The IL33 receptor ST2 contributes to mechanical hypersensitivity in mice with neuropathic pain

(2021) 14:35 Huang et al. Mol Brain https://doi.org/10.1186/s13041-021-00752-3 Open Access MICRO REPORT The IL33 receptor ST2 contributes to mechanical hypersensitivity in mice with neuropathic pain Junting Huang1,2*, Vinicius M. Gadotti2, Zizhen Zhang2 and Gerald W. Zamponi2 Abstract Pathogen infection triggers pain via activation of the innate immune system. Toll-like receptors (TLRs) and Nodlike receptors (NLRs) are the main components of innate immunity and have been implicated in pain signaling. We previously revealed that the TLR2-NLRP3-IL33 pathway mediates inflammatory pain responses during hyperactivity of innate immunity. However, their roles in neuropathic pain had remained unclear. Here we report that although knockout of TLR2 or NLRP3 does not affect spared nerve injury (SNI)-induced neuropathic pain, intrathecal inhibition of IL33/ST2 signaling with ST2 neutralizing antibodies reverses mechanical thresholds in SNI mice compared to PBS vehicle treated animals. This effect indicates a universal role of IL33 in both inflammatory and neuropathic pain states, and that targeting the IL33/ST2 axis could be a potential therapeutic approach for pain treatment. Keywords: TKR2, NLRP3, Pain, Spared nerve injury, IL33 Pathogen infection initiates host defense via activation of the pattern recognition receptors such as Toll-like receptors (TLRs) and nod-like receptors (NLRs) in the innate immune system [1–3]. This process typically induces a pain response as alarming signals to prevent further tissue damage or potential injury. Moreover, a number of different TLRs and several NLRs have been implicated in various chronic pain states [4–8], indicating an intimate interaction between innate immunity and pain. We have recently reported that activation of innate immunity through intraplantar injection of either Complete Freund’s Adjuvant (CFA), or the specific TLR2/6 heterodimer ligand FSL1 triggers pain responses by increasing interleukin33 (IL33) levels in both the paw and the dorsal root ganglia (DRG) [9]. In both cases, blocking the IL33 receptor ST2 via intrathecal delivery of *Correspondence: 1 Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, Guangdong, China Full list of author information is available at the end of the article functional neutralizing antibodies mediated analgesia. The responses to CFA and FSL1 were abolished in TLR2 receptor null mice, whereas NLRP3 null mice continued to exhibit mechanical hypersensitivity in responses to CFA, but not FSL1 [9]. Furthermore, we showed that IL33 was a key factor in priming inflammatory pain responses. Collectively, our findings suggested that that IL33 is a critical mediator of acute and chronic inflammatory pain states. Importantly, we did not observe sexual dimorphism with regard to this pathway. Previous literature has revealed that IL33 is involved in mouse models of neuropathic pain [10, 11]. We therefore asked whether TLR2, NLRP3 and IL33 signaling are important for the development of chronic pain resulting from peripheral nerve injury. We first examined the importance of TLR2 and NLRP3 in the development of mechanical hyperalgesia resulting from spared nerve injury (SNI) of the sciatic nerve in male mice. Wild type mice, TLR2 null mice and NLRP3 null mice were subjected to SNI as previously described, and mechanical paw withdrawal thresholds were © The Author(s) 2021. 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The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Huang et al. Mol Brain (2021) 14:35 Page 2 of 4 Fig. 1 Role of TLR2, NLRP3 and IL33 in SNI induced mechanical hypersensitivity in mice. a Mechanical paw withdrawal thresholds in male WT, TLR2−/− and NLRP3−/− mice subjected to SNI (n = 6–8). Data are presented as mean ± S.E.M, ****P < 0.0001, One-way ANOVA with Bonferroni’s correction. b Effect of ST2 neutralizing antibody on paw withdrawal thresholds in nerve injured male mice (n = 6). Baseline was measured 14 days after injury, and PBS or ST2 antibody were delivered on days 15, 17, and 19. Data are presented as mean ± S.E.M, *P < 0.05, ***P < 0.001, ****P < 0.0001. Two-way ANOVA with Bonferroni’s post-hoc test (i.t: intrathecal). c Effect of intrathecal delivery of the ST2 receptor neutralizing antibody on mechanical withdrawal threshold in female SNI mice compared to a PBS control solution. Data were acquired on day 14 (baseline—no drugs) and 15 (testing day—PBS or ST2 antibody) after nerve injury. *P < 0.05 (ANOVA). d Schematic representation of pathways or IL33 induced pain signaling in response to FSL1, CFA and SNI Huang et al. Mol Brain (2021) 14:35 examined [12, 13]. Briefly, mice were individually placed in a plexiglass chamber over a wire mesh floor and were habituated in chambers for a minimum of one hour prior to testing, and mechanical paw withdrawal threshold was measured using a digital plantar aesthesiometer (DPA, UgoBasile, Varese, Italy). The DPA was placed under the hind paw to allow direct stimulation of the plantar surface with the filament. Withdrawal thresholds were examined 3–5 times with interceding intervals. Compared to sham operated animals, WT mice subjected to SNI showed a significant drop in mechanical withdrawal thresholds when assessed 14 days after nerve injury (Fig. 1a). In contrast, TLR2 null mice and NLRP3 null mice still displayed mechanical hypersensitivity that was statistically indistinguishable from that of WT animals (Fig. 1a). Hence, we conclude that neuropathic pain requires neither the activation of TLR2 receptors nor the NLRP3 inflammasome, in contrast to what is observed with inflammatory pain states [9]. We then tested whether IL33 signaling at the spinal/DRG level is involved in neuropathic pain states. For this purpose, we performed SNI or sham operations, and then 14 days later we measured a baseline of paw withdrawal thresholds, followed by repeated treatment of ST2 receptor neutralizing antibody or PBS control via intrathec (...truncated)