawal of abdominal wall, licking or retraction of animal) or 0 (no response). All counts in response to an individual filament were averaged. Withdrawal responses to low forces reflect high mechanical pain sensitivity. Periorbital mechanical allodynia (PMA). Periorbital mechanical allodynia (PMA) was assessed using the up-down paradigm 59 , 60 . Briefly, mice were placed in a restraint apparatus designed for the evaluation of periorbital mechanical thresholds 61 . PMA was evaluated in the periorbital region over the rostral portion of the eye (i.e., the area of the periorbital region facing the sphenoidal rostrum) 62 before (basal threshold) and after treatments. On the day of the experiment, after 20 min of adaptation inside the chamber, a series of 7 von Frey filaments in logarithmic increments of force (0.02, 0.04, 0.07, 0.16, 0.4, 0.6 and 1.0 g) were applied to the periorbital area perpendicular to the skin, with sufficient force to cause slight buckling, and held for approximately 5 s to elicit a positive response. The response was considered positive by the following criteria: mouse vigorously stroked its face with the forepaw, head withdrawal from the stimulus, or head shaking. Mechanical stimulation started with the 0.16 g filament. Absence of response after 5 s led to the use of a filament with increased force, whereas a positive response led to the use of a weaker (i.e., lighter) filament. Six measurements were collected for each mouse or until four consecutive positive or negative responses occurred. The 50% mechanical withdrawal threshold (expressed in g) was then calculated from these scores by using a δ value of 0.205, previously determined 62 . Hindpaw Mechanical allodynia (HMA). Paw mechanical allodynia was evaluated by measuring the paw withdrawal threshold by using the up-down paradigm 59 , 63 . Mice were acclimatized (1 h) in individual clear plexiglass boxes on an elevated wire mesh platform, to allow for access to the plantar surfaces of the hind paws. von Frey filaments of increasing stiffness (0.07, 0.16, 0.4, 0.6, and 1.0, 1.4 and 2 g) were applied to the hind paw plantar surfaces of mice with enough pressure to bend the filament. The absence of a paw being lifted after 5 s led to the use of the next filament with an increased force, whereas a lifted paw indicated a positive response, leading to the use of a subsequently weaker filament. Six measurements were collected for each mouse or until four consecutive positive or negative responses occurred. The 50% mechanical withdrawal threshold (expressed in g) was then calculated. Plasmid construction All short hairpin RNAs were designed according to Vector builder and RNAi Consortium of Broad Institute libraries. pAAV[FLEXon]-CMV>EGFP:LL:rev(mCherry:miR30-mC5ar1[shRNA#1]):rev(LL):WPRE and pAAV[FLEXon]-CMV>LL:rev(EGFP:5' miR-30E:{shRNA-NLRP1a}:3' miR-30E):EGFP:5' miR-30E:{shRNA-NLRP1b}:3' miR-30E:rev(LL):WPRE were used. pAAV[FLEXon]-CMV> EGFP-LL-rev(mCherry)-mNLRP3[mir30-shRNA]-rev(LL)-WPRE was generated in two steps. In the first step two overlapping fragments, were obtained by amplifying regions from pAAV[FLEXon]-CMV>EGFP-LL-rev(mCherry)-mC5ar1 [mir30-shRNA]-rev(LL)-WPRE with P1/P4 and P2/P3 primer couples. P1 and P2 were designed to include partial shNlrp3 overlapping regions. A third PCR, using external primers P3/P4 generated the extended fragment, that was cloned (AgeI/SpeI) into pAAV[Exp]-CMV>EGFP-LL-rev(mCherry)- mC5ar1 [mir30-shRNA]-rev(LL)-WPRE. To obtain pAAV[Exp]-CMV>EGFP-LL-rev(mCherry)-mIL-1β[mir30-shRNA]-rev(LL)-WPRE two oligonucleotides (P5 and P6) containing half short hairpin sequence were used to amplify a fragment from the pAAV[Exp]-CMV>EGFP:LL:rev(mCherry:miR30-mC5ar1[shRNA#1]):rev(LL):WPRE. Purified product was phosphorylated with T4 PNK (NEB, #M0201S) according to the manufacturer instructions and finally subjected to circular ligation with a T4 ligase (Thermo Fisher #K1423). All the new-generated plasmids were validated by Sanger sequencing. Primer sequences are reported in Supplementary table S1. AAV Generation Recombinant AAV particles (rAAVs) were produced by using polyethylenimine (#23966, PEI, Polyscience) triple transfection strategy as previously described 55 . In brief, AAVpro 293T cells (#632273, Takara, RRID:CVCL_B0XW), were transfected with a 1:3 ratio of DNA:PEI 64 . To obtain rAAVs, AAVpro 293T cells were transiently transfected with 2.5 mg total DNA (plasmid expressing genes of interest, pAdDeltaF6; (#112867, Addgene) and Rep/Cap, 1:1:1 molar ratio). To infect with high efficiency Schwann cells and primary sensor neurons, Rep/Cap 2/rh10 or 2/9n were used (pAAV2/rh10 #112866 or pAAV 2/9n #112865, respectively Addgene). rAAVs virions were extracted 72 h post-transfection, purified by iodixanol gradient ultracentrifugation, concentrated, and titrated using a RT-qPCR assay (#6233 AAVpro Titration Kit, Takara) according to the manufacturer instructions. AAV production and cell lysis Detailed method is reported in Supplementary information. Iodixanol-based purification protocol Detailed method is reported in Supplementary information. Immunofluorescence Detailed method is reported in Supplementary information. Protein extraction and western immunoblot assay Detailed method is reported in Supplementary information. Proteome profiler array Mouse plasma samples were collected at different days after endometriosis induction for a cytokine array analysis using the Proteome Profiler Mouse Cytokine Array Kit, Panel A (#ARY006. R&D Systems) according to manufacturer’s instructions. Signal was developed using an imaging system (ChemiDoc; Bio-Rad). The density of specific cytokine dots was measured using HLImage ++ (v. PCM.25.5.1.a). Caspase-Glo® 1 Inflammasome Assay The caspase-1 activity was evaluated using the bioluminescent based assay Caspase-Glo® 1 inflammasome assay (#TM456, Promega) according to manufacturer’s instructions. Briefly, HSCs were grown on in 96-well poly-L-lysine-coated (8.3 μM) plates and incubated with C5a (100 ng/ml) in the presence of DF2593A (1 µM), Ac-YVAD-CHO (1µM) or veh (0.001 % DMSO) at 37 °C in 5% CO 2 and 95% O 2 . After 16 h, the cell culture medium was collected and transferred to a 96-white, opaque-walled multiwell plates (with a clear bottom) (#6005181, PerkinElmer) and incubated with Caspase-Glo ® 1 Reagent for 60 min. The luminescence signal was measured by FlexStation3 Multi-Mode Microplate Reader (Molecular Devices;) using SoftMax® Pro7 software (Molecular Devices). Results were expressed as relative light units (RLU). Caspase-1 in vitro imaging The genetically encoded fluorescent sensor Single-Cell Imaging of Caspase-1 (SCAT1) based on fluorescence resonance energy transfer (FRET) was used to detect the inflammasome-mediated caspase-1 activation in HSCs. Briefly, HSCs were plated on 96-well poly-L-lysine-coated (8.3 μM) black clear bottom (5 × 10 5 cells/well; #6055302 PerkinElmer) and transfected with cDNA of SCAT1 (130-300 ng) using jetOPTIMUS® DNA transfection reagent (#55-250, Polyplus) for 16-24 h at 37 °C in 5% CO 2 and 95% O 2 . On the day of experiments, HSCs were washed and added with HBSS at pH 7.4 at 37 °C and transferred to a chamber on the stage of a fluorescent microscope for recording (Axio Observer 7; with a fast filter wheel and Digi-4 lens to record excitations and Ultra-fast Sutter Lambda DG4 Xenon excitation source-range 300-700 nM) (Zeiss) with 20x objective. Cells were exposed to C5a (10 µg/ml) in the presence of DF2593A (1µM) or Ac-YVAD-CHO (1 µM) vehicle (0.001% DMSO). FRET changes were measured as a ratio of the acceptor fluorophore emission (530 nm) to donor emission (435 nm). The ΔF/F0 ratio was calculated for each experiment and the results were expressed as the AUC after inverting the curve. C5a and IL-1β assays C5a and IL-1β content was assayed in mouse sciatic and trigeminal nerve tissue homogenates and in mouse plasma samples using a single-analyte enzyme-linked immunosorbent assay (ELISA) kit (#ab193718 and #ab197742, Abcam, Cambridge, UK) according to the manufacturer’s protocol. Data are expressed as pg/mg of protein and pg/mL. C5a and IL-1β content was assayed in human plasma samples and HSCs cultured medium by ELISA kit (#ab193695 and #ab214025, Abcam) according to the manufacturer’s protocol. Data are expressed as pg/mL Reverse transcription-quantitative real-time PCR (RT-qPCR) Detailed method and the sets of primers for HSCs and MSCs are reported in Supplementary information and Supplementary Table S2. Trans-well migration assay Noncontact coculture transwell cell culture system between macrophages and Schwann cells was obtained using the Boyden migration assay 56 . Briefly, the noncontact cocultured cells were prepared as follows: macrophage-like U937 cells or RAW 264.7 were seeded at a density of 2.5 × 10 4 / chamber into the upper of a 24-well transwell cell culture system (6.5 mm in diameter, with 8-μm pores, (#CLS3458 ™; Corning) by using the complete media described above and allowed to grow 2 days after PMA treatment (U937 cells) or overnight (RAW 264.7) before migration assay 65 . In the meantime, HSCs and MSCs were plated on a 24-well plate. On the day of the experiment, all the cells were replaced with serum free media, and the macrophage-like U937 cells and RAW 264.7 cells cultured on the membrane of 24-well transwell insert were placed into the 24-well plate cultures containing the HSCs and MSCs to initiate the experiment. HSCs and MSCs were treated with C5a (100 ng/ml) also in the presence of DF2593A (1 µM) or veh (0.001 % DMSO) or in the presence mAb-IL-1β (1 µg/ml) or IgG control (1 µg/ml). After 16 h of incubation, migrated cells on the lower surface of the filter were fixed and stained with Diffquick staining set following the manufacturer’s directions (#726443™), and nonmigratory cells on the upper surface of the filter were wiped with a cotton swab. Random fields were counted under a light microscope using a 20x objective (1.5 x 10 5 µm 2 ). Statistical analysis Data are presented as mean ± SEM. For multiple comparisons, a one-way ANOVA followed by a post-hoc Bonferroni’s test was used. The two groups were compared using Student’s t-test. For behavioral experiments with repeated measures, a two-way mixed-model ANOVA followed by a post-hoc Bonferroni’s test was used. Statistical analyses were performed on raw data using GraphPad Prism 8 (GraphPad Software Inc.). P-values less than 0.05 (P < 0.05) were considered significant. EC50 values were determined from non-linear regression models using GraphPad Prism 8. The statistical tests used and sample size for each analysis are shown in the Fig. legends. Declarations Data Availability Materials and data generated from this study are available upon request from Francesco De Logu ( [email protected] ) or Romina Nassini ( [email protected] ). Acknowledgments Supported by grants from: Fondazione Telethon (Grant no GMR22T1070) (F.D.L), European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No. 835286) (P.G.), Ministero della Salute GR-2018-12368352 (Bando Salute 2018) (V.S.), European Union - Next Generation EU, National Recovery and Resilience Plan, Mission 4 Component 2 - Investment 1.4 - National Center for Gene Therapy and Drugs based on RNA Technology - CUP B13C22001010001 (R.N.) and NEXTGENERATIONEU (NGEU) funded by the Ministry of University and Research (MUR), National Recovery and Resilience Plan (NRRP), project MNESYS (PE0000006) – A Multiscale integrated approach to the study of the nervous system in health and disease (DR. 1553 11.10.2022) (P.G., F.D.L.). Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or the European Commission. 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Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay. J Vis Exp , (2017). Additional Declarations Yes there is potential Competing Interest. R.N., F.D.L. and P.G. are founding scientists of FloNext Srl. G.B. is fully employed at FloNext Srl, Italy. Other authors declare no competing interests. Supplementary Files 02Titizetal.Suppl.docx Supplementary information Cite Share Download PDF Status: Published Journal Publication published 25 Nov, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4045626","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":290500299,"identity":"5fc6866a-ed41-41d0-ae4a-fe07970706e7","order_by":0,"name":"Francesco De 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14:35:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4045626/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4045626/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-024-54486-6","type":"published","date":"2024-11-25T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":54671768,"identity":"2e2c8ee9-182b-4d0b-b734-2cc3d9737bf1","added_by":"auto","created_at":"2024-04-15 05:14:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":705250,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEndometriosis-associated mechanical allodynia is mediated by the complement system C5a. (a)\u003c/strong\u003e Time-dependent periorbital (PMA), hind paw (HMA) and abdominal (AMA) mechanical allodynia in endometriotic (endo) or Sham C57BL/6J female (B6) mice. Proteome profiler array \u003cstrong\u003e(b)\u003c/strong\u003e and single analyte C5a assay \u003cstrong\u003e(c)\u003c/strong\u003e in plasma samples from endo and Sham B6 mice. \u003cstrong\u003e(d)\u003c/strong\u003e C5 assay in plasma samples from endo or healthy patients (control). (\u003cstrong\u003ee) \u003c/strong\u003eTime-dependent C5a levels in plasma samples from endo and Sham B6 mice. \u003cstrong\u003e(f and g) \u003c/strong\u003eTimeline\u003cstrong\u003e \u003c/strong\u003eof 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. \u003cstrong\u003ea, e, f, g \u003c/strong\u003e2-way ANOVA, Bonferroni correction; \u003cstrong\u003ec, d\u003c/strong\u003e Student’s t test.\u0026nbsp; *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001, ****P\u0026lt;0.0001 vs. Sham \u003csup\u003e##\u003c/sup\u003eP\u0026lt;0.01, \u003csup\u003e####\u003c/sup\u003eP\u0026lt;0.0001 vs. Endo+Veh DF25.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4045626/v1/bc317f74bbb746b3a183f75d.png"},{"id":54671765,"identity":"a61c62ff-04cd-4bc2-99f3-70ef70469f69","added_by":"auto","created_at":"2024-04-15 05:14:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":734305,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeuroinflammation mediates endometriosis-associated mechanical allodynia. (a)\u003c/strong\u003e Representative images and cumulative data of the time-dependent increase of F4/80\u003csup\u003e+\u003c/sup\u003e cells in sciatic and trigeminal nerve in endometriotic (endo) or Sham C57BL/6J female (B6) mice. \u003cstrong\u003e(b)\u003c/strong\u003e Timeline\u003cstrong\u003e \u003c/strong\u003eof 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). \u003cstrong\u003e(c)\u003c/strong\u003e Representative images and cumulative data of F4/80\u003csup\u003e+\u003c/sup\u003e cells in sciatic and trigeminal nerve of endo B6 mice after AP or Veh and in Sham mice (treatment schedule as in b). (Scale bar, 50 μm, dashed lines, \u003cem\u003eperineurium\u003c/em\u003e)\u003cem\u003e.\u003c/em\u003e Data are mean ± s.e.m. \u003cstrong\u003ea, c \u003c/strong\u003e1-way ANOVA, \u003cstrong\u003eb\u003c/strong\u003e, 2-way ANOVA, Bonferroni correction; *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001, ****P\u0026lt;0.0001 vs. Sham, \u003csup\u003e####\u003c/sup\u003eP\u0026lt;0.0001 vs. Endo-MaFIA+ Veh AP.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4045626/v1/0851d73d01e3f33dd168e082.png"},{"id":54671770,"identity":"1f9de78c-a0d7-4418-9c02-2371df45ece9","added_by":"auto","created_at":"2024-04-15 05:14:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1094654,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchwann cell C5aR1 mediates endometriosis-associated mechanical allodynia and neuroinflammation\u003c/strong\u003e. Representative images of SOX10 and C5aR1 co-expression in sciatic \u003cstrong\u003e(a)\u003c/strong\u003e and trigeminal \u003cstrong\u003e(b)\u003c/strong\u003e nerve tissue from C57BL/6J female (B6) mice, (Scale bar, 50 μm). (\u003cstrong\u003ec\u003c/strong\u003e) \u003cem\u003eC5aR1\u003c/em\u003e mRNA relative expression in primary mouse and human Schwann cells (n=3 independent experiments). (\u003cstrong\u003ed\u003c/strong\u003e) Representative blot and C5aR1 protein content in primary mouse and human Schwann cells. Equally loaded protein was checked by β-actin (n=3 independent experiments). (\u003cstrong\u003ee\u003c/strong\u003e) \u0026nbsp;Schematic representation of AAV-(loxP-shRNA)-\u003cem\u003eC5aR1\u003c/em\u003e vector pre- and post-Cre switch. (\u003cstrong\u003ef\u003c/strong\u003e) Representative images and cumulative data (Rcoloc) of SOX10 and C5aR1 co-expression in sciatic and trigeminal nerve tissue in \u003cem\u003ePlp\u003c/em\u003e-AAV-\u003cem\u003eC5aR1\u003c/em\u003e and Control mice (n=4 independent experiments).\u0026nbsp;(\u003cstrong\u003eg\u003c/strong\u003e) Time-dependent periorbital (PMA), hind paw (HMA), and abdominal (AMA) mechanical allodynia in endometriotic (endo) or Sham \u003cem\u003ePlp\u003c/em\u003e-AAV-\u003cem\u003eC5aR1\u003c/em\u003e and Control mice.\u0026nbsp;(\u003cstrong\u003eh\u003c/strong\u003e) Representative images and cumulative data of F4/80\u003csup\u003e+\u003c/sup\u003e cells in sciatic and trigeminal nerve tissue in endo or Sham \u003cem\u003ePlp\u003c/em\u003e-AAV-\u003cem\u003eC5aR1\u003c/em\u003e and Control mice. (n=4 independent experiments) (Scale bar, 50 μm, dashed lines, \u003cem\u003eperineurium\u003c/em\u003e). (\u003cstrong\u003ei\u003c/strong\u003e) 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. \u003cstrong\u003ef, h, i \u003c/strong\u003e1-way ANOVA, \u003cstrong\u003eg\u003c/strong\u003e, 2-way ANOVA, Bonferroni correction; ****P\u0026lt;0.0001 vs. Sham/Control, \u003csup\u003e####\u003c/sup\u003eP\u0026lt;0.0001 vs. Endo/Control.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4045626/v1/70694908eb285b22371f1d7c.png"},{"id":54671766,"identity":"439e01a4-3340-441a-954c-fb3dc79fd853","added_by":"auto","created_at":"2024-04-15 05:14:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":904129,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchwann cell C5aR1 activation induces NLRP1 dependent release of IL-1β. (a) \u003c/strong\u003eIL-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). \u003cstrong\u003e(b)\u003c/strong\u003e Time-dependent C5a levels in plasma samples from endometriotic (endo) or Sham C57BL/6J female (B6) mice.\u003cstrong\u003e (c) \u003c/strong\u003eIL-1β assay in plasma samples from endo or healthy patients (control). (\u003cstrong\u003ed\u003c/strong\u003e) 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\u0026nbsp; Ac-YVAD (AUC, area under the curve) (cell numbers: C5a=20, Veh=16, DF25=16 and Ac-YVAD=20; n=3 independent experiments) (\u003cstrong\u003ee\u003c/strong\u003e) Time-dependent periorbital (PMA), hind paw (HMA) and abdominal (AMA) mechanical allodynia in endo or Sham \u003cem\u003ePlp-\u003c/em\u003eAAV-\u003cem\u003eNlrp1\u003c/em\u003e and Control mice\u0026nbsp;(n=8 mice per group). (\u003cstrong\u003ef\u003c/strong\u003e) IL-1β assay in sciatic and trigeminal nerve tissue homogenates in endo or Sham \u003cem\u003ePlp-\u003c/em\u003eAAV-\u003cem\u003eNlrp1\u003c/em\u003e and Control mice (n=6 independent experiments). (\u003cstrong\u003eg\u003c/strong\u003e) Representative images and cumulative data of F4/80\u003csup\u003e+\u003c/sup\u003e cells in sciatic and trigeminal nerves in endo or Sham \u003cem\u003ePlp-\u003c/em\u003eAAV-\u003cem\u003eNlrp1\u003c/em\u003e and Control mice (n=4 independent experiments). (\u003cstrong\u003eh\u003c/strong\u003e) Time-dependent PMA, HMA, and AMA in endo or Sham \u003cem\u003ePlp-\u003c/em\u003eAAV-\u003cem\u003eIL-1β\u003c/em\u003e and Control mice (n=8 mice per group). (\u003cstrong\u003ei\u003c/strong\u003e)\u0026nbsp;Il-1β assay in sciatic and trigeminal nerve tissue homogenates in endo or Sham \u003cem\u003ePlp-\u003c/em\u003eAAV-\u003cem\u003eIl-1β\u003c/em\u003e and Control mice. (\u003cstrong\u003ej\u003c/strong\u003e)\u0026nbsp;Representative images and cumulative data of F4/80\u003csup\u003e+\u003c/sup\u003e cells in sciatic and periorbital nerve in endo or Sham \u003cem\u003ePlp-\u003c/em\u003eAAV-\u003cem\u003e Il-1β\u003c/em\u003e\u0026nbsp;and Control mice (Scale bar, 50 μm, dashed lines, \u003cem\u003eperineurium\u003c/em\u003e) (n=8 mice per group). Data are mean ± s.e.m. \u003cstrong\u003ea, c, d, f, g \u003c/strong\u003e1-way ANOVA, \u003cstrong\u003eb, e, h\u003c/strong\u003e, 2-way ANOVA, Bonferroni correction; *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001, ****P\u0026lt;0.0001 vs. Sham, \u003csup\u003e#\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003eP\u0026lt;0.01, \u003csup\u003e###\u003c/sup\u003eP\u0026lt;0.001, \u003csup\u003e####\u003c/sup\u003eP\u0026lt;0.0001 vs. Endo/Control.