Peroxisome proliferator-activated receptor-gamma agonist... : PAIN (original) (raw)
1 Introduction
Peripheral neuroimmune interactions play an important role in the development of pain hypersensitivity. Notably, macrophages derived from circulating monocytes are predominately activated in the early course of acute inflammation and promote the development of hyperalgesia [1]. Macrophages can acquire distinct functional phenotypes depending on the microenvironment of inflamed sites. Two well-established polarized phenotypes are referred to as classically activated (M1) and alternatively activated (M2) macrophages [15,21]. M1 macrophages produce high levels of toxic intermediates associated with increased microbicidal activity and pronociceptive mediators such as inducible nitric oxide synthase (iNOS), whereas M2 macrophages have homeostatic functions linked to wound healing and tissue remodeling. Balance between these 2 subsets has a crucial role in regulating inflammation in the peripheral tissues.
Peroxisome proliferator-activated receptor (PPAR)γ is a member of the nuclear hormone receptor family and has been implicated in mediating many metabolic, endocrine, and cardiovascular disorders [3]. In the absence of PPARγ signaling, macrophages do not suppress inflammatory cytokine production or acquire oxidative metabolic activity associated with the M2 phenotype. Indeed, activation of PPARγ potentiates polarization of circulating monocytes to macrophages of the M2 phenotype [4]. Heme oxygenase (HO)-1, identified as a target gene for PPARs in vascular cells [13], is also implicated as a key mediator for macrophage polarization toward an M2 phenotype [35]. In addition, analysis using HO-1 knockdown RAW 264.7 macrophages and macrophages isolated from Mac-PPARγ knockout mice has revealed that HO-1 induction in macrophages is mediated by the activation of PPARγ signaling [34]. These previous reports suggest that PPARγ signaling regulates macrophage polarization towards the M2 phenotype via an HO-1-dependent mechanism.
We have recently reported that the PPARγ agonist rosiglitazone attenuates tactile allodynia by regulating macrophage activation in the early phase of neuropathic pain induced by partial sciatic nerve ligation (PSNL) [33]. In addition, rosiglitazone reduces acute hyperalgesia, with a phenotype shift from M1 to M2 at the injured sites in postincisional pain development [11]. It has been demonstrated that the increase in peripheral HO-1 also promotes cutaneous wound healing [10] and has antihyperalgesic effects at inflamed sites [24,31], suggesting that PPARγ signaling ameliorates pain hypersensitivity through HO-1 induction in macrophages. Recently, the involvement of M1/M2 balance of macrophages in the development of pain hypersensitivity has been implicated. Komori et al. demonstrated that iNOS+/arginase-1(Arg1)−M1 macrophages were infiltrated into the injured sites after PSNL [12]. Persistent hyperalgesia induced by carrageenan or spared nerve injury in kinase G protein-coupled receptor kinase 2-deficient mice was associated with an increased ratio of M1/M2 type markers in spinal cord microglia/macrophages [36]. However, it is still unknown how distinct macrophage phenotypes contribute to the regulation of pain development and exert analgesic effects. In this study, we investigated how PPARγ signaling regulates macrophage polarization and pain development in the course of complete Freund’s adjuvant (CFA)-induced inflammation focusing on the role of HO-1.
2 Materials and methods
2.1 Animals
Male C57BL6 mice aged 8–10weeks were obtained from CLEA Japan (Tokyo, Japan). The Animal Research Committee of Kagoshima University approved all experimental procedures, which were implemented according to the guidelines of the National Institutes of Health and the International Association for the Study of Pain [37]. Mice were housed in groups of 4 or 5 per cage with a 12-hour light–dark cycle.
2.2 CFA-induced inflammatory pain model
Peripheral inflammatory pain was induced by subcutaneous injection of CFA (20μL; Sigma, St. Louis, MO, USA) in the plantar surface of the left hind paws. Rosiglitazone (Cayman Chemical, Ann Arbor, MI, USA) was dissolved in 1:3 solution of dimethyl sulfoxide:phosphate-buffered saline ([PBS] pH 7.2; 0.5mg/mL). Tin protoporphyrin (SnPP; 400nmol/20μL; Tocris Bioscience, Bristol, UK), 2-chloro-5-nitro-N-phenylbenzamide (GW9662, 5ng/10μL; Sigma), or naloxone (1μg/10μL; Wako, Osaka, Japan) was locally injected 30minutes prior to CFA injection and on day 1 after CFA injection as previously described [9,23].
2.3 Depletion of local macrophages
For macrophage depletion, 10μL clodronate encapsulated in liposomes (Clophosome-A) or empty control liposomes (FormuMax, Palo Alto, CA, USA) were locally injected into the hind paw 30minutes before, and on days 1 and 2 after CFA injection.
