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NOD-like receptor protein 3 inflammasome drives postoperative mechanical pain in a sex-dependent manner

Cowie, Ashley M.a; Menzel, Anthony D.a; O'Hara, Crystala; Lawlor, Michael W.b,c; Stucky, Cheryl L.a,*

doi: 10.1097/j.pain.0000000000001555
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Postoperative pain management continues to be suboptimal because of the lack of effective nonopioid therapies and absence of understanding of sex-driven differences. Here, we asked how the NLRP3 inflammasome contributes to postoperative pain. Inflammasomes are mediators of the innate immune system that are responsible for activation and secretion of IL-1β upon stimulation by specific molecular signals. Peripheral IL-1β is known to contribute to the mechanical sensitization induced by surgical incision. However, it is not known which inflammasome mediates the IL-1β release after surgical incision. Among the 9 known inflammasomes, the NLRP3 inflammasome is ideally positioned to drive postoperative pain through IL-1β production because NLRP3 can be activated by factors that are released by incision. Here, we show that male mice that lack NLRP3 (NLRP3KO) recover from surgery-induced behavioral and neuronal mechanical sensitization faster and display less surgical site inflammation than mice expressing NLRP3 (wild-type). By contrast, female NLRP3KO mice exhibit minimal attenuation of the postoperative mechanical hypersensitivity and no change in postoperative inflammation compared with wild-type controls. Sensory neuron-specific deletion of NLRP3 revealed that in males, NLRP3 expressed in non-neuronal cells and potentially sensory neurons drives postoperative pain. However, in females, only the NLRP3 that may be expressed in sensory neurons contributes to postoperative pain where the non-neuronal cell contribution is NLRP3 independent. This is the first evidence of a key role for NLRP3 in postoperative pain and reveals immune-mediated sex differences in postoperative pain.

Males require non-neuronal and neuronal NLRP3 for both postoperative inflammation and mechanical hypersensitivity. In females, inflammation does not require NLRP3, and sensory neuron-specific NLRP3 marginally contributes to mechanical hypersensitivity.

aDepartment of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI, United States

bDivision of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, Milwaukee, WI, United States

cNeuroscience Research Center, Medical College of Wisconsin, Milwaukee, WI, United States

Corresponding author. Address: Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226, United States. Tel.: (414) 955-8373; fax: 414-456-6517. E-mail address: cstucky@mcw.edu (C.L. Stucky).

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (www.painjournalonline.com).

Received October 08, 2018

Received in revised form February 07, 2019

Accepted February 14, 2019

Online date: March 8, 2019

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1. Introduction

Postoperative pain is a significant worldwide problem. Approximately 234.2 million people across the globe undergo major surgeries each year.135 Approximately 80% of surgical patients experience acute postoperative pain. Of these patients, 74% continue to experience extreme pain after discharge and 10% to 50% develop chronic postoperative pain.21,41 A number of factors predispose patients to develop chronic postoperative pain including the severity of acute pain the first week after surgery21,40 and the sex of the patient.67 A better understanding of the mechanisms that cause acute postoperative pain is needed to develop therapies that decrease the prevalence and severity of chronic postoperative pain and increase the quality of life of surgical patients. Acute postoperative pain has both inflammatory and neuropathic qualities that stem from the tissue damage and severing of nerve endings at the surgical site. Current postoperative analgesics often target either the inflammatory or neuropathic component of postoperative pain, but not both.100 However, literature suggests targeting both neuropathic and inflammatory components may be more effective.51,70,100,103

Secreted interleukin-1β (IL-1β) is a crucial mediator of the inflammatory response74 and plays a key role in both neuropathic47,113 and inflammatory60,61,77,79,106 pain. Peripherally generated IL-1β is required for the induction of inflammation and sensitizes sensory neurons to mechanical stimuli.9,38 Postoperative patients have inflammation and mechanical hypersensitivity near the incision site.39,100 In addition, it has been shown that inhibition of IL-1β signaling at the surgical wound site decreases postoperative pain in mice and humans.60,61,107 However, the molecular sources of postoperative IL-1β are unknown. Furthermore, despite growing evidence that suggests significant sex differences exist in different types of pain is a result of distinct immune mechanisms,34,35,72,102,117,118 immune-driven sex differences have not been investigated in postoperative pain.

Inflammasomes are cytosolic receptors of the innate immune system and when activated result in IL-1β secretion. Nothing is known regarding inflammasome-specific contributions to postoperative pain. Activation of the NOD-like receptor protein 3 (NLRP3) inflammasome requires 2 signals: one signal primes the cell to express NLRP3 and pro–IL-1β, and the second signal induces inflammasome assembly and activation.31,45,55,56,74,111 Many signaling factors known to activate the NLRP3 inflammasome, such as ATP, damaged extracellular matrix components, and reactive oxygen species are released after tissue damage like that which occurs during surgical incision.1,59,127,142 Furthermore, expression of the NLRP3 inflammasome itself is upregulated in both neuropathic and inflammatory pain models including sciatic nerve ligation,95 chronic constriction sciatic nerve injury,44,125 oxaliplatin-induced nerve injury,132 paclitaxel-induced nerve injury,64 fibromyalgia,14,25,26 and inflammation of the dura.23 Therefore, it is plausible that the NLRP3 inflammasome may drive and maintain postoperative pain. As all of these pain-related studies on the NLRP3 inflammasome were performed in one sex, it is not known whether NLRP3 is differentially involved in male and female pain. Therefore, the goal of this study is to determine whether NLRP3 contributes to postoperative pain and tissue inflammation in a sex-dependent manner.

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2. Methods

2.1. Mouse lines and animal care

All animal procedures were performed in accordance with the National Institute of Health guidelines and approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin (AUA #0383). Mice were group housed with ad libitum access to food and water in a 14:10 light/dark cycle. Adult C57BL/6J (The Jackson Laboratory, Bar Harbor, ME) mice were used as control mice that normally express NLRP3 (wild-type [WT]), and B6N.129-Nlrp3tm3Hhf/J mice (The Jackson Laboratory) were used as a constitutive knockout of NLRP3 (NLRP3KO). To generate sensory neuron-specific NLRP3, conditional knockout mice (NLRP3AdvCre+) and NLRP3AdvCre− littermates C57BL/6-Nlrp3tm1.1 Mrl (Taconic Biosciences, Rensselaer, NY) mice were mated with B6;129P2-Aviltm2(cre)Fawa/J (The Jackson Laboratory)116,147 mice. This Advillin-Cre mouse line was used because Advillin is a marker for sensory neurons within the dorsal root ganglion (DRG)53,147 where it is expressed in ∼80% of sensory neurons.93,104 In addition, an extensive report of Advillin expression demonstrates that it is expressed by dorsal habenula of the epithalamus, endocrine cells of the gut, Merkel cells in the skin, and the autonomic nervous system, but, importantly, not in immune cells.62 Both male and freely cycling female mice were used for studies. Sex differences existed; thus male and female data were kept separate, and sex is noted for each experiment. For all behavior, molecular biology, and electrophysiological studies, mice were aged 8 to 16 weeks.