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4045626/v1/2892f8120bfd5029985d9c80.png"},{"id":54671769,"identity":"9571c69b-2c5d-469d-b6f3-f2dc798bc47f","added_by":"auto","created_at":"2024-04-15 05:14:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":418399,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) Neuronal TRPA1 channel mediates endometriosis-associated mechanical allodynia but not neuroinflammation. \u003c/strong\u003eTime-dependent periorbital (PMA), hind paw (HMA) and abdominal (AMA) mechanical allodynia in endometriotic (endo) or Sham \u003cem\u003eAdv-Trpa1\u003c/em\u003e and Control mice.\u0026nbsp;\u003cstrong\u003e(b) \u003c/strong\u003eIL-1β assay in sciatic and trigeminal nerve tissue homogenates in endo or Sham \u003cem\u003eAdv-Trpa1\u003c/em\u003e and Control mice (n=6 independent experiments). \u003cstrong\u003e(c) \u003c/strong\u003eRepresentative images and cumulative data of F4/80\u003csup\u003e+\u003c/sup\u003e cells in sciatic and trigeminal nerve in endo or Sham \u003cem\u003eAdv-Trpa1\u003c/em\u003e and Control mice (Scale bar, 50 μm, dashed lines, \u003cem\u003eperineurium\u003c/em\u003e) (n=4 independent experiments). Data are mean ± s.e.m. \u003cstrong\u003ea\u003c/strong\u003e, 2-way ANOVA, \u003cstrong\u003eb,c \u003c/strong\u003e1-way ANOVA, Bonferroni correction; *P\u0026lt;0.05, ***P\u0026lt;0.001, ****P\u0026lt;0.0001 vs. Sham,\u0026nbsp; \u003csup\u003e##\u003c/sup\u003eP\u0026lt;0.01, \u003csup\u003e####\u003c/sup\u003eP\u0026lt;0.0001 vs. Endo/Control.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4045626/v1/62b72f84f3d93f207d42998d.png"},{"id":69873172,"identity":"7e16d180-15f8-4387-b8f6-c38f1fe6157b","added_by":"auto","created_at":"2024-11-26 08:07:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4750197,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4045626/v1/f457d4d1-26fa-47e0-9200-dcc5ee20cbe2.pdf"},{"id":54671767,"identity":"e4d8f4cf-4b2d-45c2-a767-f39b9cb7b421","added_by":"auto","created_at":"2024-04-15 05:14:00","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3913169,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary information\u003c/p\u003e","description":"","filename":"02Titizetal.Suppl.docx","url":"https://assets-eu.researchsquare.com/files/rs-4045626/v1/1bfabc74dd4cf86532f83e2c.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nR.N., F.D.L. and P.G. are founding scientists of FloNext Srl. G.B. is fully employed at FloNext Srl, Italy. Other authors declare no competing interests.","formattedTitle":"Schwann Cell C5aR1 co-opts Inflammasome NLRP1 to Sustain Pain in a Mouse Model of Endometriosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEndometriosis, caused by superficial peritoneal/deep infiltrating implants or ovarian cysts of endometrial-like tissue\u003csup\u003e1\u003c/sup\u003e, is an enigmatic and often debilitating gynecologic condition that affects 10-15% of reproductive-aged women\u003csup\u003e2\u003c/sup\u003e. The socioeconomic burden of the disease is high, as endometriosis affects career, everyday activities, sexual and nonsexual relationships, and quality of life{\u003csup\u003e3\u003c/sup\u003e. More than 60% of women diagnosed with endometriosis suffer from abdominopelvic pain{\u003csup\u003e4\u003c/sup\u003e, and often suffer from pain conditions in anatomical sites other than the pelvis/abdomen, such as chronic back pain, fibromyalgia, vulvodynia, and migraine{\u003csup\u003e5\u003c/sup\u003e;Nagai, 2015 #86}. Migraine, the most debilitating condition for women aged 20-50 years, is frequent in women with endometriosis in reproductive age\u003csup\u003e6\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e7\u003c/sup\u003e. A correlation between fibromyalgia and endometriosis has been proposed, given that immune mechanisms appear to contribute to chronic pain in the two conditions\u003csup\u003e8\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e9\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe complement system is a broad network of soluble and cell-surface proteins distributed throughout body fluids and tissues with no, or little, basal activity\u003csup\u003e10\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e11\u003c/sup\u003e. The complement system acts not just as a first line of defense against pathogens, as it functionally connects innate and adaptive immune responses \u003cem\u003evia\u003c/em\u003e receptors expressed by myeloid and non-myeloid cells\u003csup\u003e12\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e13\u003c/sup\u003e. Activation of the complement system cascade involves a C3-convertase that cleaves C3, producing C3a and C3b. Two C3b molecules plus the Bb factor form the C5 convertase that cleaves C5 in C5a and C5b\u003csup\u003e12\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e13\u003c/sup\u003e. C5a targets a G protein coupled receptor (GPCR), C5a receptor type 1 (C5aR1), originally identified in neutrophils and monocytes/macrophages\u003csup\u003e14\u003c/sup\u003e. C5a contribution in post-operative\u003csup\u003e15\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e16\u003c/sup\u003e, inflammatory\u003csup\u003e17\u003c/sup\u003e, and neuropathic\u003csup\u003e18\u003c/sup\u003e pain has been previously proposed.\u003c/p\u003e\n\u003cp\u003eEndometriosis patients undergoing surgery are classified in four stages according to the Revised Classification of Endometriosis by the American Society of Reproductive Medicine (r-ASRM\u003csup\u003e19\u003c/sup\u003e. Women at the early (I-II) stages have shown higher C5a serum levels compared to a control group\u003csup\u003e20\u003c/sup\u003e. Macrophages play a pivotal role in neurogenesis processes occurring close to endometriotic lesions, suggesting their role in endometriosis-associated pain generation\u003csup\u003e21\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e22\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e23\u003c/sup\u003e. Activation of C5aR1 in endoneurial macrophages induces the NLRP3 inflammasome-dependent release of the pro-nociceptive mediator interleukin 1 (IL-1) \u0026beta; to maintain pain in a mouse model of peripheral nerve injury\u003csup\u003e24\u003c/sup\u003e. Recently, we showed that crosstalk between Schwann cells (SCs) and endoneurial resident monocytes/macrophages in peripheral nerves activates an oxidative stress pathway that elicits neuroinflammation and sustains chronic pain\u003csup\u003e25\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e26\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e27\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e28\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eHere, we investigated the mechanism underlying mechanical allodynia produced by endometriotic lesions in the abdomen and other areas distant from the abdomen, such as the periorbital area to mimic migraine pain and the hind paw to mimic fibromyalgia, in female mice. As abdominal mechanical allodynia was associated with mechanical allodynia in the hind paw and periorbital area, we explored the cellular and molecular pathway that diffuses mechanical hypersensitivity from endometriotic lesions to the other two anatomical areas. We found that C5a increased by endometriosis, targeting the SC C5aR1, activates the NLRP1 inflammasome, which induces the release of IL-1\u0026beta;. The increase in IL-1\u0026beta; in peripheral nerves (periorbital and sciatic nerves) promotes macrophage recruitment and the release of reactive oxygen species (ROS) that target neuronal TRPA1 to signal pain. These results implicate SC C5aR1 as a new target for the treatment of endometriosis associated pain.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eThe complement component C5a mediates endometriosis-induced allodynia. \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn C57BL/6J female (B6) mice, intraperitoneal (i.p.) injection of dissected uterus horns from donor female mice induced a time-dependent increase of endometriosis-like lesions (Supplementary Fig. 1a), which were associated with prolonged (28 days) periorbital mechanical allodynia (PMA), hind paw mechanical allodynia (HMA), and abdominal mechanical allodynia (AMA) (Fig. 1a). The simultaneous development of allodynia in anatomical areas distant from the abdomen supports the hypothesis that diffusible cellular or molecular mediators are implicated in the widespread proalgesic response. Several inflammatory cytokines/chemokines have been implicated in endometriotic pain\u003csup\u003e29\u003c/sup\u003e. The relative levels of forty cytokines/chemokines and acute phase proteins in plasma samples of endometriotic and sham B6 mice were investigated by a proteome profiler array at day 7 after surgery (Fig. 1b). The array revealed increased C5a levels in endometriotic mice (Fig. 1b), and the increase was confirmed by a C5a single analyte ELISA assay (Fig. 1c). A recent report showed that serum levels of C5a were higher in early stage (I-II) endometriotic patients than in control patients\u003csup\u003e20\u003c/sup\u003e. Here, we confirmed the higher serum levels of C5a in a group of patients with endometriosis (n=19) compared to healthy women without endometriosis (n=15) (Fig. 1d). In addition, we observed that, in endometriotic B6 mice, C5a plasma levels rose from day 1 to day 8 to then decline to basal levels at day 12-16 after surgery (Fig. 1e). \u003c/p\u003e\n\u003cp\u003eAs C5a have been implicated in several pain conditions\u003csup\u003e15\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e16\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e17\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e18\u003c/sup\u003e, we