A critical role for α7 nicotinic acetylcholine receptor signaling in myeloid lineage cells in chronic neuropathic and inflammatory pain responses

The effect of paclitaxel treatment on α7 nAChR expression in peripheral nervous tissue (DRG) was measured using qRT-PCR. WT male and female mice were treated with vehicle or a high dose of paclitaxel (8 mg/kg, i.p., total of 4 injections), which induces long-lasting cold and mechanical hypersensitivity as described by Toma et al. (2017), and tissues were collected at 7, 14, and 28 days after the initiation of paclitaxel treatment. 26 As shown in Figure 1A and 1B , α7 nAChR expression was increased significantly in the DRG of mice treated with paclitaxel as compared to the vehicle treatment group on days 7 and 14 (t(18)=9.869, p<0.001) and (t(18)=3.703, p0.5). In the spinal cord, α7 nAChR gene expression in paclitaxel-treated mice was not significantly increased compared to vehicle-treated mice on days 7 and 14 ( Figure 1D and 1E ). However, a significant increase in α7 nAChR expression was observed in the paclitaxel-treated group compared with the vehicle (t(18)=3.495, p<0.01) on day 28 ( Figure 1F ). No significant sex difference was observed across the conditions (data not shown). These results indicate that paclitaxel treatment initially upregulates α7 nAChR expression in the DRG, then later on day 28 in the spinal cord. Since the injection of paclitaxel in mice led to increased expression of α7 nAChRs in the DRGs and spinal cord, it was hypothesized that α7 nAChRs may play an important role in paclitaxel-induced CIPN. Therefore, mice null for the α7 subunit (global KO) and WT mice were injected with a high-dose or low-dose paclitaxel regimen as described above. Evoked nociceptive behaviors, including mechanical and cold sensitivity, as well as spontaneous wheel running behaviors, were measured starting on day seven after induction of CIPN with a high dose of paclitaxel. As demonstrated in Figure 2A , high-dose (8 mg/kg, i.p., 4 injections every other day) paclitaxel-treated α7 KO and WT mice experienced significant mechanical hypersensitivity as compared to vehicle-treated mice, [F(paclitaxel) (1, 28) = 693.4; p 0.05]. In the acetone test, high-dose treatment group α7 KO and WT mice again experienced significantly increased cold hypersensitivity as compared to vehicle group mice [F(paclitaxel) (1, 28) = 576.5; p < 0.0001] ( fig. 2B ). Additionally, α7 KO mice treated with high-dose paclitaxel experienced significantly elevated cold hypersensitivity when compared to WT treatment group mice [F(genotype x paclitaxel) (1, 28) = 48.83; p < 0.0001]. A three-way ANOVA analysis also revealed a significant interaction between time, genotype, and paclitaxel treatment [F(time x genotype x paclitaxel) (3, 28) = 5.210; p < 0.01]. Given that the effect of a high-dose paclitaxel regimen on mechanical hypersensitivity could not be fully characterized between WT and KO mice due to a possible floor effect, α7 KO and WT mice were tested with a low-dose paclitaxel regimen (1 mg/kg, i.p., 4 injections every other day). Low-dose paclitaxel-treated α7 KO mice showed significant mechanical hypersensitivity as measured by the Von Frey test starting on day 1 post-injection as compared to vehicle-treated mice [F(paclitaxel) (1, 360) = 91.09; p < 0.0001] ( Figure 2C ). In contrast, WT mice treated with low-dose paclitaxel did not develop significant mechanical hypersensitivity compared to the vehicle treatment group. Additionally, the hypersensitivity of low-dose paclitaxel-treated α7 KO mice was significantly greater when compared to WT paclitaxel-treated mice [F(genotype) (1, 360) = 99.91; p < 0.0001]. There was a significant relationship between time, paclitaxel treatment, and genotype [F (time x genotype x paclitaxel) (8, 360) = 8.243; p < 0.0001]. Similarly, low-dose paclitaxel-treated α7 KO mice experienced significantly increased cold hypersensitivity at day 7 as compared to the vehicle treatment group (p < 0.0001), while paclitaxel-treated WT mice did not experience a significant increase in hypersensitivity until day 14 (p < 0.05), [F(paclitaxel) (1, 40) = 67.81; p < 0.0001] ( Fig. 2D ). α7 KO low-dose paclitaxel-treatment group mice also experienced significantly greater hypersensitivity when compared to the WT treatment group [F(genotype) (1, 40) = 76.54; p < 0.0001]. In addition, there was a significant relationship between time, genotype, and paclitaxel treatment [F(time x genotype x paclitaxel) (4, 25) = 49.12; p < 0.0001]. Finally, voluntary wheel running activity was significantly diminished in high-dose paclitaxel-treated mice [F(paclitaxel) (1, 14) = 16.82; p 0.05) ( Fig. 2E ). Since global α7 KO resulted in worsening of CIPN pain measures, the role of α7 nAChRs specifically within myeloid cells in the mediation of chronic neuropathic pain was investigated. To do so, the effects of paclitaxel in mice deficient in α7 nAChRs only in myeloid lineage cells were examined. cKO mice were confirmed for the absence of the α7 transcript and protein within circulating macrophages using RT-PCR and Western blot detection methods, respectively ( Supplemental Figures 1S and 2S ). α7 cKO and WT mice were tested with a low-dose paclitaxel regimen (1 mg/kg, i.p., 4 injections every other day). α7 cKO and WT mice developed significant mechanical hypersensitivity after low-dose paclitaxel treatment as compared to vehicle mice [F(paclitaxel) (1, 58) = 374.0; p < 0.0001] ( Figure 3A ). In addition, α7 cKO mice treated with paclitaxel displayed greater mechanical hypersensitivity than WT treatment group mice [F(genotype) (1, 58) = 97.35; p < 0.0001]. Factorial analysis revealed significant effects of paclitaxel x genotype interaction [F(1, 28) = 78.92; p < 0.0001] and paclitaxel x genotype x time [F(6, 348) = 9.414; p < 0.0001]. In the acetone test, α7 cKO treatment group mice displayed significant cold hypersensitivity in comparison to the vehicle group as well as in comparison to WT mice treated with paclitaxel [F(paclitaxel x genotype) (1, 54) = 54.52; p < 0.0001] ( Figure 3B ). WT mice did not develop significant hypersensitivity in the acetone test, similar to what was observed in KO mice treated with low-dose paclitaxel. Factorial analysis revealed additional significant effects of paclitaxel [F(1, 54) = 73.13; p < 0.0001], genotype [F(1, 54) = 76.50; p < 0.0001], and paclitaxel x genotype x time [F(6, 324) = 7.168; p < 0.0001]. Spontaneous wheel running activity in α7 cKO mice on day 14 following induction of CIPN was reduced as compared to vehicle-treated mice (t(35) = 3.539; p < 0.01) ( Figure 3C ). Wheel running was also diminished in α7 cKO mice treated with low-dose paclitaxel when compared to WT paclitaxel-treated mice (t(14) = 4.447; p < 0.001). Factorial analysis revealed significant effects of genotype [F(1, 14) = 7.676; p < 0.05] and genotype x paclitaxel [F(1, 14) = 12.16; p 0.05]. In contrast, locomotor activity in mice remained unchanged in either genotype after paclitaxel administration on day 14 ( Figure 3D ). Therefore, α7 deletion in myeloid lineage cells increased the severity of mechanical and cold hypersensitivity and decreased spontaneous wheel-running activity with no effect on motor activity which may impact evoked pain measures as evidenced by unchanged locomotor activity. Low-dose paclitaxel also induced an increased inflammatory response in α7 cKO mice. Increased expression of pro-inflammatory cytokines TNF-α ( Figure 4A ), IL-6 ( Figure 4B ), and IL-1β ( Figure 4C ) is seen in the DRG of α7 cKO mice treated with paclitaxel compared to those in the vehicle group. Paclitaxel treated α7 cKO mice had significantly increased TNF-α [t(36)=4.61, p<0.01], IL-6 [t(36)=3.15, p<0.05], and IL-1β [t(36)=2.64, <0.05] when compared to WT mice. In contrast, WT mice did not display increased cytokine expression in either the treatment or vehicle group. A high-dose paclitaxel regimen was also tested (8 mg/kg, i.p., 4 injections every other day) which resulted in a similar effect to global KO mice treated with high-dose paclitaxel ( Figure 3S ). The dependency of the antinociceptive effects of α7 agonists on α7 nAChRs in myeloid lineage cells was then investigated. To do so, the effects of an active dose of two different α7 agonists in the CIPN model in WT and α7 cKO mice were tested: the full α7 orthosteric agonist PNU-282987 and the silent α7 agonist R-47, which has been shown to bind to α7 nAChRs, shifting the receptor to a desensitized state. 