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2.2. Postoperative pain model

Skin and muscle plantar incision was performed as previously described.6,7,13,27,101 Animals were anesthetized briefly with inhaled isoflurane and then with 1.5% isoflurane throughout surgery. A number-11 blade scalpel was used to make a cutaneous, longitudinal 5-mm incision starting 2 mm from the proximal edge of the heel to the distal end of the paw. The plantaris muscle was elevated with curved forceps and incised longitudinally through the belly of the muscle. The wound was closed with 2 sutures, and bacitracin cream was applied to the surgical site. When the contralateral paw was used for experiments, bacitracin was applied as a control. Sham mice were anesthetized in the same manner but were not incised, and bacitracin was applied to the plantar hindpaw.

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2.3. Isolation and culture of dorsal root ganglion neurons

Mice were briefly anesthetized through inhaled isoflurane and decapitated. Ipsilateral lumbar 3 to 6 DRGs were rapidly removed from sham, POD1, POD4, and POD7 mice and placed in cold phosphate-buffered saline (PBS). Phosphate-buffered saline was removed from DRGs, and Dulbecco modified Eagle medium/Ham F12 medium (DMEM) containing 10-mg/mL collagenase was added and incubated for 40 minutes. Next, DMEM + collagenase was removed, and DRG were incubated with DMEM containing 0.5% trypsin for 45 minutes. Mechanical dissociation was used to break up the DRG neurons and plate them onto laminin-coated glass coverslips. Then, DRG neurons were allowed to adhere to the coverslip overnight in DMEM that contained 10% heat-inactivated horse serum, 2-mmol/L L-glutamine, 1% glucose, 100-units/mL penicillin, and 100-μg/mL streptomycin 146.

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2.4. RNA isolation and cDNA synthesis

Injured and sham mice were anesthetized with inhaled isoflurane and decapitated. Lumbar 3 to 6 DRG and peri-incisional plantar hindpaw skin were dissected and immediately frozen on liquid nitrogen and stored at −80°C until use. For RNA analysis of DRG, transcardial perfusion with a 1:1 solution of 0.9% NaCl:RNAlater (Invitrogen, Carlsbad, CA) was performed. Tissues were homogenized for RNA isolation using 5-mm stainless steel beads and TissueLyser II (Qiagen, Hilden, Germany) at 20 Hz for a total of 2 minutes with a 30-second rest period between each minute. Dorsal root ganglia were homogenized in QIAzol, and skin was homogenized in RLT+ beta-mercaptoethanol. RNA was isolated from DRG using RNeasy Lipid Tissue Mini Kit (Qiagen) and from peri-incisional skin using RNeasy Fibrous Tissue Mini Kit (Qiagen). The amount of RNA added to make cDNA was standardized (285.6 ng for DRG RNA and 927.2 ng for skin RNA). Synthesis of cDNA was performed using SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA).

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2.5. Quantitative real-time PCR

For mRNA expression analysis, cDNA and TaqMan primers were mixed with TaqMan Gene Expression Master Mix (Thermo Fisher Scientific). GAPDH was used as a reference gene for DRG samples due to consistent expression,4 whereas TATA-binding protein was used as a reference gene for peri-incisional skin samples due to its stability throughout wound healing.128 GAPDH (Life Technologies, Carlsbad, CA, Assay ID: Mm99999915_g1), TATA-binding protein (Life Technologies, Mm01277042_m1), NLRP3 (Life Technologies, Mm00840904_m1), and IL-1β (Life Technologies, Mm00434228_m1) were assayed using TaqMan primers. Quantitative real-time PCR was run on a Bio-Rad C1000 CFX96 Real-Time-System Thermal Cycler (Bio-Rad Laboratories, Inc, Hercules, CA). The ΔΔCT method was used to calculate the relative fold gene expression, and the sham controls were normalized to 1.

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2.6. Protein isolation and IL-1β enzyme-linked immunosorbent assay

Injured and sham mice were anesthetized using inhaled isoflurane and decapitated. Peri-incisional plantar hindpaw skin was dissected away from the muscle and immediately frozen on liquid nitrogen and stored at −80°C until use. Tissues were homogenized at 75 mg/mL or at least 120-μL T-PER Tissue Protein Extraction Reagent was added to the tissue sample + 1X EDTA-Free Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific) protease inhibitor. To homogenize the tissue, a 5-mm stainless steel bead was added to each tube and agitated using TissueLyser II (Qiagen) at 20 Hz for a total of 2 minutes with a 30-second rest period between each minute. The tissue was further homogenized by rocking the tubes at 4°C for 20 minutes. To separate the lysate, the tubes were centrifuged for 1 minute at 2988 rpm and 4°C. The lysate was placed into a clean tube and centrifuged for 5 minutes at max speed and 4°C, and the supernatant was placed into a clean tube. Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) was used to determine protein concentration. IL-1β from peri-incisional skin was measured using Mouse IL-1 beta/IL-1F2 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) according to instructions.

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2.7. Quantification of paw thickness

Paw thickness was measured from dorsal to ventral side using a digital caliper as previously described.78 Mice were briefly anesthetized with inhaled isoflurane, and bottom of calipers was placed immediately proximal to the last pad, and the top of the calipers was placed on the dorsal side of the hindpaw. The hindpaw thickness was recorded once the calipers slightly indented the skin. To account for between-subject differences in baseline paw thickness, we calculated the percent increase in paw thickness after incision from baseline for each mouse.

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2.8. Quantification of paw immune cell infiltration

Skin and muscle incision were performed, and ipsilateral and contralateral hindpaws were cutoff at the ankle 1 or 4 days after surgery. The hindpaws were placed in Richard-Allan Scientific Buffered Zinc Formalin (Thermo Fisher Scientific) for at least 4 days. The hindpaws were removed from the formalin and cut into approximately equal thirds and placed in tissue cassettes in formalin overnight. The middle third of the hindpaw containing the incision was sectioned coronally and processed at the Children's Hospital of Wisconsin Research Institute Histology Core Facility. Sections were stained with hematoxylin and eosin. A pathologist (M.W.L.) assessed the degree, character, and distribution of inflammation and tissue damage while blinded to genotype, treatment, and any a priori hypotheses. The Children's Research Institute Imaging Core Facility used a Hamamatsu Nanozoomer HT slide scanner (Hamamatsu Photonics, K.K., Hamamatsu City, Japan) to generate images of tissue sections. The area of inflammation was manually calculated using NDP.View 2 software (Hamamatsu Photonics).