tested whether the selective allosteric C5aR1 inhibitor, DF2593A, given in two different temporal schedules would reduce PMA, HMA, and AMA in endometriotic mice. Endometriotic B6 mice treated with DF2593A (twice a day) from one day before until day 6 after the injection of the ectopic tissue, when C5a levels increase in mouse plasma, showed a significant reduction in the development of PMA, HMA, and AMA (Fig. 1f). Conversely, daily treatment with DF2593A, from day 12 to day 16, failed to reduce PMA, HMA, and AMA (Fig. 1g). Overall, the data indicate that, although transient (from day 2 to day 10 after surgery), the increase in C5a plasma levels was sufficient to initiate and sustain PMA, HMA, and AMA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNeuronal macrophages mediate endometriosis-induced mechanical allodynia\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmune cells, including macrophages and nerve fibers, interact to promote pain symptoms associated with endometriosis\u003csup\u003e30\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e31\u003c/sup\u003e. Our previous findings proposed that macrophages infiltrating peripheral nerve trunks interact with SCs to sustain mechanical allodynia in different mouse models of pain\u003csup\u003e25\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e26\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e27\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e28\u003c/sup\u003e. To understand whether macrophage accumulation plays a role in the present model of endometriosis, sciatic and trigeminal nerve trunks were examined. Due to their limited size, the nerve fibers of the endometriotic lesions were not included in these studies. In endometriotic B6 mice, the number of F4/80\u003csup\u003e+\u003c/sup\u003e macrophages inside sciatic and trigeminal nerve trunks increased in a time-dependent manner starting from day 3 after surgery (Fig. 2a). To explore the role of macrophages in endometriosis-induced mechanical allodynia, macrophage Fas-induced apoptosis (MaFIA) mice, injected with the apoptosis inducer, AP20187, which abates the number of macrophages (GFP\u003csup\u003e+\u003c/sup\u003e/F4/80\u003csup\u003e+\u003c/sup\u003ecells)\u003csup\u003e32\u003c/sup\u003e, were used. Ectopic tissue inoculation in the peritoneum of MaFIA mice induced time-dependent PMA, HMA, and AMA, as observed in B6 mice (Fig. 2b). To better understand the action of C5a on Schwann cells and macrophages, macrophages were depleted in MaFIA mice by AP20187 administration following three different time schedules: i) before the increase in C5a levels (from day -2 to day 2); ii) during the C5a increase (from day 2 to day 6); and iii) when C5a declined to baseline (from day 21 to day 25). Macrophage depletion obtained with the two later (ii and iii) (Fig. 2b,c and Supplementary Fig. 1b,c respectively), but not with the early (i) (Supplementary Fig. 1d,e) treatment schedule reduced PMA, HMA, and AMA. These findings are consistent with the transient (~48 hours) monocyte/macrophage depletion obtained in MAFiA mice\u003csup\u003e33\u003c/sup\u003e. Depletion of monocytes/macrophages in MaFIA mice after AP20187 treatment was evaluated as the number of F4/80\u003csup\u003e+\u003c/sup\u003e cells in both sciatic and trigeminal nerves (Fig. 2c, Supplementary Fig. 1c and Supplementary Fig. 1e). \u003c/p\u003e\n\u003cp\u003eThe C5aR1 inhibitor, DF2593A, given to endometriotic mice during the increase of C5a in plasma (from day 2 to day 6), reduced macrophages in sciatic and trigeminal nerve tissues (Supplementary Fig. 1f). In contrast, treatment when C5a serum levels declined (from day 12 to 16) failed to reduce the macrophage accumulation (Supplementary Fig. 1g). Collectively, our data suggest that C5a release, most likely from ectopic endometrial lesions during the first seven days after surgery, increases macrophages in nerve trunks. However, when C5a serum levels decline, increased macrophages promote PMA, HMA, and AMA in a C5a-independent manner.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSchwann cell C5aR1 mediates macrophage recruitment and endometriosis-induced mechanical allodynia.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven that, in several mouse models of pain, SCs play a crucial role in sustaining mechanical allodynia, we investigated whether these glial cells contributed to PMA, HMA, and AMA in the present model of endometriosis. Immunofluorescent staining analysis of mouse sciatic and trigeminal nerve tissues revealed that C5aR1 staining was present in 92.2% \u0026plusmn; 1.1 and 93.3% \u0026plusmn; 0.44 of nucleated cells, respectively (Supplementary Fig. 2a). SCs, which represent 45.1% of the nucleated cells in mouse peripheral nerve tissues\u003csup\u003e34\u003c/sup\u003e, are identified by SOX10 expression. Immunofluorescent staining analysis in mouse and human sciatic and mouse trigeminal nerves revealed that all SOX10+ cells co-express C5aR1 (Fig. 3a,b and Supplementary Fig. 2b). C5aR1\u003cem\u003e \u003c/em\u003emRNA and protein expression were confirmed in cultured mouse and human SCs by qRT-PCR and western immunoblot assays (Fig. 3c,d). To investigate the role of Schwann cell C5aR1 in mechanical allodynia, we used a \u003cem\u003ePlp\u003csup\u003eCre\u003c/sup\u003e\u003c/em\u003e driver that functions as a lineage tracer to express a short hairpin RNA (shRNA) for selective silencing of C5aR1 in SCs by the injection of a Cre-dependent adeno associated viral vector (AAV). A virus packaged with the AAVrh10 serotype for efficient infection of SCs was used with a \u003cem\u003eloxP\u003c/em\u003e flanked shRNA to express shRNA in SCs (AAV-\u003cem\u003eC5aR1\u003c/em\u003e) (Fig. 3e). The efficacy of the gene silencing was evaluated in sciatic and trigeminal nerves by immunofluorescent staining (Fig. 3f). Systemic (intravenous, i.v.) administration of AAV-\u003cem\u003eC5aR1\u003c/em\u003e in \u003cem\u003ePlp\u003csup\u003eCre+\u003c/sup\u003e\u003c/em\u003e but not in \u003cem\u003eControl\u003csup\u003e \u003c/sup\u003e\u003c/em\u003emice attenuated endometriosis-induced PMA, HMA, and AMA (Fig. 3g) and macrophage increase in sciatic and trigeminal nerves (Fig. 3h). To further investigate the ability of C5a to stimulate cultured mouse and human SCs to recruit macrophages, we used the Boyden chamber assay. Human and mouse SCs stimulated with C5a promoted macrophage migration through the microporous membrane that was inhibited in the presence of DF2593A (Fig. 3i). \u003c/p\u003e\n\u003cp\u003ePrevious data reported C5aR1 mRNA expression in mouse DRG neurons where C5a, by activating and sensitizing cutaneous nociceptors, amplified the capsaicin response\u003csup\u003e35\u003c/sup\u003e. To identify the role for neuronal C5aR1 in endometriotic pain, we used a \u003cem\u003eAdv\u003csup\u003eCre\u003c/sup\u003e\u003c/em\u003e driver to silence C5aR1 selectively in sensory neurons and a virus packaged with the AAV2/9n serotype for efficient infection of nociceptors. Selective silencing of C5aR1 in sensory neurons was confirmed by immunofluorescent staining in lumbar (L4-L6) dorsal root ganglion (DRG) tissues (Supplementary Fig. 2c). Intrathecal administration of AAV-\u003cem\u003eC5aR1\u003c/em\u003e in \u003cem\u003eAdv\u003csup\u003eCre+\u003c/sup\u003e\u003c/em\u003e did not attenuate endometriosis-induced PMA, HMA, and AMA and macrophage increases in nerve (sciatic and trigeminal) trunks (Supplementary Fig. 2d,e). These results indicate that, in endometriotic mice, C5a targeting of the SC C5aR1 is necessary and sufficient to induce neuroinflammation and mechanical allodynia.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eSchwann cell NLRP1 drives endometriosis-induced macrophage increases and mechanical allodynia.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC5aR1 is known to activate the NLR family pyrin domain containing 3 (NLRP3) inflammasome, thus promoting the expression of the interleukin-1\u0026beta; precursor (pro-IL-1\u0026beta;) and secretion of IL-1\u0026beta; through the cleavage by caspase-1 in different immune cells\u003csup\u003e36\u003c/sup\u003e. Here, we report that human SC C5aR1 encoded a signaling pathway that released IL-1\u0026beta;, a response attenuated by the C5aR1 inhibitor, DF2593A (Fig. 4a). IL-1\u0026beta; plasma levels from endometriotic mice were more elevated from day 10 until day 28 after surgery as compared to sham mice (Fig. 4b). Increased IL-1\u0026beta; plasma levels were also detected in women with endometriosis as compared to healthy women without endometriosis (Fig. 4c). To assess caspase-1 activation in cultured human SCs, cells were exposed to C5a that increased a bioluminescent signal due to caspase-1 activation. The increase was reduced by DF2593A and by the selective caspase-1 inhibitor, Ac-YVAD-CHO (Supplementary Fig. 3a). Confirmation of caspase-1 activation was obtained by genetically encoded fluorescent sensor (SCAT1) based on fluorescence resonance energy transfer (FRET)\u003csup\u003e37\u003c/sup\u003e. SCAT1 contains a consensus peptide preferentially cleaved by caspase-1 that reduces the FRET signal\u003csup\u003e37\u003c/sup\u003e. C5a decreased SCAT1 Venus/ECFP (V/C) ratio, a response that was prevented by DF2593A and Ac-YVAD-CHO in human SCs (Fig. 4d). Ac-YVAD-CHO inhibited the release of IL-1\u0026beta; from human SCs after C5a stimulation, supporting the role of C5aR1 in promoting inflammasome activation and IL-1\u0026beta; release (Supplementary Fig. 3b). Endometriotic mice with AAV-mediated selective silencing of C5aR1\u003cem\u003e \u003c/em\u003e(\u003cem\u003ePlp-\u003c/em\u003eAAV-\u003cem\u003eC5aR1\u003csup\u003e \u003c/sup\u003e\u003c/em\u003emice) showed reduced levels of IL-1\u0026beta; in sciatic and trigeminal nerve tissues compared to \u003cem\u003eControl\u003c/em\u003e mice (Supplementary Fig. 3c). To test the role of SC NLRP3 in endometriotic mechanical allodynia, we injected \u003cem\u003ePlp\u003csup\u003eCre+ \u003c/sup\u003e\u003c/em\u003emice with an AAV-\u003cem\u003eNlrp3\u003c/em\u003e\u003cem\u003e \u003c/em\u003efor selective silencing of NLRP3 in SCs (\u003cem\u003ePlp-\u003c/em\u003eAAV-\u003cem\u003eNLRP3\u003csup\u003e \u003c/sup\u003e\u003c/em\u003emice). However, PMA, HMA, AMA, neuroinflammation and IL-1\u0026beta; levels in sciatic and trigeminal nerve tissue in endometriotic \u003cem\u003ePlp-\u003c/em\u003eAAV-\u003cem\u003eNLRP3\u003csup\u003e \u003c/sup\u003e\u003c/em\u003emice were unaffected. (Supplementary Fig. 3d,e, f). The efficacy of the gene silencing was evaluated in sciatic and trigeminal nerve tissue by immunofluorescent staining (Supplementary Fig. 3g)\u003c/p\u003e\n\u003cp\u003eThe inflammasome family currently includes three subtypes, NLRP1, NLRP3, and NLRC4, which recognize specific classes of pathogens or risk signals\u003csup\u003e38\u003c/sup\u003e. The qRT-PCR analysis of mRNA expression in both human and mouse SCs revealed mainly the presence of NLRP1 and NLRP3 transcripts (Supplementary Fig. 3h). Given the very low level of NLRC4 expression, we tested the role of SC NLRP1 in endometriotic pain by infecting \u003cem\u003ePlp\u003csup\u003eCre+ \u003c/sup\u003e\u003c/em\u003emice with an AAV-\u003cem\u003eNlrp1\u003c/em\u003e for selective silencing of NLRP1 in SCs (\u003cem\u003ePlp-\u003c/em\u003eAAV-\u003cem\u003eNLRP1)\u003c/em\u003e. \u003cem\u003ePlp-\u003c/em\u003eAAV-\u003cem\u003eNLRP1 \u003c/em\u003emice showed reduced PMA, HMA, and AMA IL-1\u0026beta; levels and neuroinflammation in sciatic and trigeminal nerve tissue compared to \u003cem\u003eControl\u003c/em\u003e mice (Fig. 4e-g). The efficacy of NLRP1gene silencing was evaluated in sciatic and periorbital nerve tissue by immunofluorescent staining (Supplementary Fig. 3i)\u003c/p\u003e\n\u003cp\u003ePrevious studies indicated a crucial role of IL-1\u0026beta; in sustaining inflammation through macrophage recruitment\u003csup\u003e39\u003c/sup\u003e. To support the role of IL-1\u0026beta; in increasing macrophage number in nerve trunks, mouse and human SCs and macrophages were exposed to a neutralizing monoclonal antibody against IL-1\u0026beta; (anti-IL-1\u0026beta; mAb) and C5a in a Boyden chamber assay. The anti-IL-1\u0026beta; mAb reduced macrophage migration evoked by C5a (Supplementary Fig. 4a). The anti-IL-1\u0026beta; mAb treatment also reduced PMA, HMA, and AMA and macrophage numbers in sciatic and trigeminal nerves in endometriotic mice (Supplementary Fig. 4b,c). To identify the SC role in releasing IL-1\u0026beta; following C5a stimulation in endometriotic mice, we selectively silenced IL-1\u0026beta; in SCs by infecting \u003cem\u003ePlp\u003csup\u003eCre+ \u003c/sup\u003e\u003c/em\u003emice with an AAV-\u003cem\u003eIL-1\u0026beta; (Plp-\u003c/em\u003eAAV-\u003cem\u003e IL-1\u0026beta; \u003c/em\u003emice)\u003cem\u003e.\u003c/em\u003e \u003cem\u003ePlp-\u003c/em\u003eAAV-\u003cem\u003e IL-1\u0026beta;\u003c/em\u003e mice showed reduced PMA, HMA, and AMA (Fig. 4h), IL-1\u0026beta; levels (Fig. 4i), and neuroinflammation in sciatic and trigeminal nerve tissue (Fig. 4j) compared to \u003cem\u003eControl\u003c/em\u003e mice. The efficiency of gene silencing was evaluated in sciatic and trigeminal nerve tissue by immunofluorescent staining (Supplementary Fig. 4d). Overall, these data support the view that C5a from endometriotic lesions targets SC C5aR1, which activates inflammasome NLRP1 to release IL-1\u0026beta;, which in turn increases macrophages within peripheral nerve trunks to sustain mechanical allodynia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNeuronal TRPA1 mediates endometriosis-induced mechanical allodynia.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe previously reported that targeting the transient receptor ankyrin 1 (TRPA1) in primary sensory neurons by oxidative stress signals mechanical allodynia in several mouse models of pain\u003csup\u003e25\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e26\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e27\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e28\u003c/sup\u003e. Here, endometriotic \u003cem\u003eAdv\u003csup\u003eCre \u003c/sup\u003e\u003c/em\u003emice with selective deletion of TRPA1 in sensory neurons (\u003cem\u003eAdv-TRPA1)\u003c/em\u003e showed attenuation of PMA, HMA, and AMA compared to \u003cem\u003eControl\u003c/em\u003e mice (Fig. 5a). Conversely, IL-1\u0026beta; levels, as well as neuroinflammation in sciatic and trigeminal nerve tissue, were unaffected (Fig. 5b,c). These data show that neuronal TRPA1 is the final common pathway that, from endometriotic lesions and \u003cem\u003evia\u003c/em\u003e SCs, signals mechanical allodynia.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere, we provide evidence that, in the present mouse model of endometriosis, peritoneal lesions, by increasing serum levels of C5a, not only exert local effects in mesenteric afferent neurons, but also spread a macrophage-dependent proalgesic mechanism throughout the body, reaching distant anatomical regions, including the periorbital or hind paw areas. We reveal that C5a, in both proximal and remote areas, such as the abdomen and periorbital area/hind paw, respectively, targets the SC C5aR1, where it encodes a proalgesic mechanism that results in neuroinflammation and the ensuing widespread mechanical allodynia. Another unexpected finding of the present study highlights the role of the inflammasome NLRP1, instead of the classical NLRP3, normally activated by C5a, as we found that SC C5aR1 \u003cem\u003evia\u003c/em\u003e NLRP1 enhances the caspase-1 processing of pro-IL-1\u0026beta;. The resulting IL-1\u0026beta; release increases the macrophage number that, \u003cem\u003evia\u003c/em\u003e oxidative stress and neuronal TRPA1 activation, promotes and sustains mechanical allodynia.\u003c/p\u003e\n\u003cp\u003eC5a has been implicated in various pain conditions, including post-operative\u003csup\u003e16\u003c/sup\u003e, inflammatory\u003csup\u003e17\u003c/sup\u003e, and neuropathic pain\u003csup\u003e24\u003c/sup\u003e. To date, although little is known about the association between the complement system components and endometriotic pain, fluctuation of C5a plasma levels during the different disease stages of endometriosis has been documented in female patients\u003csup\u003e20\u003c/sup\u003e\u003csup\u003e,\u0026nbsp;\u003c/sup\u003e\u003csup\u003e40\u003c/sup\u003e. Time course analysis of C5a plasma levels in the present mouse model of endometriosis showed similar fluctuations in female mice with endometriotic lesions, which were identified as the initial stimulus responsible for the abdominal and diffuse proalgesic condition.\u003c/p\u003e\n\u003cp\u003eA considerable proportion of women with endometriosis-associated pain reported a neuropathic-like pain component\u003csup\u003e41\u003c/sup\u003e. Recently, it has been observed that neuropathic-like pain caused by peripheral nerve injury in mice was modulated by activation of neuron associated C5aR1+ macrophages\u003csup\u003e24\u003c/sup\u003e. In our study, results obtained with the C5aR1 inhibitor and transgenic MaFIA mice confirm that increases in plasma C5a and nerve trunk macrophages are essential for the development of endometriosis-associated pain-like responses. We also found that endometriotic lesions promote mechanical allodynia diffused to various tissues by orchestrating a series of temporally distinct and strictly coordinated phases. The role of C5a was confined to its initial sharp increase. Although C5aR1 expression has not been reported in SCs located at neuromuscular junctions\u003csup\u003e42\u003c/sup\u003e, we found that both cultured primary human and mouse SCs and SOX10+ cells in mouse and human peripheral nerve fibers expressed C5aR1. The present data obtained by C5aR1 selective silencing highlighted the presence and the role of SC C5aR1 in endometriosis-associated mechanical allodynia and neuroinflammation.\u003c/p\u003e\n\u003cp\u003eRecent studies suggest that the C5a-C5aR1/C5aR2 interaction regulates the NLRP3 inflammasome in several immune system cells, including macrophages\u003csup\u003e43\u003c/sup\u003e\u003csup\u003e,\u0026nbsp;\u003c/sup\u003e\u003csup\u003e44\u003c/sup\u003e and T cells\u003csup\u003e45\u003c/sup\u003e. Furthermore, NLRP3 has been implicated in the pathobiology of chronic neuropathic and inflammatory pain\u003csup\u003e46\u003c/sup\u003e\u003csup\u003e,\u0026nbsp;\u003c/sup\u003e\u003csup\u003e47\u003c/sup\u003e\u003csup\u003e,\u0026nbsp;\u003c/sup\u003e\u003csup\u003e48\u003c/sup\u003e. Inflammation is one of the key features of endometriosis, and the inhibition of NLRP3 inflammasome has been associated with reduction of IL-1\u0026beta; secretion from endometrial stromal cells\u003csup\u003e49\u003c/sup\u003e\u003csup\u003e,\u0026nbsp;\u003c/sup\u003e\u003csup\u003e50\u003c/sup\u003e and mast cells\u003csup\u003e51\u003c/sup\u003e. However, the present observation that selective silencing in SCs of the NRLP3 subtype in endometriotic mice was ineffective, whereas silencing of the NRLP1 SC subtype reduced IL-1\u0026beta; release and mechanical allodynia, highlights the NRLP1 involvement in the proalgesic pathway promoted by endometriosis.\u003c/p\u003e\n\u003cp\u003eThe time course of the various soluble and cellular mediators implicated in endometriosis-induced mechanical allodynia is complex and under a strict sequential order; it encompasses the contribution of macrophages and DRG afferents but recognizes the central role of SCs. The most parsimonious hypothesis indicates as the originator of endometriosis associated pain the transient increase in C5a plasma levels, which targets the SC C5aR1. C5aR1 encodes an intracellular signaling pathway that consists in the NRLP1-dependent caspase-1 activation and the ensuing cleavage of pro IL-1\u0026beta; and IL-1\u0026beta; release. IL-1\u0026beta; increases macrophages within the nerve trunks that, \u003cem\u003evia\u003c/em\u003e their oxidative stress burst, target the neuronal TRPA1.