22 , 46 Male and female mice were injected with a high-dose paclitaxel regimen or vehicle, and 14 days after the last injection of paclitaxel, they were injected with PNU-282987 ( Figure 5A ), R-47 ( Figure 5B ), or vehicle and tested at different time points. Mice injected with paclitaxel developed robust mechanical hypersensitivity at time zero when compared to vehicle-treated mice [F(paclitaxel) (1, 70) = 590.2; p < 0.0001]. One hour after the full agonist PNU-282987 injection, both α7 cKO and WT mice treated with paclitaxel exhibited reduced hypersensitivity, with mice returning to their baseline threshold value. By hour three, hypersensitivity had returned to a level comparable to that of pretreatment. The α7 cKO and WT mice treated with paclitaxel and PNU-282987 did not display a significant difference in response to PNU [F(genotype) (1, 70) = 0.002252; p >0.05]. Overall, PNU-282987 treatment effectively reduced mechanical hypersensitivity in both α7 cKO and WT mice. In contrast, the oral administration of silent agonist R-47 resulted in reduced hypersensitivity in WT-paclitaxel mice but not in α7 cKO-treated mice. The WT mice returned to baseline threshold values at hour two, while α7 cKO mice experienced no difference in their hypersensitivity when compared to α7 WT-paclitaxel [F(genotype) (1, 70) = 71.09; p < 0.0001]. Factorial analysis revealed additional significant effects of time [F(time) (4, 70) = 275.5; p < 0.0001], treatment [F(R-47) (1, 70) = 58.18; p < 0.0001], and time x treatment x genotype [F (time x R-47 x genotype) (4, 70) = 14.90; p < 0.0001]. These results show that α7 cKO mice treated with paclitaxel did not experience a significant reduction in hypersensitivity after administration of the α7 silent agonist R-47. WT mice, on the other hand, experienced complete resolution of CIPN-induced hypersensitivity at 2 hours post-administration of R-47. Both threshold-bound (p<0.01; genes lists in Tables 1S - 5S ) enrichment analysis in Enrichr ( Tables 1 - 3 ; Table 6S - 7S ) and threshold-free analysis in GSEA ( Figures 4S – 8S ; Tables 8S - 12S ) were performed. For main effect of Genotype, 395 differentially expressed genes were identified, including 115 upregulated genes and 280 downregulated in cKO vs. WT (p<0.01; Table 1S ). Top pathways from Enrichr identified Schwan cell/myelination- and muscle- related terms (“skeletal”; down in cKO; Table 1 ). GSEA further supported downregulation of muscle-related genes (“development”, “system”, “contraction”) and neuronal physiology genes in cKO (membrane potential, ion transport, action potential, Ca2+ transport/levels ( Figure 4S ; Table 8S ). Additional downregulated terms were related to sensory perception (thermal stimulus, pain), pain behavioral response, and wound healing ( Figure 4S ; Table 8S ). To summarize, cKO show a downregulation of muscle contractile gene sets that could reflect cytoskeletal remodeling and Schwan cell myelination that might be explained by shared enrichment terms for myeloid cell membrane lipid processing and trafficking. These terms combined with the enriched cell types implicate an impairment in tissue repair and remodeling that could contribute to the enhanced neuropathic pain phenotype in cKO. For main effect of Treatment, 356 differentially expressed genes were identified, including 150 upregulated genes and 206 downregulated genes (p<0.01; Table 2S ). Top Enrichr-derived pathways included a downregulation of circadian clock/rhythm/regulation/signaling, actin cytoskeleton and microtubules, matrix metalloproteinases, protein ubuiquitination, TGF-beta and SMAD signaling, immune-related (antigen presentation in Class I MHC proteins, NK cells), protein ubiquitination-associated congugation/ligase/transferase, Hedgehog signaling, and RAC1 GTPase cycle ( Table 2 ). The only paclitaxel-induced upregulated terms were related to endothelial cells (skin, vascular; Table 2 ). In contrast to threshold-bound Enrichr analysis, GSEA enrichment of the paclitaxel effects were driven by increased gene expression, including mitochondria translation and ATP synthesis, oxidative phosphorylation, amino acid metabolism, purine and NTP synthesis, and small molecule catabolism ( Figure 5S ; Table 9S ). Additional terms showing paclitaxel-induced increased expression include wound healing, synapse assembly, muscle (system, contraction), endothelial cell migration, external encapsulating structure organization, and cellular oxidant detoxification. To summarize, paclitaxel induced an increased gene expression signature indicative of nucleic acid synthesis, energy production/usage, a detoxifying response, changes in muscle function, and potentially a compromised encapsulation of the spinal cord and its connective tissue and synaptic transmission ( Table 2 ; Figure 5S ; Table 9S ). Threshold-bound analysis of paclitaxel treatment in cKO mice only indicated that cKO was responsible for a majority of the enrichment. Here, 688 differentially expressed genes were identified, including 275 upregulated genes and 413 downregulated genes (p<0.01; Table 3S ). Upregulated terms included glycolysis/glucogenesis, mTORc signaling, xenobiotic metabolism, smooth muscle, gastrointestinal and lung cells, and vasculature and endothelial cells/precursors (hemangioblasts)/gap junction/tight junction, and astrocytes ( Table 6S ). Downregulated terms were limited to PIP synthesis/metabolism, circadian rhythm/signaling, neuronal brain/system/projection/synaptic signaling transmission/dendrites/, BDNF-GABA neurotransmission, NMDAR-AMPK signaling, trafficking of microtubule connexins, hedgehog signaling (down), circadian rhythm (down), synaptic transmission (down, including GABAergic synapse) ( Table 6S ). Threshold-free GSEA of Treatment in cKO supported strongest enrichment for upregulated transcripts associated with glucose metabolism, fructose-dependent glycolysis, energy production and electron transport, external encapsulating structure organization, reactive oxygen species metabolism, blood pressure regulation, cell substrate adhesion, response to oxygen levels, cell chemotaxis, and morphogenesis of epithelium ( Table 10S ). Taken together, cKO is implicated in increased energy demand and a robust metabolic response, namely increased glycolysis and glucose metabolism and increased cytoskeletal remodeling. Treatment effects in WT mice were more limited, comprising only 151 differentially expressed genes, including 91 upregulated genes and 60 downregulated genes (p<0.01; Table 4S ). The threshold-bound approach (p<0.01) via Enrichr identified only cell type terms, including primarily immune cell types (all upregulated), including prolierating NK/T, CD4+ T central/effector memory, and CD56- NK3/4, gamma-delta T) ( Table 7S ). Downregulated predicted cell types included glutamatergic, myeloid dendritic type 1, endothelial, and ciliated ( Table 7S ). Together, the results for WT mice imply a paclitaxel-induced immune expansion and neuronal suppression. For threshold-free analysis via GSEA, paclitaxel in WT mice induced upregulation of transcripts