2.4 Pain behavior
All behavioral experiments were performed by the same tester in a blinded manner. Withdrawal latencies to heat stimuli were assessed by applying a focused radiant heat source to the unrestrained mouse placed on a heat-tempered glass floor using the Paw Thermal Stimulator (UCSD, San Diego, CA, USA). A thermal stimulus was then applied to the plantar surface of each hind paw. Each mouse was tested at an interval of 2–3minutes. The latencies to thermal stimuli were calculated as the mean of 3 trials. A cut-off time was set at 20.5seconds to avoid tissue damage. To evaluate tactile allodynia, calibrated von Frey filaments (0.08–2.0g) were applied to the plantar surface of the hind paw from underneath the mesh floor. The 50% paw withdrawal threshold was determined using the up-down method [6]. Behavioral experiments were performed before the administration of reagents to hind paws on day 1 and day 2.
2.5 Immunohistochemistry
Mice were deeply anesthetized with sodium pentobarbital (50mg/kg intraperitoneally [i.p.]) and perfused transcardially with saline. Tissues were fixed in 4% paraformaldehyde overnight at 4°C and placed in 30% sucrose solution for 24hours at 4°C. Sections (30μm thick) were incubated overnight with primary antibodies to a pan-macrophage marker, F4/80 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), HO-1 (1:100; Santa Cruz Biotechnology), iNOS (1:200; Abcam, Cambridge, UK), or CD206 (1:500; Santa Cruz Biotechnology) at 4°C overnight and then incubated for 1hour at room temperature with secondary antibody labeled with Alexa Fluor 488 or Alexa Fluor 546 (1:500; Invitrogen, Carlsbad, CA, USA) followed by nuclear staining with DAPI (Vector Laboratories, Burlingame, CA, USA). Fluorescent images were obtained using LSM700 imaging systems (Carl Zeiss, Aalen, Germany). The intensity of F4/80 immunofluorescence at clodronate-treated sites, the number of total F4/80+, F4/80+iNOS+, or F4/80+CD206+ cells with clearly visible cell bodies in the skin were evaluated using Image J 1.43u 2010 software (National Institutes of Health, Bethesda, MD, USA).
2.6 Isolation of peritoneal macrophages
Mice were injected i.p. with 3mL 4% thioglycollate (Sigma). After 3days, peritoneal macrophages were collected by peritoneal lavage with 8mL cold PBS. Cells were incubated overnight in 6-well tissue culture plates with Dulbecco’s modified Eagle’s medium. Nonadherent cells were removed with PBS by repeated washing. Cells were stimulated with 10ng/mL interferon (IFN)-γ (PeproTech, Rocky Hill, NJ, USA) and 10ng/mL lipopolysaccharide (LPS; Sigma) for 48hours.
2.7 Quantitative polymerase chain reaction (PCR)
Total RNA of hind paw or peritoneal macrophages was extracted using Sepazol reagent (Nacalai Tesque, Kyoto, Japan). The synthesis of first-strand cDNA was performed using High Capacity RNA-to-cDNA (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer’s instructions. Quantitative polymerase chain reaction (PCR) was performed on an ABI Prism StepOnePlus real-time PCR System (Applied Biosystems) using Taqman Fast Advanced Master Mix (Applied Biosystems) according to the manufacturer’s instructions. Target gene expression was normalized to glyceraldehyde 3-phosphate dehydrogenase.
2.8 Statistical analysis
Values are presented as mean±SEM. Differences among groups were analyzed using 1-way or 2-way analysis of variance (ANOVA) followed by Bonferroni post hoc analysis (GraphPad Prism 5.0, La Jolla, CA, USA). P<0.05 was considered significant.
3 Results
3.1 Local administration of rosiglitazone ameliorates CFA-induced mechanical hyperalgesia
To evaluate the effects of rosiglitazone on the development of CFA-induced inflammatory hyperalgesia, rosiglitazone was injected locally into the hind paw 30minutes before and on day 1 after CFA injection. Hyperalgesia to mechanical stimuli was dose-dependently attenuated on days 5 and 7 after the procedure in mice that received rosiglitazone, which was reversed to the level of vehicle-injected mice by coadministration of selective PPARγ receptor antagonist, GW9662 [Fig. 1, left, 2-way ANOVA interaction: F(20,145)=2.09, P<0.01]. In contrast to the effects of rosiglitazone on mechanical stimuli, rosiglitazone had little effect on withdrawal latency to heat stimuli [Fig. 1, right, 2-way ANOVA interaction: F(20,145)=1.83, _P_=0.068].
Local administration of rosiglitazone dose-dependently alleviated hypersensitivity to mechanical stimuli. Withdrawal threshold to mechanical stimuli (A) and withdrawal latency to thermal stimuli (B) were examined. * P < 0.05. Each bar represents the mean ± SEM (n = 6–8 for each group). CFA, complete Freund’s adjuvant.