\u003c/p\u003e\n\u003cp\u003eThe role of C5a as a soluble and diffusible pain mediator explains the epidemiological observation that other painful conditions, like migraine or fibromyalgia, coexist with endometriosis\u003csup\u003e6\u003c/sup\u003e\u003csup\u003e,\u0026nbsp;\u003c/sup\u003e\u003csup\u003e7\u003c/sup\u003e\u003csup\u003e,\u0026nbsp;\u003c/sup\u003e\u003csup\u003e9\u003c/sup\u003e\u003csup\u003e,\u0026nbsp;\u003c/sup\u003e\u003csup\u003e52\u003c/sup\u003e. The expression and the ability to promote mechanical allodynia and neuroinflammation of the C5a receptor, C5aR1, in SCs explains how endometriosis features chronic pain as a major symptom of the disease. One limitation of our study is that we documented all the cellular and molecular steps of the endometriotic inflammatory and proalgesic pathways in sciatic and trigeminal nerve trunks, implicated in fibromyalgia and migraine pain, respectively, but in contrast, due to the limited size of mesenteric nerve fibers, we could not assess the steps underlying mechanical allodynia in the abdomen, the primary site of endometriotic pain. However, functional experiments in mice with selective silencing in SCs suggest that abdominal mechanical allodynia is produced by the same cellular and molecular mechanisms identified in sciatic and trigeminal nerves. Development of therapies targeted to the cellular and molecular mediators of the C5a-C5aR1/NLRP1/IL-1\u0026beta; pathway in SCs may be a future option for the treatment of pain in endometriosis patients.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eExperimental model and content details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFemale mice were used throughout (25\u0026ndash;30\u0026thinsp;g, 5\u0026ndash;8 weeks old). The following strains of mice were used: C57BL/6\u0026thinsp;J (Charles River, RRID: IMSR_JAX:000664), B6.Cg-Tg(Plp1-CreERT)3Pop/J mice (\u003cem\u003ePlp-Cre\u003csup\u003eERT\u003c/sup\u003e\u003c/em\u003e, RRID: IMSR_JAX:005975 Jackson Laboratory) expressing a tamoxifen-inducible Cre in Schwann cells (Plp1, proteolipid protein myelin 1). Both positive and negative mice for \u003cem\u003eCre\u003csup\u003eERT\u003c/sup\u003e\u003c/em\u003e (\u003cem\u003ePlp-Cre\u003csup\u003eERT\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e+ \u003c/sup\u003eor\u003cem\u003e Plp-Cre\u003csup\u003eERT\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e-\u003c/sup\u003e (control) respectively) were treated with intraperitoneal (i.p.) 4-hydroxytamoxifen (4-OHT, 1\u0026thinsp;mg/100\u0026thinsp;\u0026mu;L in corn oil once a day consecutively for 3 days) before the infection with AAV for selective silencing of the different genes in Schwann cells.\u003c/p\u003e\n\u003cp\u003eTo selectively delete \u003cem\u003eTrpa1\u003c/em\u003e in primary sensory neurons, homozygous 129S-Trpa1\u003csup\u003etm2Kykw/J \u003c/sup\u003e(floxed Trpa1, \u003cem\u003eTrpa1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e, RRID:IMSR_JAX: 008649 Jackson Laboratory) mice were crossed with hemizygous Advillin-Cre mice (Adv-Cre). Mice positive or negative for Cre and homozygous for floxed Trpa1 (\u003cem\u003eAdv-Trpa1\u003c/em\u003e and control respectively) were used. The successful Cre-driven deletion of TRPA1 mRNA was confirmed using reverse transcription quantitative real-time PCR (RT-qPCR). Some \u003cem\u003eAdv-Cre\u003c/em\u003e\u003csup\u003e+ \u003c/sup\u003eor\u003cem\u003e Adv-Cre\u003c/em\u003e\u003csup\u003e-\u003c/sup\u003e (control) were used for the infection with AAV for selective gene silencing in primary sensory neurons.\u003c/p\u003e\n\u003cp\u003eTo evaluate the involvement of macrophages, transgenic Macrophage Fas-Induced Apoptosis (MaFIA) mice (C57BL/6-Tg(Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6)2Bck/J, stock No: 005070, RRID:IMSR_JAX:005070, Jackson Laboratories) were used. These transgenic mice express a mutant human FK506 binding protein 1A, 12kDa (FKBP12)-Fas inducible suicide/apoptotic system, driven by the mouse Csf1r promoter conjugated with a green fluorescent protein (GFP), which preferentially binds the B/B dimerizing agent (B/B-HmD, AP20187). Treatment of mice with AP20187 induces the dimerization of the suicide protein to activate the cytoplasmic FKBP12-Fas fragments, leading to the apoptosis of transgene‐expressing cells and consequent macrophage depletion \u003csup\u003e32\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe group size of n=8 mice for behavioral experiments was determined by sample size estimation using G Power [v3.1 \u003csup\u003e53\u003c/sup\u003e] to detect the size effect in a \u003cem\u003epost-hoc\u003c/em\u003e test with type 1 and 2 error rates of 5% and 20%, respectively. Allocation concealment of mice into the vehicle(s) or treatment groups was performed using a randomization procedure (http://www.randomizer.org/). The assessors were blinded to the identity of the animals (genetic background) or allocation to treatment groups. None of the animals were excluded from the study. Mice were housed in a temperature- and humidity-controlled \u003cem\u003evivarium\u003c/em\u003e (12 h dark/light cycle, free access to food and water, 5 animals per cage). At least 1 h before behavioral experiments, mice were acclimatized to the experimental room and behavior was evaluated between 9:00 am and 5:00 pm. Animals were anesthetized with a mixture of ketamine and xylazine (90 mg/kg and 3 mg/kg, respectively, i.p.) and euthanized with inhaled CO\u003csub\u003e2\u003c/sub\u003e plus 10-50% O\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003eAll behavioral experiments followed Animal Research: Reporting of \u003cem\u003eIn Vivo\u003c/em\u003e Experiments (ARRIVE) guidelines and were in accordance with European Union (EU) guidelines for animal care procedures and the Italian legislation (DLgs 26/2014) application of the EU Directive 2010/63/EU. The study was approved by the National Committee for the Protection of Animals used for Scientific Purposes of the Italian Ministry of Health (research permits # 452/2020-PR).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman samples \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe use of blood plasma samples collected from human individuals diagnosed with endometriosis and healthy control was approved by the Local Ethics Committee of the Florence University Hospital (Area Vasta Toscana Centro, CEAVC) (EndoTRP study, 15211.oss and subsequent amendments), according to the Helsinki Declaration and good clinical practice guidelines, and all patients gave their informed consent. Participants did not receive any form of compensation. The blood plasma samples derived from nineteen endometriosis diagnosed patients [female, median age 30 years (range 22-43)] and fifteen healthy individuals [female, median age 27 years (23-42)]. Considering the exploratory nature of the blood sampling analyses, the sample size was not determined based on statistical considerations but enrolling all consecutive outpatients and healthy controls.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMouse Schwann cells (MSC).\u003c/strong\u003e MSC were isolated from sciatic nerves of C57BL/6J mice \u003csup\u003e54\u003c/sup\u003e. Briefly, the \u003cem\u003eepineurium\u003c/em\u003e was removed, and nerve explants were cut into 1 mm segments and enzyme-dissociated in Hank's Balanced Salt Solution (HBSS, 2 hr, 37 \u0026deg;C) added with collagenase (0.05%) and hyaluronidase (0.1%). Cells were collected by centrifugation (150xg, 10 min, room temperature RT) and the pellet was resuspended and cultured in Dulbecco's Modified Eagle Medium (DMEM) containing fetal calf serum (10%), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 mg/ml), neuregulin (10 nM) and forskolin (2 \u0026mu;M). Cytosine arabinoside (Ara-C, 10 mM) was added three days later, to remove fibroblasts. The culture medium was replaced every 3 days. Cells were cultured at 37 \u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e and 95% O\u003csub\u003e2\u003c/sub\u003e for 15 days before experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman Schwann cells (HSCs).\u003c/strong\u003e HSCs (#P10351; Innoprot) were grown in Schwann cell medium (#P60123, Innoprot) at 37\u003csup\u003e◦\u003c/sup\u003eC with 5 % CO\u003csub\u003e2\u003c/sub\u003e and 95 % O\u003csub\u003e2\u003c/sub\u003e and discarded and replaced after 12 passages \u003csup\u003e55\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe murine macrophage cell lineage, RAW 264.7 (#TIB-71\u0026trade;; ATCC, RRID: CVCL_0493), was cultured and maintained in DMEM supplemented with FBS (10%), penicillin (100 U/mL), streptomycin (100 mg/mL) and L-glutamine (2 mM) at 37 ◦C in 5% CO\u003csub\u003e2\u003c/sub\u003e and 95% O\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003eThe human monocytic cell line, U937 (#CRL-1593.2\u0026trade;; ATCC, RRID:CVCL_0007) was maintained at 37 ◦C, 5% CO\u003csub\u003e2\u003c/sub\u003e and 95% O\u003csub\u003e2 \u003c/sub\u003ein Roswell Park Memorial Institute (RPMI-1640) medium supplemented with heat-inactivated FBS (10 %), L-glutamine (2 mM), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; 10 mM), and sodium pyruvate (1 mM). The differentiation of U937 cells (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mL) towards macrophage-like lineage was promoted by the exposure to 200 ng/mL of phorbol 12-myristate 13- acetate (PMA) for 2 days before experiments \u003csup\u003e56\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAAVpro293T cell line. \u003c/strong\u003eAAVpro 293T cells (#632273, Takara, RRID:CVCL_B0XW), were maintained in DMEM high glucose supplemented with 10% heat inactivated FBS, 4 mM L-glutamine, 1 mM penicillin/streptomycin and 1 mM sodium pyruvate at 37 \u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e and 95% O\u003csub\u003e2\u003c/sub\u003e. The day before transfection, cells were plated in DMEM supplemented with 2% FBS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInduction of endometriosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEndometriosis was induced in mice as previously reported \u003csup\u003e57\u003c/sup\u003e. Briefly, donor mice, received a subcutaneous injection of estradiol benzoate (3 \u0026mu;g/100\u0026thinsp;\u0026mu;L) to stimulate the endometrium growth. Seven days later, the uteri of the donor mice were dissected and divided longitudinally. Uterine horns from each donor mouse were minced in tissue fragment smaller than 1 mm in Hank\u0026rsquo;s balanced salt solution (HBSS). Each dissociated uterine horn was then injected intraperitoneally (50 mg/500 \u0026mu;L i.p.) in recipient mice. One donor mouse was used for every 2 endometriosis mice. Sham mice received an intraperitoneal injection of 500 \u0026mu;L of HBSS. Behavioral experiments were performed during a 28-day period. After 28 days from the injection of the endometrium suspension, mice were sacrificed, and tissues collected. Lesion implantation was also quantified in a time-course manner by counting the number of lesions in endometriotic mice at day 7, 14, 21, and 28.