associated with mitochondrial and cytoplasmic translation, electron transport chain, ATP synthesis, oxidative phosphorylation, NTP synthesis, cellular respiration, and muscle contraction ( Figure 7S ; Table 11S ). Downregulated terms comprised GTPase signaling, cognition, synapse assembly, and homophillic membrane cell adhesion. Together, combined analysis suggests paclitaxel-induced mitochondrial bioenergetic activation and protein translation and immune expansion in WT mice, and further supports suppression of neuronal and cytoskeletal gene expression. For Genotype x Treatment interactive analysis, 460 differentially expressed genes were identified, including 167 upregulated genes and 293 downregulated genes (p<0.01; Table 5S ). Threshold-bound analysis via Enrichr analysis identified a downregulation of terms in cKO mice associated with IL-9, chemokines/receptors/activity, cytokine activity/signaling, eosinophil/NK cell/granulocyte, chemotaxis, and GPCRs (Oprm1, Drd3, Htr1f, Adra2c, Ptger3, Npy2r). Terms associated with upregulation in cKO mice included SLC transporters/disorders (up in Cre), CCR1 targets, metabolic epileptic disorders, myogenesis, glycolysis, glucose metabolism/transport, carbohydrate metabolism, and glycan complexes, cell cycle/M phase/mitotic spindle/mitosis/kinetochore ( Table 3 ). Threshold-free GSEA indicated a consistent enrichment of energy usage and production, including mitochondrial expression/translation, ATP synthesis and electron transport and respiration, oxidative phosphorylation, RNA metabolism, and RNP biogenesis ( Figure 8S ; Table 12S ). To summarize the Genotype x Treatment interactive analysis, paclitaxel in WT induces mitochondrial and immune cell activation and decreased neuronal transcription affecting neuronal function (synapse assembly, glutamatergic neurons). In contrast, paclitaxel in cKO indicates increased glcyolysis and cell cycle pathways and decreased chemokine/cytokine/chemotaxis signaling. The results suggest a shift from a chemokine-based inflammatory state induced by paclitaxel in WT to a hyperglycolytic-based metabolic phenotype in cKO (perhaps mediated by microglia) leading to increased cellular proliferation that could underlie enhanced behavioral hypersensitivity. Two mouse models were used to assess the role of α7 nAChRs in myeloid cells in acute pain: the paw incision model of post-surgical pain and the LPS model of acute inflammatory pain. In this model, α7 cKO and WT mice developed time-dependent hyperalgesia and hind paw edema measured on days 1, 3, 7, 14, and 21 following surgery ( Figure 6 ). α7 cKO surgery mice and WT surgery mice followed a similar time course of hyperalgesia post-surgery ( Figure 6A ). The surgery group, α7 cKO and WT mice, developed similar nociceptive responses to thermal stimuli, which differed significantly from their sham counterparts [F(surgery) (1, 36) = 171.6; p < 0.0001]. Their return to baseline occurred between 14 and 21 days. Similarly, the paw edema was most pronounced on day one following surgery for both α7 cKO and WT mice ( Figure 6B ). The genotypes did not differ in severity or time to recovery. Analysis with three-way ANOVA revealed the significance of time and surgery [F(time x surgery) (5, 36) = 19.47; p 0.05]. Therefore, although paw incision induces significant thermal hyperalgesia and paw edema, cKO and WT mice do not display a significant difference in response to the acute incision pain caused by paw incision. A separate cohort of α7 cKO mice and their WT littermates were injected with intraplantar LPS. The response to mechanical stimulation as measured by the von Frey assay was assessed, and the paw diameter was measured at one, six, 24, and 48 h after LPS injection. In this model, as observed in the surgical model, mice developed time-dependent hypersensitivity expressed as threshold in grams ( Figure 6C ) and paw edema ( Figure 6D ). As shown in Figure 6C , α7 cKO LPS mice and WT LPS mice exhibited a similar time course of mechanical hypersensitivity following injection. The LPS group α7 cKO and WT mice developed similar responses to the mechanical stimuli, which differed significantly from their sham counterparts [F(LPS) (1, 28) = 126.6; p < 0.0001], and their return to baseline occurred within 48 hours. Additionally, the paw diameter was most pronounced at hour 6 post-injection, but the LPS group α7 cKO and WT mice did not differ significantly in severity or time to recovery ( Figure 6D ). This is very similar to what was seen in the surgical model, where analysis with three-way ANOVA revealed significance of time and LPS [F(time x LPS) (4, 112) = 22.86; p 0.05]. No significant sex difference was observed across treatment and genotype (data not shown). Therefore, although LPS injection induces significant mechanical hypersensitivity and paw edema, cKO and WT mice do not display a significant difference in response to the acute inflammation induced by LPS injection. Overall, α7 deletion in myeloid lineage cells did not appear to affect the severity of LPS-induced inflammation. After evaluating the impact of the deletion of α7 nAChRs in myeloid cells in acute pain models, their role in chronic inflammatory pain models were investigated. For that, the CFA chronic inflammatory pain model as described by AlSharari et. al. (2013) was used. 21 First, the effect of CFA on gene expression in mice was examined. Male and female C57BL/6J mice were injected with CFA (20%) or vehicle, and DRG tissues were collected on day three after injection. Mice treated with CFA experienced a significant increase in α7 nAChR mRNA expression in the DRG when compared to vehicle-treated mice [t(14) = 5.986, p 0.0001] ( Figure 7 ). α7 cKO and WT mice were injected with a low dose of 20% CFA in their right hind paws and their hypersensitivity to thermal stimuli was measured using the Hargreaves method and their paw diameter was evaluated ( Figure 8 ). Both genotypes, α7 cKO and WT, developed hypersensitivity to thermal stimuli following CFA administration; however, α7 cKO mice had a significantly greater degree of hyperalgesia on days one and three after injection than WT mice treated with CFA [F(CFA x genotype) (1, 30) = 25.32; p < 0.0001] ( Figure 8A ). cKO mice continued to experience significant hypersensitivity compared to vehicle-treated mice until day 14 (p < 0.05). Additionally, the increase in paw diameter for the α7 cKO group treated with CFA was significantly increased as compared to the WT group treated with CFA [F(CFA x genotype) (1, 30) = 38.51; p<0.0001] ( Figure 8B ). The latter returned to baseline by day 14, while the α7 cKO mice continued to display significantly increased paw edema through day 21 (p < 0.0005). Three-way ANOVA revealed a significant relationship between time, CFA injection, and genotype [F(CFA x time x genotype) (5, 30) = 2.763, p=0.0362]. No significant sex differences were observed (data not shown). These results show that α7 deletion in myeloid lineage cells worsened the severity and duration of CFA-induced hypersensitivity and inflammation. Expression levels of the pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 were measured in α7 cKO mice and their WT littermates receiving CFA. DRG tissue was collected on day three following CFA injection. IL-1β and TNF-α expression in the DRG were significantly increased in α7 cKO mice as compared to WT mice (t(12)=2.27, p<0.05) and (t(14)=3.22, p<0.01), respectively ( Figure 9A and 9B ). IL-6 expression was not significantly increased in the DRG at this time point ( Figure 9C ). Overall, α7 deletion in myeloid lineage cells resulted in increased expression of pro-inflammatory cytokines IL-1β and TNF-α peripherally in the DRG at an early time point which was selected based on the time course of the thermal hyperalgesia.