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2.9. Behavior

A designated mouse behavioral room was used for all behavioral studies of the same assay, and testing was performed at approximately the same time in the morning under normal light. A female experimenter (blinded to genotype and treatment) performed all behavioral studies, and male and female mice were tested separately. Males were initially tested, and because incised male NLRP3KO mice consistently recovered by POD3, they were not tested on POD5 and POD6. They were again tested on POD7 and/or POD10 to ensure that the incised WT mice recovered to baseline levels. Female mice and male and female NLRP3AdvCre mice were tested after the data collection was completed for male mice. The female NLRP3KO mice and the NLRP3AdvCre+ mice were tested on POD5 and POD6 because they did not recover to baseline levels by POD4. Therefore, to definitively observe when the female NLRP3KO mice and the NLRP3AdvCre+ mice recovered, we continued testing on these additional days. Based on previous behavioral studies from our laboratory, at least 8 mice per group were used, and mice were randomly assigned to treatment groups.

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2.10. Mechanical threshold

Mice were habituated in individual Plexiglas enclosures on top of wire mesh for 30 minutes before testing. Calibrated von Frey monofilaments (0.09-19.6 mN) were applied to the plantar hindpaw, and the up–down method20,33 was used to calculate the 50% withdrawal thresholds. The stimuli were applied to the medial-posterior aspect of the plantar hindpaw, the most sensitive area post surgery.13,101 The contralateral, sham-operated hindpaw was used as a control because the contralateral hindpaw was unaffected by surgery on the ipsilateral hindpaw (Figs. 1B and C and Figs. 2A and B). A response was considered to be lifting of the hindpaw. For intraplantar administration of compounds before testing, animals were briefly anesthetized with inhaled isoflurane, and 20 μL of a drug or vehicle were administered subcutaneously using a 29-G needle. Each animal received the same drug that was administered to the ipsilateral incised hindpaw in the contralateral sham hindpaw. For peripheral inhibition of TRPA1, 9.4 mM of the selective TRPA1 antagonist HC-030031 (Sigma, St. Louis, MO) dissolved in 0.5% dimethylsulfoxide and 0.25% Tween 80 in PBS or vehicle (0.5% dimethylsulfoxide and 0.25% Tween 80 in PBS) was administered daily 60 minutes before testing.7,76

Figure 1

Figure 1

Figure 2

Figure 2

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2.11. Electrophysiology

The experimenter was blinded to group identities throughout data collection and analyses.

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2.12. Ex vivo tibial skin-nerve preparation

Ex vivo tibial skin-nerve preparations84,105 of sham, POD1, and POD4 animals were used to assess the effect of incision and NLRP3 on primary afferent firing in response to mechanical stimulation. This preparation was used in order to measure afferent firing from the same anatomical location as the stimulation site for the behavioral assays used. Animals were briefly anesthetized using inhaled isoflurane and cervically dislocated. The hindleg was shaved with commercial clippers, and sutures were removed from incised hindpaws. The glabrous skin, muscle, and tibial nerve were quickly dissected and placed in a 32 ± 0.5°C, oxygenated circulating buffer (123-mM NaCl, 3.5-mM KCl, 2.0-mM CaCl2, 0.7-mM MgSO4, 1.7-mM NaH2PO4, 5.5-mM glucose, 7.5-mM sucrose, 9.5-mM sodium gluconate, and 10-mM HEPES; pH 7.45 ± 0.05). The edges of the skin were pinned down with insect needles, the remaining tendons and interossei muscles were removed, and the tibial nerve was placed on a mirror in a chamber containing enough mineral oil to cover the nerve. The nerve was teased into small fascicles, and individual fascicles were placed on a recording electrode. A blunt glass probe was used to mechanically stimulate the entire skin area to search for receptive fields of mechanically sensitive fibers. Conduction velocity of each fiber was used to characterize them into C fibers (<1.2 m/s) or Aδ fibers (1.2 to 10 m/s).73 Aβ fibers were not recorded from because they are not sensitized to mechanical stimulation by incision.50 Only slowly adapting Aδ fibers were recorded and analyzed because these include myelinated nociceptors as shown by previous tibial skin-nerve studies from our laboratory.84 To enhance the probability of recording from afferents that were sensitized by incision, fibers with cutaneous receptive fields 2 mm or less away from the incision site were recorded because fibers in this region have been shown to be sensitized to mechanical stimuli after incision, whereas fibers located more than 2 mm away from the incision site have been shown to not differ from sham controls.68 After identification of a fiber, baseline activity was recorded for 5 minutes before subjecting the receptive field to the mechanical stimulation protocol. Next, a continuous force ramp from 0 to 100 mN over 10 seconds was used to determine action potential firing thresholds. After 1-minute rest, square wave mechanical stimulations (2, 5, 10, 20, 40, 100, 150, and 200 mN) for 10 seconds each were applied to the receptive field via a 0.8-mm probe in 1-minute intervals; these intervals were used to prevent sensitization or desensitization of a fiber's cutaneous terminals. All mechanical stimuli were applied using a custom-designed closed-loop feedback-controlled mechanical stimulator (created by Dr. Larry Fennigkoh, P.E. and Jonathan Kokott, Milwaukee, WI). The stimulator consisted of 3 motorized and linear stages (T-LSM200A; Zaber Technologies, Inc, Vancouver, Canada) configured as a Cartesian (x,y,z) gantry equipped with an ultra–low-force transducer (F30; Harvard Apparatus, Holliston, MA) mounted to the vertical, z-axis of the gantry as previously described.84 Labchart (ADInstruments, Colorado Springs, CO) was used to record and analyze data.

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2.13. In vitro calcium imaging of dorsal root ganglion neurons

Calcium imaging was performed on cultured ipsilateral lumbar 3 to 6 DRG neurons from sham, POD1, POD4, and POD7 WT and NLRP3KO animals. Neurons were washed with extracellular buffer (150-mM NaCl, 10-mM HEPES, 8-mM glucose, 5.6-mM KCl, 2-mM CaCl2, and 1-mM MgCl2, pH 7.40 ± 0.03, and 320 ± 3 mOsm) for 30 minutes, incubated with 2.5-mg/mL Fura-2-AM (Life Technologies) in 2% bovine serum albumin for 45 minutes and then washed with extracellular buffer again for 30 minutes. All incubations were performed at room temperature. Fura-2-AM–loaded coverslips were mounted onto a perfusion chamber, placed on an inverted fluorescent microscope, and superfused with extracellular buffer at a constant rate (6 mL/min). Fluorescence images were captured at 340 nm and 380 nm with a cooled Andor Zyla sCMOS camera (Oxford Instruments, Abingdon, United Kingdom) to calculate the bound to unbound ratio (340/380). NIS Elements software (Nikon, Melville, NY) was used to detect and analyze intracellular calcium changes. Cells that exhibited ≥20% increase in 340/380 ratio from baseline were considered to be responsive to application of 300-nM capsaicin (Sigma-Aldrich), 100-μM cinnamaldehyde (Sigma-Aldrich) or 50-mM KCl. A 30-second application of KCl was used as a measure of cell viability. To assess the effect of incision on TRPA1 function, cinnamaldehyde was applied for 2 minutes. To evaluate the effect of incision on TRPV1 function, capsaicin was applied for 2 minutes. The percentage of responsive neurons and magnitude of response (percent change from baseline) were recorded for each treatment. Neurons were divided into small diameter (<27 μm) and large diameter (≥27 μm) groups to approximate C-fiber-type and A-fiber-type somata, respectively.32 There were no changes in large diameter neurons (data not shown), and therefore, only data from small diameter neurons are presented.