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTreatment protocols \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIf not otherwise indicated, all reagents were obtained from Merck Life Science SRL. Mice received systemic administration of C5a complement antagonist (DF2593A, 1 mg/kg, intragastric, i.g.) or vehicle (0.5% carboxymethyl cellulose, CMC) twice a day, by two different time schedules: from one day before the endometriotic tissue injection to day 6 or from day 12 to day 16.\u003c/p\u003e\n\u003cp\u003eAnti-IL1\u0026beta; (#BE02463, clone B122, Bio X Cell, RRID:AB_2687727) monoclonal antibody (mAb) or IgG isotype control were administered (50 \u0026micro;g/200 \u0026mu;L, i.p.) at day 8 and day 14 after endometriosis/sham induction. MaFIA mice were treated with B/B homodimerizer (AP20187, 2 mg/kg, i.p., once a day) or vehicle (10% PEG-400, 1.7% tween 80 in 0.9% NaCl) by three different time schedules: from day -2 to day 2; from day 2 to day 6, and from day 21 to day 25. \u003cem\u003ePlp-Cre\u003csup\u003e+\u003c/sup\u003e\u003c/em\u003e and control mice were infected with an intravenous (1 mL/kg, 1x10\u003csup\u003e12\u003c/sup\u003e v/g, i.v.) injection of different AAVs. \u003cem\u003eAdv-Cre\u003csup\u003e+\u003c/sup\u003e\u003c/em\u003e and control mice were infected with an intrathecal (1x10\u003csup\u003e12\u003c/sup\u003e v/g, i.th., 5 μL) injection of AAV. Animals were used 3 weeks after AAVs infection. Sciatic and trigeminal nerve tissues were harvested for evaluating AAVs infection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBehavioral assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbdominal Mechanical allodynia (AMA).\u003c/strong\u003e The mechanical pain sensitivity of the abdominal region was evaluated with von Frey filaments by methods described previously \u003csup\u003e58\u003c/sup\u003e . Mice were habituated in small compartments on a perforated grid for 1 h before the test. Subsequently von Frey filaments were applied to the abdomen (between diaphragm and genitals). The test was performed by a trained blinded observer. The individual filaments were tested in an ascending order covering 0.008, 0.04, 0.16, and 0.4 g forces. Each force was applied 10 times to the abdominal surface. The maximal duration of each force application was 2 s, and the inter-stimulus interval was 2\u0026ndash;3 min. Following each challenge, the withdrawal response was quantified either as 1 (withdrawal of abdominal wall, licking or retraction of animal) or 0 (no response). All counts in response to an individual filament were averaged. Withdrawal responses to low forces reflect high mechanical pain sensitivity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePeriorbital mechanical allodynia (PMA). \u003c/strong\u003ePeriorbital mechanical allodynia (PMA) was assessed using the up-down paradigm \u003csup\u003e59\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e60\u003c/sup\u003e. Briefly, mice were placed in a restraint apparatus designed for the evaluation of periorbital mechanical thresholds \u003csup\u003e61\u003c/sup\u003e. PMA was evaluated in the periorbital region over the rostral portion of the eye (i.e., the area of the periorbital region facing the sphenoidal rostrum) \u003csup\u003e62\u003c/sup\u003e before (basal threshold) and after treatments. On the day of the experiment, after 20 min of adaptation inside the chamber, a series of 7 von Frey filaments in logarithmic increments of force (0.02, 0.04, 0.07, 0.16, 0.4, 0.6 and 1.0 g) were applied to the periorbital area perpendicular to the skin, with sufficient force to cause slight buckling, and held for approximately 5 s to elicit a positive response. The response was considered positive by the following criteria: mouse vigorously stroked its face with the forepaw, head withdrawal from the stimulus, or head shaking. Mechanical stimulation started with the 0.16 g filament. Absence of response after 5 s led to the use of a filament with increased force, whereas a positive response led to the use of a weaker (i.e., lighter) filament. Six measurements were collected for each mouse or until four consecutive positive or negative responses occurred. The 50% mechanical withdrawal threshold (expressed in g) was then calculated from these scores by using a \u0026delta; value of 0.205, previously determined \u003csup\u003e62\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHindpaw Mechanical allodynia (HMA). \u003c/strong\u003ePaw mechanical allodynia was evaluated by measuring the paw withdrawal threshold by using the up-down paradigm \u003csup\u003e59\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e63\u003c/sup\u003e. Mice were acclimatized (1 h) in individual clear plexiglass boxes on an elevated wire mesh platform, to allow for access to the plantar surfaces of the hind paws. von Frey filaments of increasing stiffness (0.07, 0.16, 0.4, 0.6, and 1.0, 1.4 and 2 g) were applied to the hind paw plantar surfaces of mice with enough pressure to bend the filament. The absence of a paw being lifted after 5 s led to the use of the next filament with an increased force, whereas a lifted paw indicated a positive response, leading to the use of a subsequently weaker filament. Six measurements were collected for each mouse or until four consecutive positive or negative responses occurred. The 50% mechanical withdrawal threshold (expressed in g) was then calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmid construction \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll short hairpin RNAs were designed according to Vector builder and RNAi Consortium of Broad Institute libraries. pAAV[FLEXon]-CMV\u0026gt;EGFP:LL:rev(mCherry:miR30-mC5ar1[shRNA#1]):rev(LL):WPRE and pAAV[FLEXon]-CMV\u0026gt;LL:rev(EGFP:5' miR-30E:{shRNA-NLRP1a}:3' miR-30E):EGFP:5' miR-30E:{shRNA-NLRP1b}:3' miR-30E:rev(LL):WPRE were used. pAAV[FLEXon]-CMV\u0026gt; EGFP-LL-rev(mCherry)-mNLRP3[mir30-shRNA]-rev(LL)-WPRE was generated in two steps. In the first step two overlapping fragments, were obtained by amplifying regions from pAAV[FLEXon]-CMV\u0026gt;EGFP-LL-rev(mCherry)-mC5ar1 [mir30-shRNA]-rev(LL)-WPRE with P1/P4 and P2/P3 primer couples. P1 and P2 were designed to include partial shNlrp3 overlapping regions. A third PCR, using external primers P3/P4 generated the extended fragment, that was cloned (AgeI/SpeI) into pAAV[Exp]-CMV\u0026gt;EGFP-LL-rev(mCherry)- mC5ar1 [mir30-shRNA]-rev(LL)-WPRE. To obtain pAAV[Exp]-CMV\u0026gt;EGFP-LL-rev(mCherry)-mIL-1\u0026beta;[mir30-shRNA]-rev(LL)-WPRE two oligonucleotides (P5 and P6) containing half short hairpin sequence were used to amplify a fragment from the pAAV[Exp]-CMV\u0026gt;EGFP:LL:rev(mCherry:miR30-mC5ar1[shRNA#1]):rev(LL):WPRE. Purified product was phosphorylated with T4 PNK (NEB, #M0201S) according to the manufacturer instructions and finally subjected to circular ligation with a T4 ligase (Thermo Fisher #K1423). All the new-generated plasmids were validated by Sanger sequencing. Primer sequences are reported in Supplementary table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAAV Generation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRecombinant AAV particles (rAAVs) were produced by using polyethylenimine (#23966, PEI, Polyscience) triple transfection strategy as previously described \u003csup\u003e55\u003c/sup\u003e. In brief, AAVpro 293T cells (#632273, Takara, RRID:CVCL_B0XW), were transfected with a 1:3 ratio of DNA:PEI \u003csup\u003e64\u003c/sup\u003e. To obtain rAAVs, AAVpro 293T cells were transiently transfected with 2.5 mg total DNA (plasmid expressing genes of interest, pAdDeltaF6; (#112867, Addgene) and Rep/Cap, 1:1:1 molar ratio). To infect with high efficiency Schwann cells and primary sensor neurons, Rep/Cap 2/rh10 or 2/9n were used (pAAV2/rh10 #112866 or pAAV 2/9n #112865, respectively Addgene). rAAVs virions were extracted 72 h post-transfection, purified by iodixanol gradient ultracentrifugation, concentrated, and titrated using a RT-qPCR assay (#6233 AAVpro Titration Kit, Takara) according to the manufacturer instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAAV production and cell lysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDetailed method is reported in Supplementary information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIodixanol-based purification protocol\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDetailed method is reported in Supplementary information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDetailed method is reported in Supplementary information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein extraction and western immunoblot assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDetailed method is reported in Supplementary information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProteome profiler array \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMouse plasma samples were collected at different days after endometriosis induction for a cytokine array analysis using the Proteome Profiler Mouse Cytokine Array Kit, Panel A (#ARY006. R\u0026amp;D Systems) according to manufacturer\u0026rsquo;s instructions. Signal was developed using an imaging system (ChemiDoc; Bio-Rad). The density of specific cytokine dots was measured using HLImage\u003csup\u003e++\u003c/sup\u003e (v. PCM.25.5.1.a).