All studies used male and female C57BL/6J mice ≥8 weeks of age (Jackson Laboratory, Bar Harbor, ME). Male and female animals were 8–10 weeks of age at the start of experimentation and were group-housed (4 per cage) in a 21°C humidity-controlled Association for Assessment and Accreditation of Laboratory Animal Care-approved animal care facility with free access to food and water. Experiments were performed during the light cycle and were approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University. All studies were carried out in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. Mice null for the α7 subunits (global knock-out: KO) first described by Orr-Urtreger et. al. (1997), heterozygous (HET), and their wild-type (WT) littermates were obtained from The Jackson Laboratory. 23 They were bred in an animal care facility at Virginia Commonwealth University. Mutants and WT controls were obtained from crossing HET mice for all experiments. This breeding scheme permits rigorous control for anomalies that may occur when crossing only mutant animals. Generation of Lyz2-Cre Driven α7 Conditional KO Mice. Lyz2Cre mice are commonly used to knock down a gene in myeloid cells, which include macrophages, via Cre-lox methodology. 24 , 25 Lyz2-Cre driven α7 cKO mice were generated using two previously developed lines, the Lyz2-Cre and α7 flox/flox mice strains, purchased from JAX labs (Bar Harbor, ME, USA). Lyz2-Cre transgenic mice (aka LysM-Cre mice), which express Cre recombinase under the control of the lysosome 2 promoter, were cross-bred with α7flox/flox mice, in which exon 4 of the endogenous a7 gene is flanked by two loxP sites. This resulted in the generation of littermates that were either LysM-Cre negative/α7 flox/flox (cre-, control mice) or LysM-Cre positive/α7 flox/flox (cre+, cKO), in which the loxP flanked region of exon 4 is ligated via Cre-mediated cleavage, which results in the production of an unstable mRNA and thus creates a “null” allele. Genotyping of LysM-Cre gene in mouse tissue samples (tail tip) was performed by Transnetyx (Cordova, TN, USA) and confirmed using sequence specific probes (Lyzs-2 KO: 5'-GCCAGCAGCTGACCCA-3', 5'-GGTCAGTAAATTGGACACCTTCCT-3', 5'-CACCATGCCCAAGAAGA-3'; Chrna7–4 FL: 5'-CCACATGTGCATTGGAGAAGACA-3', 5'-CATATTCAATAACCCTTAATATAACTTCGTATAATGTATGCTA-3', 5'-TTTGGACCTAGCGATACCG-3'; Chrna7–4 WT: 5'-GGTGCCAGACCACATGTGCA-3', 5'-GATCTTGGTCAAAATGCAACAGACACA-3', 5'-CAGATAAGAACCTAGGTCC-3'). Following genotype confirmation, confirmation that exon 4 deletion in the α7 allele was present in the target cells, macrophages, and relevant tissues involved in the neurotransmission of pain was desirable. Peritoneal macrophage (FAC sorted), lumbar dorsal root ganglia (L4–6 DRGs), dorsal horn of the spinal cord (SC), and liver (which is rich in myeloid lineage cells) samples were collected from cre- and cre+ mice (1 male and 1 female from both genotypes). Purified DNA was extracted from these samples, and PCR amplification was performed using a 3-primer protocol (primer 1: 5'-TTCTGCCTTATGAAAACAAACGG-3'; primer 2: 5'-CAGATGGGCAATGATCTTCACTTG-3'; and primer 3: 5'-CAACAGACACACGGTGTACAATGG-3'). 1275-bp and 617-bp PCR products were generated for the floxed α7 (Chrna7-CKO) allele, while a 381-bp product was produced for the CRE-mediated KO (Chrna7-KO) allele. CRE-mediated deletion of exon 4 (encoding amino acid residues 81–116) produced a translational 3'). To investigate the role of α7 nAChRs in chronic neuropathic pain, a well-established model of paclitaxel-induced peripheral neuropathy was used. 26 Paclitaxel (Athenex, NDC 70860–200-50, Richmond, VA, USA) was obtained from VCU Health Pharmacy. Paclitaxel was dissolved in a 1:1:18 mixture of Kolliphor (Sigma-Aldrich, St. Louis, MO, USA), ethanol (Sigma-Aldrich), and distilled water. Paclitaxel was administered intraperitoneally every other day, with four administrations in total, in two regimens: A low dose at 1 mg/kg or a high dose at 8 mg/kg, which were selected based on previous work by Toma et al. (2017). 26 Control mice received the vehicle (1:1:18, ethanol, kolliphor, and distilled water) at a volume of 10 mL/kg, i.p., and followed the same injection schedule. Male and female α7 KO and WT mice, as well as α7 cKO and WT mice, received both regimens and were tested at multiple time points after drug injections. Two models were used to study the role of α7 nAChRs in myeloid cells in acute pain: the paw incision and the Escherichia coli-derived Lipopolysaccharides (LPS) models. The paw incision procedure was performed as described by Cowie (2019). 27 Briefly, mice were anesthetized with 5.0% isoflurane and maintained using a constant stream of 2.5% isoflurane in oxygen using a face mask and vaporizer. A 0.5- to 1.0-cm incision was made through the skin and fascia of the right hind paw. After the incision, the flexor muscle was elevated and cut longitudinally with a scalpel, keeping the origin and insertion intact. Two sutures (5–0 nylon, discontinuous) were used to close the wound, with only skin sutured. Corresponding sham groups received an identical duration of isoflurane, and the surgical site was disinfected without incising the paw. The thickness of the right paw was measured both before and after the paw incision at the end of thermal sensitivity assays using a digital caliper (Traceable Calipers, Friendswood, TX, USA). Data were recorded to the nearest ± 0.01 mm. Escherichia coli-derived LPS (026:B6) was purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). LPS was resuspended in sterile saline at a concentration of 2.5 μg/ 20 μL of sterile saline and injected intraplantarly into the dorsal surface of hind paws. Male and female α7 cKO and WT mice were injected intraplantarly with 20 μl of LPS, and mechanical sensitivity and paw diameter were measured at different time points after LPS according to Spulbur et. al. (2012). 28 The Freund's adjuvant (CFA) was chosen as the chronic pain model. CFA is composed of inactivated and dried Mycobacterium tuberculosis and adjuvant and was purchased from Sigma-Aldrich (St. Louis, MO, USA). Male and female α7 cKO and WT mice were injected intraplantarly with 20 μl of CFA (20%, diluted in mineral oil; Sigma-Aldrich), and thermal sensitivity was measured at different time points after CFA. The silent agonist R-47, ( R )-N-(4-Methoxyphenyl)-2-((pyridin-3-yloxy)methyl)piperazine-1-carboxamide Dihydrochloride, was synthesized by following the previously reported experimental procedure. 29 R-47 was dissolved in distilled water (D.W) and given by gavage (p.o) at 15 mg/kg according to previous work by Toma et al. (2019). 22 PNU282987 [N-(3R)-1 Azabicyclo [2.2.2] oct-3-yl-4-chlorobenzamide] was obtained from the National Institute on Drug Abuse (NIDA) supply program (Bethesda, MD) and was dissolved in physiological saline and given at 20 mg/kg, s.c. as described by Donvito et. al. (2017). 