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2.14. Statistical analysis

A biostatistician was consulted for statistical analyses of data sets. Data are shown as the mean ± SEM. An a priori decision was made to perform sex and genotype comparisons using the same animals to decrease the number of animals required for this study. Area under the curve (AUC) was used for hindpaw thickness and behavioral mechanical threshold data. Area under the curve was calculated using the trapezoidal rule, 0.5 × Δt x-axis × {(Ybaseline + Yt1) + (Yt1 + Yt2)…}.94,138,145 Area under the curve graphs display change from baseline. For data that were not normally distributed, a nonparametric chi-square test was applied, and the P value was adjusted for multiple comparisons by dividing the P value by the number of comparisons run (ie, for calcium imaging data, the percentage of cells responding). Analysis of variance (ANOVA) assumes that variances are normally distributed; when variances were not normally distributed, the values were transformed using a log transformation. For behavioral studies conducted over time, repeated-measures 2-way ANOVA with conservative multiple comparisons followed by Tukey post hoc test were conducted. For hindpaw thickness measurement, 3-way ANOVA followed by Tukey post hoc test was conducted. For qPCR, ELISA, area of immune cell infiltration, AUC, calcium imaging magnitude of response, and skin nerve (mechanical threshold and conduction velocity), ordinary 2-way ANOVA followed by an appropriate post hoc test (Sidak test for AUC, calcium imaging, ELISA, and area of immune cell infiltration data and Tukey test for qPCR and skin nerve data) were performed. For actional potential firing frequency in response to incremental increases in force, repeated-measures mixed-model analysis with Sidak post hoc test was performed on data from all surgical treatments (sham, POD1, and POD4); POD1 and POD4 were treated as different groups and were displayed in separate graphs for clarity. Sample sizes for each experiment are listed in the figure legends. Significance for all experiments was considered to be P < 0.05. Statistical tests were performed with GraphPad Prism or IBM SPSS.

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3. Results

3.1. NLRP3 mediates postoperative swelling and immune cell infiltration in males, but not females

Swelling and inflammation are major problems associated with incisional wounds. There are several factors in wounds that activate the NLRP3 inflammasome including extracellular matrix components and reactive oxygen species.28,91,121,123,136,141 To determine whether activation of the NLRP3 inflammasome and subsequent release of IL-1β drive wound swelling and inflammation, we incised hindpaws of both sexes of WT and NLRP3 global knockout (NLRP3KO) mice. Postoperative hindpaw edema (as measured by paw thickness) was significantly lower in male NLRP3KO mice than male WT mice throughout the wound healing timeframe (Fig. 1A). Notably, NLRP3KO paw swelling returned to preincision levels by postoperative day 2 (POD2) while WT paw swelling did not resolve until POD7 (Fig. 1A). In female mice, global deletion of NLRP3 had no significant effect on postoperative hindpaw swelling (Fig. 1A). The hindpaw edema was alleviated in male NLRP3KO mice significantly faster than in female NLRP3KO mice. This suggests that NLRP3 drives postoperative hindpaw edema in males, but not females.

Consequently, immune cell infiltration was also assessed in peri-incisional tissues on POD1 and 4. In males, at POD1, there was a similar degree and distribution of immune cell infiltration in both WT and NLRP3KO hindpaws. The immune cell infiltration extended from the plantar incision site, around the periphery of the paw, extended dorsally through the muscle, and mainly consisted of mast cells (white arrowhead) and neutrophils (red arrowhead) (Figs. 1B–D, left side). However, by POD4, there were pronounced genotype differences. Wild-type hindpaws had an increased area of immune cell infiltration that was not apparent in NLRP3KO hindpaws. Necrotic muscle tissue, activated macrophages (black arrowhead), and lymphocytes (blue arrowhead) were all observed in WT paws but were less evident in NLRP3KO hindpaws (Figs. 1B–D, right side).

In females, at POD1 and POD4, there was a similar degree of immune cell infiltration in WT and NLRP3KO hindpaws, indicating that deletion of NLRP3 had no effect on immune cell infiltration across the time course of inflammation (Figs. 2A–C). In addition, WT females had less immune cell infiltration than WT males (Fig. 2D). Despite WT females having less immune cell infiltrate than WT males at POD4, the hindpaw thickness of incised WT males and females did not differ at POD4 (Fig. 1A). These results may be discrepant because swelling occurs as a result of both immune cell infiltrate and buildup of fluid (capillaries leak water, salt, and protein). Estrogen has been shown to result in increased edema.120 Therefore, while females have less immune cell infiltrate than males, they may have a higher amount of fluid at the site of injury. An example of how the area of inflammatory cell infiltrate was quantified is shown in Supplemental Digital Content Figure 1 (available at http://links.lww.com/PAIN/A770). Overall, these data indicate that NLRP3 mediates surgical site inflammation predominately in male mice, and that female mice have less surgical site infiltration than male mice.

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3.2. Sustained postoperative IL-1β production is dependent on NLRP3 in males but is independent of NLRP3 in females

Because global ablation of NLRP3 decreased hindpaw edema and the area of immune cell infiltration after incision in males, we hypothesized that NLRP3 and its product IL-1β would be upregulated in dorsal root ganglia (DRG) that contain neurons that innervate the surgical site and in tissue at the surgical site. Quantitative real-time PCR was used to assess NLRP3 and IL-1β mRNA expression in both the lumbar DRG and peri-incisional skin of male and female mice. In males, NLRP3 mRNA was significantly increased at POD1 in WT DRG as compared to sham and NLRP3KO DRG (Fig. 3A). In contrast to males, NLRP3 mRNA was not elevated to a statistically significant level in females when males and females were compared. However, when sexes were analyzed independently, significant increases were observed in WT females at POD4 (Fig. 3A, 2-way ANOVA with a Tukey post hoc test; *P = 0. 0.0159). Consistent with the upregulation of NLRP3 after incision in male WT DRG, IL-1β mRNA was also significantly upregulated at POD1 in WT DRG from male mice, but not in DRG from female mice. However, when sexes were analyzed independently, significant increases were observed in WT females at POD1 (Fig. 3B, 2-way ANOVA with a Tukey post hoc test; **P = 0. 0.0065).