3.2 Analgesic effects of rosiglitazone on mechanical stimuli was reversed by HO-1 inhibitor
To examine the involvement of PPARγ downstream enzyme, HO-1 in CFA-induced hyperalgesia, an HO-1 inhibitor, SnPP was coadministered with 30μg rosiglitazone before CFA injection and on day 1 after CFA injection. Intraplantar injection of SnPP with rosiglitazone significantly decreased the mechanical threshold compared with a single injection of rosiglitazone [Fig. 2A, 2-way ANOVA interaction: F(15,115)=2.33, P<0.05]. Therefore, we evaluated the expression level of Ho-1 by real-time PCR after CFA injection. CFA induced a 5.2-fold increase in Ho-1 induction in vehicle-treated hind paws on day 1 compared with intact hind paws [Fig. 2B, 2-way ANOVA interaction: F(6,57)=3.20, P<0.0001]. Rosiglitazone markedly increased Ho-1 mRNA expression that reached a maximum level on day 2 (9.9-fold), which was reversed by the PPARγ antagonist GW9662. In addition, HO-1 was mainly expressed by F4/80+ macrophages in CFA-injected inflamed sites on day 2 (Fig. 2C).
Rosiglitazone exerted analgesic effects through the induction of heme oxygenase (HO)-1. (A) Coadministration of HO-1 inhibitor tin protoporphyrin (SnPP) blocked the analgesic effects of rosiglitazone on mechanical stimuli. (B) Gene induction of HO-1 was increased after complete Freund’s adjuvant (CFA) injection in rosiglitazone-treated sites. (C) HO-1 was highly expressed in infiltrated macrophages 2 days after CFA injection in rosiglitazone-treated sites. Green, F4/80; red, HO-1; blue, DAPI. Scale bar, 50 μm. * P < 0.05. Each column represents the mean ± SEM (n = 6–8 for each group).
To test whether rosiglitazone exerted its analgesic effects via macrophages, clodronate liposomes were locally injected on the day of CFA injection and days 1 and 2 after CFA injection. Macrophage depletion was evaluated by immunostaining of F4/80+ macrophages on day 2 (Fig. 3A). F4/80 intensity was decreased by intraplantar injection of clodronate liposomes both in vehicle- and rosiglitazone-treated paws [1-way ANOVA: F(3,17)=14.94, P<0.0001] (Fig. 3B). Macrophage depletion alone did not change the sensitivity to mechanical and thermal stimuli (Fig. 3C). However, the analgesic effects of rosiglitazone on mechanical stimuli were reversed by depletion of infiltrated macrophages [2-way ANOVA interaction: F(15,105)=4.41, P<0.0001]. Therefore, we speculated that macrophages with a distinct phenotype, which were increased by the administration of rosiglitazone but not by vehicle, might contribute to the mechanism of PPARγ/HO-1-dependent analgesia.
Depletion of local macrophages resulted in decreased analgesic effects of rosiglitazone. Macrophage depletion after subcutaneous injection of clodronate liposomes was evaluated by immunostaining of F4/80+ local macrophages. (B) Intensity of F4/80 immunoreactivity was reduced by clodronate liposomes (n = 5–6 for each group). (C) Withdrawal threshold to mechanical stimuli were examined. * P < 0.05. Each point represents the mean ± SEM (n = 5–10 for each group). CFA, complete Freund’s adjuvant; rosi, rosiglitazone.
3.3 Rosiglitazone promotes infiltration of M2 macrophages in an HO-1-dependent manner
To test our hypothesis that M1/M2 balance of macrophages at inflamed sites might regulate CFA-induced inflammatory pain development, macrophages in inflamed paws were counted by immunostaining with F4/80 and an M1-specific marker, iNOS, or an M2-specific marker, CD206. In addition, to examine whether PPARγ signaling regulates macrophage polarity through HO-1 induction, SnPP was locally administered with rosiglitazone (Fig. 4). Induction of the M1 marker iNOS is negatively regulated by PPARγ agonists [26]. The total number of F4/80+ macrophages was significantly reduced at rosiglitazone-treated sites 2days after CFA injection, and reversed by coadministration of SnPP [Fig. 4C; 2-way ANOVA interaction, F(2,29)=5.49, P<0.05]. The number of F4/80+iNOS+ M1 macrophages was reduced by administration of rosiglitazone compared with vehicle-treated sites and to rosi+SnPP-treated sites on day 2 [F(2,29)=26.19, P<0.01]. Consistent with decreased M1 macrophages by rosiglitazone, the number of F4/80+CD206+ M2 macrophages was significantly increased by rosiglitazone compared with vehicle, and effectively reversed by SnPP on day 7 [F(2,29)=8.43, P<0.01]. These data indicate that the increase in M2 macrophages is dependent on HO-1, and macrophage-mediated analgesic effects might be mediated by M2 macrophages increasing in the late phase after rosiglitazone injection. Therefore, late onset of rosiglitazone might be explained by delayed accumulation of M2 macrophages compared with M1 macrophages.