30 Pain-like behaviors in mice were measured using stimulus-evoked reflexive assays (mechanical, cold and thermal assays) and non-stimulus evoked/spontaneous pain assays such as the voluntary wheel running test. Locomotor activity in mice was also measured to differentiate a specific effect on the target pain-like behaviors. Mechanical sensitivity thresholds were determined according to the method of Chaplan et. al. (1994) and as adapted in Toma et. al. (2017). 26 , 31 Mice were placed in clear plastic containers on an elevated surface with mesh metal flooring and allowed to acclimate for 30 min before testing. A series of calibrated von Frey filaments (Stoelting, Wood Dale IL, USA) with logarithmically incremental stiffness ranging from 0.07 to 3.6 expressed as diameter sensitivity (ds) log 10 of 10 x force (in milligrams) was applied to the paw with a modified up-down method. 31 , 32 The mechanical threshold was expressed as log 10 of 10 x force (in milligrams), which indicates the force of the von Frey filament to which the animal reacted, defined as paw withdrawal, licking, or shaking. Behavioral testing on animals was performed in a blinded manner. This test was conducted as previously described. 26 Mice were placed in clear plastic containers on an elevated surface with mesh metal flooring and allowed to acclimate for 30 min before testing. A volume of 10 μl of acetone was projected from a pipette onto the plantar surface of each hind paw. Time spent licking, lifting, and/or shaking the hind paw was recorded by a stopwatch over one minute. Paw withdrawal latency (PWL) to noxious thermal stimuli was performed using the Hargreaves method. 33 Briefly, mice were placed in clear plastic containers on an elevated glass surface and allowed to acclimate for 30 min before testing. A radiant heat source was applied to the plantar surface of the hind paw of the mice, and the PWL (around 11 seconds for naive mice) was calculated as the average of three measures of the time from onset of radiant heat exposure to withdrawal of the hind paw. The PWL was then used to determine the degree of hyperalgesia. The thickness of treated or injured paws was measured before and after injections or incisions at different time points using a digital caliper (Traceable Calipers, Friendswood, TX). Data were recorded to the nearest ±0.01 mm and expressed as the change in paw thickness (ΔPD = difference in the ipsilateral paw diameter before and after injection paw thickness). cKO and WT male and female mice treated with paclitaxel (1 mg/kg, 4 injections every other day) or vehicle (n=8/group) were individually placed in boxes (20 × 24 × 16.5 cm, Omnitech, Columbus, OH) where locomotor activity was recorded by MED-PC IV software for 30 min. The test was performed 7 days after the last injection of paclitaxel or vehicle. Locomotor activity scores were defined as the number of interruptions of the photobeam cells measured for the total test period. Data were expressed as the mean of the number of photocell interruptions. The activity in the voluntary wheel running test of global and cKO and WT male and female mice treated with paclitaxel (8 mg/kg, 4 injections every other day) or vehicle (n=8/group) was determined by measuring the distance traveled using the wheel running test as previously reported. 34 Briefly, mice were placed in wheels with a diameter of 21.5 cm and a width of 5 cm. An electronic sensor recorded the number of complete rotations of the wheel during a 2-h session (10 a.m.–12 p.m.). Wheel running was performed 7 days after the last injection of paclitaxel or vehicle. The distance traveled was determined by multiplying the number of rotations performed by the wheel's circumference (0.68 cm). Thioglycollate Medium was used to enhance the number of circulating activated macrophages. Powder Thioglycollate broth was diluted according to the manufacturer’s protocol (Sigma-Aldrich, St. Louis, MO, USA). After the appropriate aging time of the media (after the color change), each mouse was injected with 2 ml of 3% thioglycollate medium i.p. using a 10 ml syringe with a 26G needle. Three days after the media injection, mice were euthanized by cervical dislocation, and peritoneal cells were collected via peritoneal lavage as previously described. 35 Firstly, RBC lysis buffer (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to eliminate red blood cells within the cell suspension. The magnetic sorting method (Miltenyi Biotec, CA, USA) was used to separate macrophages from the other cells according to the manufacturer’s protocol. Briefly, the cell number was counted using the manufacturer’s protocol. Moving forward the protocol was followed based on per 10 7 cells count. The cell suspension was centrifuged at 300 × g for 5 min. Supernatant was aspirated, and the cell pellet was resuspended in 36 μL of buffer. Then, 4 μL of FcR Blocking reagent and 10 μL of Macrophage (Peritoneum) Biotin-Antibody Cocktail were added. The suspension was mixed well and incubated in the refrigerator (2−8 °C) for 10 min. The cells were washed by adding 2 mL of buffer and centrifuged at 300×g for 5 min. After the centrifuge, supernatant was aspirated, and cell pellets were resuspended in 80 μL of buffer. 20 μL of Anti-Biotin MicroBeads were added to the solution and incubated for 15 min in the refrigerator (2−8 °C) after mixing the solution. The cells were washed by adding 2 mL of buffer and centrifuged at 300×g for 5 min. After the centrifuge, the supernatant was aspirated, and the cell pellets were resuspended in 500 μL of buffer. The cell suspension was run through the magnetic columns to separate bead label all other cell types from macrophages. Mice were euthanized by cervical dislocation, and peritoneal cells were collected via peritoneal lavage as previously described. 35 FAC sorting was performed similar to that described by Prat et al. (2023). 36 Before staining with extracellular fluorescent antibodies, cell suspensions were incubated with ACK lysis buffer (ThermoFisher Scientific, Waltham, MA) to remove red blood cells. Cell suspensions were then incubated with TruStain FcX (anti-mouse CD16/32, cat# 101320, BioLegend, San Diego, CA, USA) to block Fc receptors and prevent non-specific binding. Next, cell suspensions were then incubated with fluorescent antibodies [CD19 APC (cat# 152410); CD90.2-APC (cat# 105312); CD45-APC FIRE (cat# 103154); F4/80-BV421 (cat# 123132); and CD11b-PE/Cy7 (cat# 101216), BioLegend)] to identify peritoneal macrophage (CD90.2-/CD19-/CD45+/CD11b+/F4/80+) cell populations. Samples were sorted using a BD FACSAria Fusion SORP High-Speed Cell Sorter (BD Biosciences, San Jose, CA) instrument. RNA isolation from nervous tissue (lumbar DRG and spinal cord dissected as previously reported in Caillaud, 2021) was performed using the Invitrogen ™ PureLink ™ RNA Mini Kit Ambion (ThermoFisher Scientific, Waltham, MA, USA). 