Figure 3

Figure 3

In WT male mice, NLRP3 mRNA was highly upregulated in peri-incisional skin (Fig. 3C). NLRP3 mRNA from WT females was upregulated in peri-incisional skin to a similar extent as males (Fig. 3C). IL-1β mRNA was upregulated in the skin of both WT and NLRP3KO male mice after incision, although the increase was much smaller in NLRP3KO male mice (Fig. 3D). IL-1β mRNA expression in the skin showed a different pattern in females than males. In females, IL-1β mRNA was highly upregulated in peri-incisional skin to a similar degree in both genotypes (Fig. 3D). The effects of genotype and sex on postoperative IL-1β protein levels were consistent with those effects on IL-1β mRNA levels. In males, IL-1β protein levels were upregulated in both genotypes initially at POD1. By POD4, IL-1β protein was no longer upregulated in skin from NLRP3KO mice but remained high in skin from WT mice (Fig. 3E). In females, IL-1β protein was significantly upregulated in skin from both genotypes at both POD1 and POD4 (Fig. 3E). Although the effects of NLRP3 deletion on IL-1β protein were comparable with the effects on mRNA levels, there were some discrepancies between the protein and mRNA levels in females. Discrepancies between IL-1β mRNA and protein levels are not uncommon, and it has been suggested that post-translational or post-transcriptional regulation and sex can influence IL-1β protein expression.65,108,115 Here, it seems that IL-1β is post-translationally regulated in females because the IL-1β protein levels are not as elevated as the mRNA levels. In addition, the pattern of IL-1β protein level changes is similar to the changes in the area of immune cell infiltration between sexes and genotypes in Figure 2D, where females have similar levels of immune cell infiltration as male NLRP3KO mice. Therefore, differences in the area of immune cell infiltration may also account for differences in IL-1β protein levels. Together, these data indicate that in males, most incision-induced IL-1β production is dependent on the NLRP3 inflammasome, whereas in females, postoperative IL-1β production is largely independent of NLRP3.

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3.3. Deletion of NLRP3 prevents postoperative mechanical hypersensitivity in males and attenuates the mechanical hypersensitivity in females

Because global deletion of NLRP3 resulted in a diminished immune response after incision in male, but not female mice, we next asked whether NLRP3 is involved in postoperative mechanical hypersensitivity. Two hours after incision, male WT mice developed pronounced mechanical hypersensitivity that subsided by POD7. By contrast, male NLRP3KO mice developed a lower magnitude of hypersensitivity that resolved by POD3 (Fig. 4A). Similar to WT males, WT females developed mechanical hypersensitivity 2 hours after surgery that resolved by POD7. In female NLRP3KO mice, the mechanical hypersensitivity was similar to WT females in onset but was attenuated by POD3 and completely resolved by POD7 (Fig. 4B). Thus, although there were no differences in postoperative mechanical hypersensitivity between male and female WT mice, incised male and female NLRP3KO mice showed marked differences in mechanical hypersensitivity (Fig. 4C). Male NLRP3KO mice did not develop mechanical hypersensitivity to the same extent as female NLRP3KO mice immediately after surgery (Fig. 4C). Area under the curve analysis showed that in males, deletion of NLRP3 substantially reduced the overall mechanical hypersensitivity after incision but had little effect in females (Fig. 4C).

Figure 4

Figure 4

Overall, these data indicate that NLRP3 is essential for the development and maintenance of postoperative mechanical hypersensitivity in males but is significantly less involved in females. Furthermore, since IL-1β production is dependent on NLRP3 expression in non-neuronal and potentially neuronal cells (Fig. 3), it appears that the decrease in IL-1β by global deletion of NLRP3 alleviates the postoperative mechanical hypersensitivity in males. We assessed IL-1β levels only in skin because incision of the skin alone has been shown to drive the mechanical hypersensitivity after incision through POD4, whereas muscle incision predominantly drives the spontaneous pain-like behaviors.12,139,140 However, IL-1β in muscle may contribute to the mechanical pain-like behavior longer than 4 days after incision. It has been demonstrated that skin and muscle incision produces a sustained pain phenotype that lasts approximately 7 days, whereas the skin-only incision model produces hypersensitivity that lasts only 4 days.7,139 Furthermore, IL-1β is increased in skin and muscle to a similar magnitude, but the peak of expression occurs at a later time-point in muscle.119 Therefore, IL-1β in muscle may contribute to the prolonged hypersensitivity (≥POD4) in the skin and muscle incision model that we used here.

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3.4. Deletion of sensory neuron NLRP3 attenuates postoperative mechanical hypersensitivity in both sexes

Next, we asked why global deletion of NLRP3 has a greater effect on mechanical hypersensitivity and edema in males than in females. It has been demonstrated that sex differences exist in sensory neuron sensitization after injury,108 and that in addition to immune cells, sensory neurons also express NLRP3.23,64,144 Thus, we asked whether the sex differences in the contribution of NLRP3 to postsurgical pain and edema could be due to cell type–specific expression of NLRP3.

To determine whether sensory neuron NLRP3 mediates postsurgical pain, we selectively deleted NLRP3 from Advillin-Cre–expressing cells (ie, predominately sensory neurons, but not immune cells). Surgical incision induced marked mechanical hypersensitivity in both male (Fig. 5A) and female (Fig. 5B) NLRP3AdvCre− mice that resolved by POD7, similar to WT mice (Fig. 4). Deletion of sensory neuron NLRP3 (NLRP3AdvCre+) attenuated postoperative mechanical hypersensitivity in both male and female mice. Mechanical hypersensitivity in males was attenuated by POD3 (Figs. 5A and C) and in females, was attenuated by POD4 (Figs. 5B and C). Area under the curve analysis revealed that overall, NLRP3AdvCre+ male and female mice were not different (Fig. 5C). In male mice, selective deletion of NLRP3 from sensory neurons did not alleviate incision-induced mechanical hypersensitivity to the same extent as global NLRP3 deletion (Fig. 5D). Conversely, in females, there were no differences between global and sensory neuron NLRP3 deletion (Fig. 5E). These data indicate that in females, some of the postoperative hypersensitivity may be mediated by sensory neuron–restricted NLRP3, whereas in males, most of the postoperative hypersensitivity is mediated by heterologously expressed NLRP3.