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCaspase-Glo\u0026reg; 1 Inflammasome Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe caspase-1 activity was evaluated using the bioluminescent based assay Caspase-Glo\u0026reg; 1 inflammasome assay (#TM456, Promega) according to manufacturer\u0026rsquo;s instructions. Briefly, HSCs were grown on in 96-well poly-L-lysine-coated (8.3 \u0026mu;M) plates and incubated with C5a (100 ng/ml) in the presence of DF2593A (1 \u0026micro;M), Ac-YVAD-CHO (1\u0026micro;M) or veh (0.001 % DMSO) at 37 \u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e and 95% O\u003csub\u003e2\u003c/sub\u003e. After 16 h, the cell culture medium was collected and transferred to a 96-white, opaque-walled multiwell plates (with a clear bottom) (#6005181, PerkinElmer) and incubated with Caspase-Glo\u003csup\u003e\u0026reg;\u003c/sup\u003e 1 Reagent for 60 min. The luminescence signal was measured by FlexStation3 Multi-Mode Microplate Reader (Molecular Devices;) using SoftMax\u0026reg; Pro7 software (Molecular Devices). Results were expressed as relative light units (RLU).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCaspase-1 \u003cem\u003ein vitro\u003c/em\u003e imaging\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genetically encoded fluorescent sensor Single-Cell Imaging of Caspase-1 (SCAT1) based on fluorescence resonance energy transfer (FRET) was used to detect the inflammasome-mediated caspase-1 activation in HSCs. Briefly, HSCs were plated on 96-well poly-L-lysine-coated (8.3 \u0026mu;M) black clear bottom (5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well; #6055302 PerkinElmer) and transfected with cDNA of SCAT1 (130-300 ng) using jetOPTIMUS\u0026reg; DNA transfection reagent (#55-250, Polyplus) for 16-24 h at 37 \u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e and 95% O\u003csub\u003e2\u003c/sub\u003e. On the day of experiments, HSCs were washed and added with HBSS at pH 7.4 at 37 \u0026deg;C and transferred to a chamber on the stage of a fluorescent microscope for recording (Axio Observer 7; with a fast filter wheel and Digi-4 lens to record excitations and Ultra-fast Sutter Lambda DG4 Xenon excitation source-range 300-700 nM) (Zeiss) with 20x objective. Cells were exposed to C5a (10 \u0026micro;g/ml) in the presence of DF2593A (1\u0026micro;M) or Ac-YVAD-CHO (1 \u0026micro;M) vehicle (0.001% DMSO). FRET changes were measured as a ratio of the acceptor fluorophore emission (530 nm) to donor emission (435 nm). The \u0026Delta;F/F0 ratio was calculated for each experiment and the results were expressed as the AUC after inverting the curve.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC5a and IL-1\u0026beta;\u003c/strong\u003e\u003cstrong\u003e assays \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC5a and IL-1\u0026beta; content was assayed in mouse sciatic and trigeminal nerve tissue homogenates and in mouse plasma samples using a single-analyte enzyme-linked immunosorbent assay (ELISA) kit (#ab193718 and #ab197742, Abcam, Cambridge, UK) according to the manufacturer\u0026rsquo;s protocol. Data are expressed as pg/mg of protein and pg/mL. C5a and IL-1\u0026beta; content was assayed in human plasma samples and HSCs cultured medium by ELISA kit (#ab193695 and #ab214025, Abcam) according to the manufacturer\u0026rsquo;s protocol. Data are expressed as pg/mL\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReverse transcription-quantitative real-time PCR (RT-qPCR) \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDetailed method and the sets of primers for HSCs and MSCs are reported in Supplementary information and Supplementary Table S2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrans-well migration assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNoncontact coculture transwell cell culture system between macrophages and Schwann cells was obtained using the Boyden migration assay \u003csup\u003e56\u003c/sup\u003e. Briefly, the noncontact cocultured cells were prepared as follows: macrophage-like U937 cells or RAW 264.7 were seeded at a density of 2.5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e / chamber into the upper of a 24-well transwell cell culture system (6.5 mm in diameter, with 8-\u0026mu;m pores, (#CLS3458 \u0026trade;; Corning) by using the complete media described above and allowed to grow 2 days after PMA treatment (U937 cells) or overnight (RAW 264.7) before migration assay \u003csup\u003e65\u003c/sup\u003e. In the meantime, HSCs and MSCs were plated on a 24-well plate. On the day of the experiment, all the cells were replaced with serum free media, and the macrophage-like U937 cells and RAW 264.7 cells cultured on the membrane of 24-well transwell insert were placed into the 24-well plate cultures containing the HSCs and MSCs to initiate the experiment. HSCs and MSCs were treated with C5a (100 ng/ml) also in the presence of DF2593A (1 \u0026micro;M) or veh (0.001 % DMSO) or in the presence mAb-IL-1\u0026beta; (1 \u0026micro;g/ml) or IgG control (1 \u0026micro;g/ml). After 16 h of incubation, migrated cells on the lower surface of the filter were fixed and stained with Diffquick staining set following the manufacturer\u0026rsquo;s directions (#726443\u0026trade;), and nonmigratory cells on the upper surface of the filter were wiped with a cotton swab. Random fields were counted under a light microscope using a 20x objective (1.5 x 10\u003csup\u003e5\u003c/sup\u003e \u0026micro;m\u003csup\u003e2\u003c/sup\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are presented as mean \u0026plusmn; SEM. For multiple comparisons, a one-way ANOVA followed by a post-hoc Bonferroni\u0026rsquo;s test was used. The two groups were compared using Student\u0026rsquo;s t-test. For behavioral experiments with repeated measures, a two-way mixed-model ANOVA followed by a post-hoc Bonferroni\u0026rsquo;s test was used. Statistical analyses were performed on raw data using GraphPad Prism 8 (GraphPad Software Inc.). P-values less than 0.05 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were considered significant. EC50 values were determined from non-linear regression models using GraphPad Prism 8. The statistical tests used and sample size for each analysis are shown in the Fig. legends. \u003cstrong\u003e\u003cbr /\u003e\u003c/strong\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMaterials and data generated from this study are available upon request from Francesco De Logu ([email protected]) or Romina Nassini ([email protected]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupported by grants from: Fondazione Telethon (Grant no GMR22T1070) (F.D.L), European Research Council (ERC) under the European Union\u0026rsquo;s Horizon 2020 research and innovation programme (grant agreement No. 835286) (P.G.), Ministero della Salute GR-2018-12368352 (Bando Salute 2018) (V.S.), European Union - Next Generation EU, National Recovery and Resilience Plan, Mission 4 Component 2 - Investment 1.4 - National Center for Gene Therapy and Drugs based on RNA Technology - CUP B13C22001010001 (R.N.) and NEXTGENERATIONEU (NGEU) funded by the Ministry of University and Research (MUR), National Recovery and Resilience Plan (NRRP), project MNESYS (PE0000006) \u0026ndash; A Multiscale integrated approach to the study of the nervous system in health and disease (DR. 1553 11.10.2022) (P.G., F.D.L.). Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or the European Commission. Neither the European Union nor the European Commission can be held responsible for them.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.T., L.L., D.S.M.de A., M.M., V.S., M.C., P.P., M.Mo., G.D.S., B.P., S.V., L.F.I., G.B., E.B., I.S., A.M., M.T., M.Di T., F.P., P.G., R.N., F.D.L. designed and conducted the experiments, performed the analysis, discussed the results, and wrote the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eR.N., F.D.L. and P.G. are founding scientists of FloNext Srl. G.B. is fully employed at FloNext Srl, Italy. 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Although the imbalance of proinflammatory mediators, including the complement component C5a, has been implicated in endometriosis-associated pain symptoms, the mechanisms causing widespread pain and the role of C5a remain unclear. Female mice with endometriotic lesions displayed widespread pain and increased plasma C5a levels, similarly, observed in women with endometriosis. We hypothesized Schwann cells involvement in endometriotic pain. Silencing the C5a surface receptor (C5aR1) in mouse Schwann cells abolished C5a-induced activation of NLRP1 inflammasome and the ensuing interleukin-1β (IL-1β) release. IL-1β, from Schwann cells, recruited macrophages in sciatic/trigeminal nerve trunks. Macrophages induced oxidative stress, targeting proalgesic TRPA1, causing widespread mechanical allodynia. This pathway, initiated by C5aR1, engages Schwann cell signaling, including NLRP1/IL-1β/macrophages/oxidative stress/TRPA1, sustaining pain in an endometriosis mouse model.\u003c/p\u003e","manuscriptTitle":"Schwann Cell C5aR1 co-opts Inflammasome NLRP1 to Sustain Pain in a Mouse Model of Endometriosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-15 05:13:55","doi":"10.21203/rs.3.rs-4045626/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d0078e86-e72e-4479-8c97-a1f11e92f829","owner":[],"postedDate":"April 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":30600764,"name":"Health sciences/Health care/Therapeutics/Pain management"},{"id":30600765,"name":"Health sciences/Medical research/Experimental models of disease"},{"id":30600766,"name":"Health sciences/Medical research/Translational research"},{"id":30600767,"name":"Biological sciences/Cell biology/Mechanisms of disease"},{"id":30600768,"name":"Health sciences/Medical research/Preclinical research"}],"tags":[],"updatedAt":"2024-11-26T08:07:40+00:00","versionOfRecord":{"articleIdentity":"rs-4045626","link":"https://doi.org/10.1038/s41467-024-54486-6","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2024-11-25 05:00:00","publishedOnDateReadable":"November 25th, 2024"},"versionCreatedAt":"2024-04-15 05:13:55","video":"","vorDoi":"10.1038/s41467-024-54486-6","vorDoiUrl":"https://doi.org/10.1038/s41467-024-54486-6","workflowStages":[]},"version":"v1","identity":"rs-4045626","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4045626","identity":"rs-4045626","version":["v1"]},"buildId":"WvIrzKhiLBfengagbw6Ux","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}