11 The RNA concentration was measured using a QuBit 3.0 fluorimeter (ThermoFisher Scientific, Waltham, MA, USA). To obtain cDNA, reverse transcription was performed on 20 ng/μL of RNA using the iScript ™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Quantitative real-time PCR (qRT-PCR) was used to quantify Chrna7 (α7) (MmuCIP0034095_m1 chrna7), TNF-α (Mm00443258_m1 Tnf), IL-1β (Mm00434228_m1 Il1b), and IL-6 (Mm00446190_m1 Il6) mRNA expression. Expression of α7, TNF-α, IL-1β, and IL-6 mRNA was normalized using the reference gene: GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) (Mm99999915_g1 Gapdh, Thermo Fisher Scientific). qRT-PCR was performed using a QuantStudio ™ 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) and the qRT-PCR kit TaqMan ™ Gene Expression Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). A cycle threshold (Ct) value for each well was determined using a threshold of 10−1. This value was chosen as it was clearly above the background fluorescence level. A melting curve analysis was performed, which showed single product-specific melting temperatures. No primer dimers were generated during the 40 RT-PCR amplification cycles. Samples were quantified in triplicate for analysis, and values were standardized according to the reference genes. Membrane proteins were solubilized from MAC-sorted peritoneal macrophages as described in King et. al. (2017) and Nordman et. al. (2014). 17 , 37 Briefly, membrane proteins were solubilized by incubation of cells overnight at 4°C with a 0.1% Triton lysis buffer solution (Triton X-100, 150 mM NaCl, 20 mM Tris HCl, 2 mM EDTA, and 10% glycerol, supplemented with protease inhibitors (Roche, Complete Mini 11836153001) and PhosSTOP (Sigma Aldrich 4906845001)). The soluble fraction was collected by centrifuging for 20 minutes at 12,000 RPM and 4°C. Total concentration of protein was measured using a Bradford assay. Proteins were separated onto a NuPAGE 4–12% Bis-Tris gradient gel (Thermo Fisher NP0322BOX) and transferred onto a nitrocellulose membrane (Thermo Fisher IB301002). Membranes were blocked with 5% milk before applying primary antibodies: α7 nAChR (Alomone ANC-007) and GAPDH (Cell Signaling 5174) at 4°C for 2 h. Horseradish peroxidase-conjugated secondary antibodies were obtained from Jackson Immunoresearch (West Grove, PA, USA). Proteins were visualized using SuperSignal West Femto (Thermo Fisher 34094) or SuperSignal West Pico PLUS (Thermo Fisher 34577) using the G:BOX Imaging System and GeneSYS software (Syngene, Frederick, MD, USA). SeeBlue Plus2 (Thermo Fisher LC5925) was used as a molecular weight marker on the gel. Band density analysis was measured using Image J (NIH, Bethesda, MD, USA). RNA extractions, sequencing, and data processing were performed similarly to Beierle et. al. (2022). 38 Briefly, cKO and WT male and female mice were treated with paclitaxel (1 mg/kg, 4 injections every other day) or vehicle (n=6 per genotype per sex per treatment, 24 mice total). On day 7 after the first paclitaxel injection, RNA was extracted from L4-L6 spinal cord tissue (RNA later preserved) using Trizol and ethanol precipitation, filtered for cleanup using an Rneasy kit (Qiagen ® ), and DNAse-treated to purify RNA nucleotides, and eluted with RNA-free water. Library preparation (poly-A selection) and RNA-seq were performed at the University of Chicago Genomics facility using Illumina NovaSEQ6000. Reads (100 bp PE, ~30 million clusters per sample) were demultiplexed, aligned, counted, quality-checked and visualized using scruff were trimmed using Timmomatic. 39 , 40 Reads were aligned to the mm10 mouse reference genome using STAR and counted via featureCounts. 41 , 42 Differential gene expression analysis was performed using DESeq2 (v1.38.0) in R (v4.2.0). 43 A multifactorial model was fitted with Genotype (Cre vs. WT), Treatment (Paclitaxel vs. Vehicle), and Sex as factors. Main effects (Genotype, Treatment) and interaction terms (Genotype x Treatment) were tested using the Wald test with Benjamini-Hochberg correction for multiple comparisons. Stratified analyses were also performed to examine treatment effects within each genotype separately. Variance explained by Sex was controlled for in reporting main effects and interactions, due to the small sample size and the lack of reliable Sex effects at the behavioral level. The FASTQ files are publicly accessible at NCBI’s Sequence Read Archive (PRJNA1418145). Processed data including read counts and metadata are publicly available at Gene Expression Omnibus ( GSE318938 ). For enrichment analysis, a threshold-bound approach and a threshold-free appropach for assessing the effects of Genotype, Treatment (collapsed vs. stratified across each genotype), and Genotype x Treatment interaction were used. For analyses involving a threshold (p<0.01), the online database and tool, EnrichR to input gene lists was used. 44 Significantly enriched pathways (p adjusted 200 gene set libraries, including those under “Pathways” (Reactome Pathways 2024, Wikipathways 2024 Human/Mouse, Bioplanet 2019, KEGG 2021 Human, Elsevier Pathway Collection, MsigDB Hallmark 2020) “Ontologies” (GO Biological/Cellular/Molecular, MGI Mammalian Phenotype Level 4 2024, KOMP2 Mouse Phenotypes 2022), and “Cell Types” (CellMarker 2024, Descartes 2021, Mouse Gene Atlas, Azimuth Cell Types 2021). For the threshold-free approach, Gene Set Enrichment Analysis (GSEA) was run using the fgsea package (v1.24.0) 45 with gene sets from the Molecular Signatures Database (MSigDB) to derive biological pathways associated with main effect of Genotype, main effect of Treatment, and Genotype x Treatment interaction. Genes were ranked by the Wald statistic from DESeq2. Two gene set collections were used - Hallmark gene sets (50 curated gene sets representing well-defined biological states) and Gene Ontology Biological Process (GO:BP) terms. GSEA was run with a minimum gene set size of 15 and maximum of 500. Pathways with FDR-adjusted p-values of < 0.05 were considered significantly enriched. Both positive (upregulated) and negative (downregulated) normalized enrichment scores (NES) for each enriched pathway are reported visually with dot plots for the top 20 enrichment terms that also visually highlight gene list size as well as strength of enrichment with a color-coded p-value range. Patients and the public were not involved in the creation of this work. All data are expressed as mean ± SEM (standard error of the mean). Normality and equal variance were verified by Shapiro–Wilk and Brown–Forsythe tests, respectively. Data were then compared using a t-test or a one-way, two-way, or three-way analysis of variance (ANOVA) with repeated measures, followed by post-hoc Tukey’s comparison test. All statistical analyses were performed with GraphPad statistical software (GraphPad Software, Inc., La Jolla, CA, USA). Differences were considered significant when p was <0.05, <0.01, or <0.001.