Figure 5

Figure 5

In addition to mechanical hypersensitivity, hindpaw edema was decreased in male NLRP3AdvCre+ by POD3 in comparison with NLRP3AdvCre− mice but was still elevated compared with baseline (BL) (Supplemental Digital Content Figure 2A, available at http://links.lww.com/PAIN/A770). Edema in female NLRP3AdvCre+ mice did not differ from that observed in NLRP3AdvCre− mice (Supplemental Digital Content Figure 2A, available at http://links.lww.com/PAIN/A770). Male and female comparisons revealed that male mice, regardless of genotype, had more hindpaw edema after incision than females (Supplemental Digital Content Figure 2A, available at http://links.lww.com/PAIN/A770). However, AUC analysis revealed that there were no overall differences between genotypes or sexes for the NLRP3AdvCre mice (Supplemental Digital Content Figure 2A, available at http://links.lww.com/PAIN/A770). The sex and genotype changes in hindpaw edema were similar to the changes in mechanical hypersensitivity. In males, global deletion of NLRP3 resulted in reduced hindpaw swelling compared with selective deletion of NLRP3 from sensory neurons (Supplemental Digital Content Figure 2B, available at http://links.lww.com/PAIN/A770). In females, there were no differences in hindpaw edema between global and sensory neuron-specific deletion of NLRP3 (Supplemental Digital Content Figure 2C, available at http://links.lww.com/PAIN/A770). Quantitative real-time PCR was used to confirm that NLRP3 was decreased in DRG. NLRP3 was upregulated at POD4 in female NLRP3AdvCre− DRG as compared to sham NLRP3AdvCre− DRG and NLRP3AdvCre+ DRG at POD4 (Supplemental Digital Content Figure 3A, available at http://links.lww.com/PAIN/A770). As expected, since the expression of NLRP3 must be induced,8 there were no differences in NLRP3 expression in sham samples. POD4 samples were used to determine the percent knockdown of NLRP3 because NLRP3 was upregulated at this time-point. As expected, there was a significant knockdown (66.9%) of NLRP3 in NLRP3AdvCre+ DRG as compared to NLRP3AdvCre− DRG (Supplemental Digital Content Figure 3B, available at http://links.lww.com/PAIN/A770). Taken together, these data indicate that postoperative edema is driven by heterologously expressed NLRP3 in males, whereas most edema is independent of NLRP3 in females.

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3.5. NLRP3 is required for postoperative mechanical sensitization of cutaneous nociceptor terminals in both sexes

We next asked whether NLRP3 is required for the sensitization of cutaneous nociceptors after incision. To address this, we used an ex vivo tibial skin-nerve preparation and examined mechanically sensitive C and Aδ fibers surrounding the incision site. In preparations from males, C fibers from incised WT animals displayed a significant reduction in mechanical threshold at POD1 and POD4, whereas C fibers from incised NLRP3KO animals were unaffected (Fig. 6A). At POD1, C fibers from incised WT animals displayed increased firing frequencies to low (5-20 mN) and high (200 mN) receptive field stimulation in comparison with sham controls (Figs. 6B–D). By contrast, C fibers from incised NLRP3KO animals at POD1 were not sensitized to mechanical stimuli (Figs. 6B–D). The mechanical firing frequency of C fibers from incised WT animals at POD4 was similar to that at POD1 as similar increases in firing frequency to low and high mechanical stimulation occurred (Figs. 6E and F). As at POD1, C-fiber-firing frequency at POD4 in NLRP3KO preparations was similar to sham controls (Figs. 6E and F). Of note, there were no baseline differences in C-fiber mechanical responsivity between genotypes. Thus, NLRP3 does not play a role in baseline mechanotransduction. In contrast to C fibers, though consistent with previous findings,50,68 mechanically sensitive myelinated nociceptors were not affected by incision. Neither incision nor global deletion of NLRP3 altered Aδ-fiber mechanical thresholds (Supplemental Digital Content Figure 4A, available at http://links.lww.com/PAIN/A770) or firing frequencies in response to mechanical stimulation (Supplemental Digital Content Figure 4B–F, available at http://links.lww.com/PAIN/A770).

Figure 6

Figure 6

For skin nerve preparations from females, C fibers at POD4 were specifically recorded because this was the time-point at which behavioral differences were observed between genotypes. C fibers from incised female WT animals displayed a significant reduction in mechanical thresholds at POD4, whereas C fibers from incised NLRP3KO animals were unaffected (Fig. 7A). C fibers from incised WT animals displayed increased firing frequencies to high (100-200 mN) forces compared with sham controls (Figs. 7B–D). By contrast, C-fiber mechanical firing frequencies in incised NLRP3KO preparations were not statistically different from sham controls (Figs. 7B–D). Postoperative male and female mice could not be directly compared due to the sex differences in C-fiber mechanical firing frequency in sham conditions. C-fiber mechanical thresholds of sham animals did not differ between genotypes or sexes nor did the mechanical firing frequencies in response to low (2-40 mN) forces (Supplemental Digital Content Figure 5A and D, available at http://links.lww.com/PAIN/A770). However, at intense forces (100-200 mN), C fibers from sham females displayed higher firing frequencies than C fibers from sham males regardless of genotype (Supplemental Digital Content Figure 5B–D, available at http://links.lww.com/PAIN/A770). This is consistent with behavioral data showing that female rats display lower thresholds to noxious stimuli than male rats.57 In addition, it has been shown that there are more mechanically sensitive primary muscle afferents in sham female than male mice.108

Figure 7

Figure 7

To demonstrate that NLRP3 that is expressed in sensory neurons (but not non-neuronal cells) contributes to the postoperative mechanical hypersensitivity observed in females, we compared the C-fiber mechanical response properties of female NLRP3KO and NLRP3AdvCre+ preparations. Consistent with our behavioral data, there were no differences in mechanical thresholds (Fig. 8A) or firing frequencies in response to mechanical stimulation (Figs. 8B–D) between NLRP3KO and NLRP3AdvCre+ preparations. Collectively, these data demonstrate that (1) plantar incision sensitizes only C fibers but not Aδ fibers to mechanical stimuli, (2) NLRP3 expression is required for the mechanical sensitization of C fibers after incision, (3) cutaneous afferents from females are more responsive to high forces than afferents from males, and (4) NLRP3 specifically in sensory neurons contributes to postoperative mechanical hypersensitivity in females.

Figure 8

Figure 8

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3.6. NLRP3 mediates the enhanced TRPA1 but not TRPV1 function in sensory neurons

Transient Receptor Potential Ankyrin 1 (TRPA1) and Transient Receptor Potential Vanilloid 1 (TRPV1) are nonselective cation channels that are expressed on nociceptive neurons.42,85 Previous studies implicate involvement of both TRPA152,121,130,134 and TRPV15,7,99,129,130 in postoperative pain-like behaviors. In addition, these channels may be downstream targets of NLRP3 inflammasome activation because IL-1β has been shown to sensitize or upregulate expression of both TRPA154,80,88 and TRPV1.16,106,109 Therefore, we used calcium imaging to test whether TRPA1 and TRPV1 function is affected in DRG neurons by surgical incision and if the effects of incision can be prevented by global deletion of NLRP3.

The percentage of small-diameter WT DRG neurons that responded to cinnamaldehyde, a potent TRPA1 agonist,3 increased at POD4 and returned back to sham levels by POD7. No significant changes occurred in neurons from NLRP3KO mice (Fig. 9A). The magnitude of response to cinnamaldehyde was unaltered in either genotype or at any time-point (Supplemental Digital Content Figure 6A, available at http://links.lww.com/PAIN/A770). These data indicate that NLRP3 is required for the increased proportion of TRPA1-sensitive neurons after incision.