Chronic pain and neuroinflammation are critical public health concerns, and therapeutic solutions are urgently required. α7 nAChRs are possible targets for the mediation of pain and inflammation in human and mouse models. 15 In this study, the role of α7 nAChRs in CIPN and inflammatory pain was investigated using genetic mouse models to evaluate the receptor globally and specifically within myeloid-lineage cells. Mice deficient in α7 nAChR in myeloid cells exhibited a marked increase in various aspects of CIPN and chronic inflammation-related pain-like behaviors, accompanied by increased expression of pro-inflammatory cytokines in the peripheral sensory nervous system. This suggests the presence of an α7 nAChR-dependent endogenous cholinergic anti-inflammatory pathway in myeloid cells contributing to mechanisms of chronic pain. The role of α7 nAChRs in chronic neuropathic pain was examined using a well-established mouse model of CIPN. 26 Paclitaxel treatment increased expression of α7 nAChRs initially in the DRG and subsequently in the spinal cord, demonstrating a time-dependent increase in expression of α7 nAChRs from the periphery to the CNS. These changes prompted exploration of the functional role of α7 nAChRs in CIPN. Thus, the effects of paclitaxel in global α7 KO mice were studied. Significantly increased cold and mechanical sensitivity as well as reduced voluntary wheel running activity was found in α7 KO mice. These results were limited by the lack of further characterization of CIPN effects on sensory nerve compound action potential amplitude or intra-epidermal nerve fiber density; however, the behavioral evidence of CIPN was consistent with that observed in C57BL/6J mice as described previously, offering sufficient confirmation that CIPN was established. 26 This suggests α7 nAChRs play an important role in CIPN, with global loss of α7 expression resulting in worsening of CIPN measures. α7 nAChRs are expressed in neuronal and non-neuronal cells, including myeloid cells like macrophages and microglia. 15 Myeloid cells were selected as the likely location of α7 nAChR action based on previous work demonstrating elevated myeloid cells in injured nerves and DRG at the time of injury or during chronic inflammation. 47 – 50 Although the current study does not determine if paclitaxel-induced changes in α7 nAChR expression in the DRG and spinal cord occur specifically within myeloid cells, results from α7 cKO mice strongly implicate involvement of α7 nAChR expression in myeloid cells in the pathophysiology of CIPN. cKO mice experienced increased cold and mechanical sensitivity following low-dose paclitaxel administration. Additionally, wheel running activity was diminished in paclitaxel-treated α7 cKO mice while locomotor activity was unaffected, indicating that the decrease in voluntary exercise was not due to diminished spontaneous locomotor activity of mice in their environment. Pro-inflammatory cytokines TNF-α, IL-6, and IL-1β were significantly elevated in the DRG of paclitaxel-treated α7 cKO mice as compared to vehicle and WT mice, correlating with the behavioral responses observed. Global KO and cKO mice exhibited similar behavioral responses, suggesting α7 nAChR’s primary mechanism is through myeloid lineage cells. Transcriptome analysis of spinal cord in cKO at baseline suggests a vulnerable biological state as indicated by downregulated Schwan cell terms suggesting reduced lipid processing and trafficking in myeloid cell membranes, reduced cytoskeletal remodeling, and reduced neuronal physiological function. These observations predict an impairment of tissue repair and remodeling in response to paclitaxel. In contrast, WT mice showed the predicted indices of mitochondrial activation and oxidative phosphorylation, neuronal suppression and immune expansion. Paclitaxel administration induced additional, distinct perturbations in the cKO transcriptome, including upregulation of glycogenesis, metabolism, endothelial cells and vasculature, and encapsulating structure organization, cell substrate adhesion, suggesting an increased demand for cytoskeletal remodeling as compensation for pre-existing deficits in cKO. The Genotype x Treatment analysis further supported this distinction. Indices of downregulated chemokine/cytokine/chemotaxis were observed in cKO, along with upregulated glycolysis and cytoskeletal remodeling, and increased energy demand/usage. The paclitaxel-induced spinal cord transcriptome implicates the recruitment of additional biological pathways in cKO mice that may underlie the behavioral hypersensitivity phenotype. The enrichment of multiple gene pathways related to vascular function and endothelial cells in the spinal cord of cKO mice supports previous findings that α7 nAChR function impacts endothelial cell function and angiogenesis. 51 These gene pathways contribute to neuropathic pain development; therefore, the enrichment of vascular function and endothelial cell pathways in the spinal cord may represent an initial CNS disruption that reflects enhanced nociception prior to later increases in cytokine activity. 52 Activation of α7 nAChR is a key negative regulator of the RAGE pathway through initiation of the JAK2/STAT3 pathway, thereby reducing neuroinflammation and alleviating pain behaviors in chronic pain. 53 RAGE directly influences IL-1, IL-6, and TNF-α expression, is expressed in vascular endothelial cells, which are crucial for angiogenesis, and is associated with vascular remodeling. 54 – 56 Spinal RAGE activation participates in the early stages of CIPN in mice. 57 Therefore, α7 cKO and subsequent cytokine upregulation in the DRG may alter RAGE function to influence vascular remodeling in CNS tissues. Future transcriptome studies in the DRG are needed to confirm this possibility. Based on previous reports, α7 agonists including PNU-282987 and R-47 reduce pain-like behaviors in CIPN mouse models. 22 , 30 , 58 Full agonists like PNU-282987 initially trigger opening of the ion channel but ultimately transition the receptor into non-conducting, desensitized conformations. 15 In contrast, silent agonists like R-47 selectively bind α7 nAChRs causing conformational change in the receptor which modulates signal transduction without channel opening or inducing a current. 15 , 20 , 22 Here, the effect of these two agonists was investigated in a CIPN model using cKO mice. Interestingly, the full agonist PNU-282987 reversed CIPN-induced mechanical hypersensitivity similarly in α7 cKO and WT mice, while the silent agonist R-47 had no effect in α7 cKO mice. These findings may be explained by the hypothesis that full agonists target α7 nAChRs in neuronal and non-neuronal tissues, such that their anti-CIPN effect remains intact even in cKO mice, while silent agonists maintain higher pharmacological activity in nonconducting conformations of α7 nAChRs in non-neuronal cells like myeloid cells. 15 , 20 Activity of α7 nAChRs within immune cells, such as myeloid cells, regulates the expression and release of inflammatory cytokines through intracellular signaling pathways, ultimately reducing inflammation. 20 It is hypothesized that α7 nAChRs on immune cells exist in a desensitized state since no currents are observed after agonist application, indicating that α7 nAChR desensitized conformation signaling is metabotropic. 59 – 60 R-47 treatment prevented and reversed CIPN in a mouse model; therefore, since R-47 had no effect on cKO mice, this suggests activation of α7 nAChRs specifically within myeloid lineage cells drives their anti-CIPN effects. 22 The interaction between α7 nAChRs in myeloid cells and α7 silent agonists presents a promising direction for therapeutic targeting in neuropathic pain treatment. Since neuroinflammation is implicated in CIPN, it was hypothesized that α7 nAChRs may play a similar role in inflammatory pain. 7 , 61 However, in acute pain and inflammation models, no differences in thermal hyperalgesia or mechanical hypersensitivity were found between WT and α7 cKO mice. The change in α7 nAChR expression in the DRG was not assessed, nor was expression of pro-inflammatory cytokines further characterized, as there was no difference in pain-like behaviors between genotypes. A previous study of post-surgical pain in WT and global α7 KO mice similarly demonstrated no difference in thermal hyperalgesia, while the antinociceptive effect of α7 agonist choline was reduced in KO mice. 62 Although the absence of α7 nAChRs in myeloid cells did not enhance the evoked nociceptive hypersensitivity of the α7 cKO mice, α7 agonists produce an antinociceptive effect in acute inflammatory models. 63 – 65 These results suggest that removing the endogenous tone of α7 nAChRs in myeloid cells does not worsen pain-like behavior in a post-surgical or acute inflammatory setting while receptor activation with agonists may shift the cellular environment from pro- to anti-inflammatory. 5 , 18 , 62 In contrast to the acute pain and inflammation models, the CFA-induced chronic inflammation model resulted in increased α7 nAChR expression in the DRG. The absence of α7 nAChRs in myeloid cells exacerbated thermal hypersensitivity and paw edema in cKO mice, and this response was sustained longer than in WT mice. At day 3 after CFA administration, IL-1β and TNF-α expression levels were increased in the DRG, consistent with the increased thermal hypersensitivity and paw edema observed in α7 cKO mice at this time point. Overall these results indicate a key role for α7 nAChRs in myeloid lineage cells in the mediation of chronic inflammatory pain similar to chronic neuropathic pain. Limitations of this work include a lack of targeted analysis of protein level changes in α7 nAChRs and inflammatory cytokine expression in the DRG and spinal cord. Previous work, although not specifically in the DRG or spinal cord, has indicated decreased protein levels of IL-1β, IL-6 and TNF-α following treatment with α7 nAChR agonists. 66 Therefore, it may be anticipated that mRNA expression would correlate with protein levels in this model of CIPN and chronic inflammation. The evaluation of full and silent α7 nAChR agonists does not fully elucidate their different mechanisms of action in α7 cKO mice. Previous studies suggest that nicotinic agonists impact macrophage and microglial migration and proliferation in inflammatory states, decrease pro-inflammatory cytokines TNF-α, IL-6, and IL-1β, and induce anti-inflammatory cytokines such as IL-10 in an α7 nAChR-dependent fashion. 66 – 69 Future work may assess the change in mRNA and protein level expression of pro-inflammatory cytokines in the DRG and spinal cord after treatment with full and silent agonists. Additionally, the role of specific myeloid cells was not examined, and future studies may seek to differentiate them using a specific cre-driver. In conclusion, using a novel cKO mouse model, this study demonstrates that α7 nAChR signaling within myeloid lineage cells plays an important role in CIPN and chronic inflammatory pain. Targeting these receptor subtypes using silent agonists or similar strategies represents a promising therapeutic strategy for CIPN and chronic inflammatory pain conditions.

Chronic pain is increasingly common worldwide. Mediated by myriad pathways and rooted in countless biological, psychological, and social origins, chronic pain can be debilitating and result in significant healthcare costs and lost productivity. 1 In 2021, the Centers for Disease Control and Prevention (CDC) estimated that 20.9% of U.S. adults experienced chronic pain. 2 Chronic pain often results in overtreatment with analgesics that cause undesirable side effects and dependence. 3 Due to its prevalence and impact on the population, it is crucial to understand the biological mechanisms and mediators of chronic pain in order to find new treatments that are efficacious and carry fewer adverse effects. Chronic nociceptive pain is due to the activation of nociceptors following injury, mechanical irritation, or inflammation. When inflammation is acute, it contributes to the normal process of healing. 4 Chronic inflammation is deleterious, compromising the normal functioning and homeostasis of the body. 5 Chronic peripheral neuropathy is associated with many different conditions, including diabetes, trauma, and the use of certain chemotherapeutics. 6 Paclitaxel (Taxol ® ), a chemotherapeutic agent efficacious in breast, ovarian, and non-small cell lung cancers, is associated with chemotherapy-induced peripheral sensory neuropathy (CIPN), due to its neurotoxicity on peripheral nerves. 7 CIPN can limit treatment, resulting in reduced dosing of the chemotherapeutic agent or discontinuation. 8 Several recent studies have focused on reducing and treating CIPN, but there is a continued need to establish adequate therapeutic targets. 9 – 11 In recent decades, studies have shown a role for nicotinic acetylcholine receptors (nAChRs) in the modulation of pain and inflammation. 12 , 13 Neuronal nAChRs are ligand-gated ion channels composed of a pentameric arrangement of α and β subunits in homomeric and heteromeric combinations. 14 Homopentameric α7 nAChRs exhibit high calcium permeability and rapid desensitization following activation. 15 Additionally, α7 nAChRs are found in neuronal and non-neuronal immune cells of the myeloid lineage, such as macrophages and microglia. 15 The function of α7 nAChRs in modulating inflammatory processes has been reinforced by pharmacological findings that the anti-inflammatory effects of nicotine on macrophages are blocked by α7 nAChR antagonists. 12 Studies have demonstrated that α7 nAChRs are coupled to metabotropic signaling pathways, including Gαi and JAK2/STAT3/nuclear factor kappa B (NF-kB), leading to reduced synthesis and release of pro-inflammatory cytokines in macrophages and microglia. 12 , 16 , 17 These receptors appear effectively targeted by various ligands, including orthosteric agonists, allosteric modulators, and a growing class of “silent” agonists that favor the non-conducting metabotropic nAChR state within non-neuronal cells, including immune cells. 18 – 20 Global knockout (KO) of the α7 nAChR in mice results in increased hypersensitivity to thermal and mechanical stimuli following treatment in inflammatory chronic pain models induced by complete Freund’s adjuvant (CFA). 21 Additionally, α7 nAChRs appear to play a role in neuropathic pain as evidenced by a study showing that α7 hypersensitive nAChR mice (α7 nAChR knock-in (KI)) experience decreased hypersensitivity to thermal and mechanical stimuli following neuropathic injury. 21 More recently, it was reported that the an α7 nAChR silent agonist R-47 prevents and reverses CIPN in a mouse model; however, the cell types mediating the beneficial effects of R-47 in regulating neuropathic and inflammatory pain have not been elucidated. 22 In this study, mice with deletion of Chrna7 in myeloid cells (conditional KO or cKO) were engineered to elucidate their role in chronic pain. This study confirms an important role of these α7 nAChRs in chronic neuropathic and inflammatory mouse pain models based on behavioral, physiological, and molecular measures.