Figure 9

Figure 9

Next, we used capsaicin, a potent TRPV1 agonist,19 to investigate TRPV1 function. The percentage of both WT and NLRP3KO capsaicin-sensitive neurons was significantly increased at POD1 compared with sham controls, and the increases were no longer found at POD4 or POD7 (Fig. 9B). The magnitude of response to capsaicin was unaltered in either genotype or at any time-point (Supplemental Digital Content Figure 6B, available at http://links.lww.com/PAIN/A770). These data demonstrate that incision causes an increase in the proportion of TRPV1-sensitive neurons at early time-points after incision through factors that are independent of NLRP3 activation.

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3.7. NLRP3 may be required for TRPA1-mediated postoperative mechanical hypersensitivity in males, whereas postoperative mechanical hypersensitivity is in females is independent of TRPA1

Because the proportion of TRPA1-responsive sensory neurons was increased after incision and this was dependent on NLRP3 expression, we next asked whether peripheral inhibition of TRPA1 would decrease behavioral postoperative mechanical hypersensitivity. In male mice, daily intraplantar administration of the TRPA1 antagonist HC-030031 attenuated the mechanical hypersensitivity of WT mice after incision (Fig. 10A). However, daily HC-030031 treatment in NLRP3KO male mice had no effect on postoperative mechanical hypersensitivity (Fig. 10B). Comparison of HC-030031–treated WT and NLRP3KO mice revealed that peripheral TRPA1 inhibition in WT mice had a similar effect on mechanical hypersensitivity as global deletion of NLRP3 (Fig. 10C). In female mice, intraplantar delivery of HC-030031 had no effect on the postoperative mechanical hypersensitivity in either WT or NLRP3KO female mice (Figs. 11A and B). Comparison of female WT and NLRP3KO mice showed that there was an overall trend for NLRP3KO mice to have attenuated mechanical hypersensitivity (Fig. 11C). The trend may be due to damage from repeated injections into the incision site. These data imply that the mechanical hypersensitivity observed in males, but not females, after incision may occur through peripheral TRPA1 and is dependent on NLRP3.

Figure 10

Figure 10

Figure 11

Figure 11

In summary, these studies reveal that in male mice, non-neuronal and potentially neuronal NLRP3 are required for maintenance of postoperative inflammation and mechanical hypersensitivity, and that upregulation of NLRP3 may drive mechanical hypersensitivity through sensitization of TRPA1. By contrast, in females, most postoperative edema is independent of NLRP3, and only NLRP3 expressed in sensory neurons contributes to the postoperative mechanical hypersensitivity. The postoperative mechanical hypersensitivity in females is predominately driven independent of NLRP3 (Fig. 12).

Figure 12

Figure 12

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4. Discussion

In this study, we demonstrate that in males, maintenance (POD4) of postoperative IL-1β production is predominately due to the activation of NLRP3 in non-neuronal cells and potentially sensory neurons. NLRP3 expression induces postoperative mechanical hypersensitivity and increases the number of TRPA1-sensitive neurons. However, in females, only NLRP3 that may be expressed in sensory neurons contributes to postoperative mechanical hypersensitivity. The contributions of non-neuronal cells to postoperative mechanical hypersensitivity are independent of NLRP3. This suggests that additional inflammasomes, or inflammasome-independent IL-1β production, contribute to the postoperative mechanical hypersensitivity in females.

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4.1. NLRP3 is the predominant source of postoperative IL-1β and exacerbates inflammation in males

Multiple factors released by surgical incision are capable of priming10,37,66,90,96,124 or activating48,121,127,136 NLRP3, which subsequently drive IL-1β release. We demonstrate that incision-induced upregulation of IL-1β occurs in both sexes. However, the source and levels of the IL-1β increase is sex dependent. In males, the sustained IL-1β production after incision is largely regulated by the NLRP3 inflammasome. However, in females, most IL-1β production occurs through a mechanism independent of NLRP3. Because sensory neurons express high levels of the IL-1β receptor IL-1R19 and IL-1β plays a prominent role in postoperative mechanical pain,22,61,79 it follows that reduction in the amount of IL-1β by global deletion of NLRP3 in males may lead to less stimulation of IL-1R1 on sensory neurons and alleviation of postoperative mechanical hypersensitivity.

Although NLRP3 is associated with sterile inflammation,18 the type of inflammation that occurs with surgical incision, there are other potential sources of IL-1β in females. IL-1β can be produced by other inflammasomes or be generated independent of the inflammasomes in the context of sterile inflammation. For example, the absent in melanoma 2 (AIM2)86,131 and NLRP1149 inflammasomes, which release IL-1β, are involved in sterile cutaneous inflammation. Alternatively, mechanisms independent of inflammasomes, such as caspase-8,49,75 caspase-1,30,77 and neutrophil proteases,61,69,74 activate IL-1β and are involved in sterile inflammation and wound healing. Although these cited studies were performed in male mice or mice of unspecified sex, it is possible that one or more of these IL-1β–producing proteins drive IL-1β production in females after surgery. Overall, more studies on IL-1β and surgical wounds in females are needed to fully elucidate the neuroimmune mechanisms that underlie female postoperative pain.

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4.2. Activation of NLRP3 profoundly mediates postoperative mechanical pain in males

Here, we demonstrate that deletion of NLRP3 decreases the intensity of mechanical hypersensitivity and the duration of mechanical pain-like behavior in males. Deletion of NLRP3 in females resulted in a modest attenuation in postoperative mechanical hypersensitivity but did not alter its duration. Our data indicate that the differential contributions of NLRP3 to the mechanical pain behavior in each sex is cell-type dependent. NLRP3 in sensory neurons potentially contributes to pain in both sexes at delayed time-points (ie, POD3 and later), whereas non-neuronal NLRP3 contributes to mechanical hypersensitivity in males immediately after injury. In addition, deletion of sensory neuron NLRP3 attenuates mechanical hypersensitivity after incision in vivo and ex vivo in a similar manner as global deletion of NLRP3 in females. This indicates that sensory neuron NLRP3 may have a direct functional role in neuronal sensitization after injury, potentially through direct action on ion channels or autocrine cytokine signaling. For example, in macrophages, TRP channel activation can activate NLRP3.112 The aforementioned sex differences may be due to sexual dimorphisms that exist in the immune system.34,35,83,102,108,117,118 Murine mast cells, neutrophils, and macrophages all express NLRP346,87 and may contribute to the sexual dimorphism noted in the global NLRP3KO studies. In addition, the time course of mast cell, neutrophil, and macrophage activation/recruitment after injury may reveal the contribution of NLRP3 in each of these cell types to the incision-induced mechanical hypersensitivity in males.

In males, dermal mast cells regulate inflammation immediately after cutaneous wounding by releasing inflammatory mediators, increasing vascular permeability, and recruiting neutrophils.87,137 Neutrophil recruitment is generally followed by macrophage recruitment, which occurs 1 to 2 days after injury.29,36,114 Additional support for this non-neuronal NLRP3 time course comes from studies in which mast cells92,142 and neutrophils17,122 were demonstrated to contribute to mechanical hypersensitivity for only 2 to 24 hours after surgery and studies where macrophages (indirectly) sustain the mechanical hypersensitivity after incision (ie, 24-48 hours).43 Therefore, the immediate attenuation of mechanical pain observed in NLRP3KO males, but not females, may be a result of the loss of NLRP3 in mast cells and neutrophils while the loss of NLRP3 in macrophages and sensory neurons may drive the maintenance of attenuation (POD3 and later).

In females, mast cell, neutrophil, and macrophage recruitment is like that of males where there is increased immune infiltration in the same sequence after injury. However, the extent of infiltration and inflammation patterns differ in females. After cutaneous injury, mast cells release less histamine2 and as a result a lower number of neutrophils are recruited to the site of injury in females, perhaps through the suppressive effects of estrogen.11 Similarly, Price et al. recently showed that a reduced number of macrophages are recruited to postoperative injury sites in female mice (Price, T.J., Ph.D., written personal communication, March 2018; abstract Ref. 58). Here, we show that females have less immune cell infiltration of the surgical site than males. In addition to decreased innate immune cell recruitment in females after injury, high levels of estrogen found in females skew macrophages towards the M2 phenotype (anti-inflammatory) while high levels of testosterone found in males promote the M1 phenotype (proinflammatory). In males, macrophages have higher expression of TLR4, the signal that is upstream to activation of NLRP3.63,71,81 Furthermore, sex-dependent alterations in inflammasome expression may account for the differences between the NLRP3KO males and females. Studies on the effects of sex hormones on inflammasome expression are emerging and demonstrate that high levels of estrogen attenuate NLRP3 expression and function after injury.24,148 Although females experience lower immune cell infiltration than males, elevated cytokine levels are sustained longer than males in response to injury, which may compensate for the lower immune cell infiltration.11 Furthermore, it is known that females have more tissue-resident immune cells. Therefore, males may require more recruitment of immune cells in response to injury to mount the same immune response.63 Overall, the reduced efficacy of global NLRP3 deletion for postoperative mechanical hypersensitivity in females may result from high levels of estrogen and sexual dimorphism in the innate immune system.

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4.3. NLRP3 acting through TRPA1 mediates the sensitization of cutaneous C fibers after incision in males

TRPA1 is a nonselective cation channel expressed on C fibers and immune cells42,85 that mediate inflammation-induced mechanical hyperalgesia.82,130,134 Here, we show that in males, the sustained sensitization of cutaneous C fibers and the increase in TRPA1-sensitive neurons require NLRP3. Together with the substantial decrease in IL-1β in NLRP3KO mice, this suggests that IL-1β may sensitize sensory neurons and increase the proportion of TRPA1-sensitive neurons after incision. Potentially, the mechanical sensitization of C fibers in part occurs through the effects of NLRP3-mediated production of IL-1β on TRPA1. TRPA1 antagonism in male WT mice produced a similar attenuation of the postoperative mechanical hypersensitivity as global deletion of NLRP3. IL-1β can increase TRPA1 expression in osteoarthritic chondrocytes88 and in neurons through IL-1R1, which induces NF-κB and p38/MAPK signaling pathways.54,89,133 Furthermore, prevention of IL-6 activity, a byproduct of NF-κB and p38/MAPK signaling, attenuates mechanonociception through downregulation of sensory neuron TRPA1.80 Together, these data indicate that mechanical sensitization of sensory neurons after surgical incision may require NLRP3-mediated IL-1β production, which can lead to upregulation of sensory neuron TRPA1 through various pathways.

A different mechanism for postoperative mechanical hypersensitivity exists in females. The mechanical hypersensitivity in female mice was unaffected by peripheral TRPA1 antagonism. TRP channels are regulated in a sex-dependent manner, where TRPA1, TRPV1, and TRPM8 function differ between males and females after inflammatory injury.97,98,108 In fact, estrogen prevents oxidative stress-induced sensitization of TRPA1,143 the type of stress that leads to caspase, and NLRP3 activation. Therefore, mechanisms independent of TRPA1 drive the mechanical hypersensitivity in females.

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4.4. Future applications for NLRP3 inhibition and studies on sexual dimorphism in tissue injury

Global deletion of NLRP3 prevents the maintenance of postoperative pain in males, which may be a consequence of reduced peri-incisional IL-1β. General blockade of IL-1β signaling, like that obtained with FDA-approved Anakinra (IL-1 receptor antagonist), increases infections due to the necessity of IL-1β for bacterial infection clearance.15,110 Therefore, reduction of IL-1β, but not complete loss, through inhibition of NLRP3 may avoid these complications while decreasing mechanical pain. We show for the first time that sexual dimorphism exists in postoperative pain, and that it is most likely mediated by differences in male and female immune systems. An extensive study is needed to fully elucidate the inflammatory mechanisms that drive male and female postoperative pain. Overall, our study demonstrates a crucial role for NLRP3 in postoperative mechanical pain and, given the negative side effects of current postoperative therapeutics like opioids, provides a rationale for the use of NLRP3 inhibitors in male patients after surgery.

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Conflict of interest statement

The authors have no conflicts of interest to declare.

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Acknowledgements

The authors thank Dr. Bonnie Dittel, Dr. Katelyn Sadler, Dr. Jessica Ross, and Dr. Francie Moehring for editing and providing advice on organization of the manuscript. The authors thank Dr. Aniko Szabo, Director of the Medical College of Wisconsin Biostatistics Consulting Service, for advice and assistance with statistical analysis throughout the manuscript. The authors also thank the Medical College of Wisconsin Histology Core for tissue sectioning and staining and the Medical College of Wisconsin Imaging Core for slide scanning. The authors thank Neil Smith for production and design of the diagram in Figure 12.

This work was supported by the National Institute of Neurological Disorders and Stroke grants NS040538 and NS070711 to C.L. Stucky and F31GM123778 to A.M. Cowie. The Research and Education Component of the Advancing a Healthier Wisconsin Endowment at the Medical College of Wisconsin provided partial support.

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Supplemental digital content

Supplemental digital content associated with this article can be found online at http://links.lww.com/PAIN/A770 and http://links.lww.com/PAIN/A771.

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Supplemental video content

Video content associated with this article can be found online at http://links.lww.com/PAIN/A771.

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Keywords:

Interleukin-1β; Incision; TRPA1; TRPV1; Hypersensitivity; Allodynia; Sexual dimorphism; Mechanical; TLR4; Mechanosensation

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