Increase in the infiltration of F4/80+CD206+ M2 macrophages by rosiglitazone was reversed by coadministration with tin protoporphyrin (SnPP). Infiltration of F4/80+iNOS+ M1 macrophages (A) and F4/80+CD206+ M2 macrophages (B) was evaluated on days 2 and 7. (C) The number of total F4/80, F4/80+iNOS+, or F4/80+CD206+ macrophages was counted. Green, F4/80; red, iNOS or CD206. Scale bar, 50 μm. * P < 0.05. Each column represents the mean ± SEM (n = 6–9 for each group). CFA, complete Freund’s adjuvant; rosi, rosiglitazone.
For further assessment of macrophage polarity at CFA-injected inflamed sites, gene expression of M1/M2-related markers was evaluated by quantitative PCR (Fig. 5). Consistent with the increase in iNOS+ M1 macrophages at CFA-injected sites, mRNA of iNOS (Nos2) and other M1 markers, integrin, αX (ItgaX) was also increased after CFA injection in vehicle-injected hind paw. In rosiglitazone-treated sites, Nos2 and ItgaX mRNA was inhibited [2-way ANOVA interaction, Nos2: F(2,32)=6.84, P<0.01; ItgaX: F(2,32)=4.51, P<0.01], whereas the expression of M2 markers, Mrc1 [CD206, F(2,32)=3.91, P<0.05] and Arg1 [F(2,32)=4.80, P<0.05] were markedly upregulated on day 2. It has been reported that peripheral immune cells produce endogenous opioids such as β-endorphin, dynorphin, and enkephalin, and regulate inflammatory pain [17,32]. To investigate whether analgesic effects of rosiglitazone might be due to increased production of endogenous opioids in immune responses, possibly produced by M2 macrophages infiltrated in late phase after CFA injection, gene induction of Pdyn (prodynorphin), Penk (proenkephalin), and the precursor of β-endorphin, Pomc, was examined. Of these endogenous opiates, the expression level of Penk was 3-fold higher in rosiglitazone-treated sites compared with vehicle-treated sites 2 days after CFA injection [F(2,30)=8.90, P<0.01].
Rosiglitazone (rosi) altered the gene induction of M1/M2-related markers and endogenous opioids at the incisional sites. * P < 0.05. Each column represents the mean ± SEM (n = 6–7 for each group). CFA, complete Freund’s adjuvant.
3.4 Rosiglitazone alters macrophage polarity from M1 to M2 with increased induction of HO-1 in peritoneal macrophages
To investigate whether rosiglitazone directly alters macrophage polarity through HO-1, isolated macrophages from mouse peritoneal lavage were directly stimulated by rosiglitazone followed by activation with IFN-γ and LPS. Rosiglitazone significantly increased gene expression of HO-1 48hours after incubation, which was reversed by addition of PPARγ antagonist GW9662, indicating that rosiglitazone directly induces Ho-1 expression by acting on PPARγ receptors expressed by macrophages [Fig. 6 1-way ANOVA, P<0.01, Ho-1: F(3,20)=7.387]. Changes in expression of M1 marker Nos2 [iNOS, F(3,28)=48.11, P<0.0001] and ItgaX [F(3,24)=4.249, P<0.05], M2 marker Mrc1 [CD206, F(3,21)=9.045, P<0.01] and Arg1 [F(3,21)=11.00, P<0.001] by rosiglitazone were reversed by addition of SnPP. Therefore, alterations of macrophage polarity by rosiglitazone might be mediated by HO-1. In addition, we also evaluated whether rosiglitazone directly increased production of endogenous opioids from macrophages. Although the expression of Pdyn and Pomc was not detected, Penk was significantly increased in rosiglitazone-treated peritoneal macrophages [F(3,28)=6.283, P<0.01], consistent with increased expression of Penk in rosiglitazone-injected hind paws (Fig. 5).
Rosiglitazone (rosi) increased gene induction of HO-1 as well as endogenous opioid proenkephalin in interferon (IFN)-γ/lipopolysaccharide (LPS)-stimulated peritoneal macrophages. Gene induction was quantified by real time polymerase chain reaction. * P < 0.05. Each column represents the mean ± SEM (n = 5–8 for each group). CFA, complete Freund’s adjuvant.
3.5 Analgesic effects of rosiglitazone on CFA-induced hyperalgesia is mediated by the activation of opioid receptor
We next examined whether rosiglitazone ameliorated CFA-induced hyperalgesia through induction of proenkephalin, by coadministration of pan-opioid receptor antagonist naloxone with rosiglitazone to CFA-injected hind paws [2-way ANOVA interaction: F(15,120)=1.87, P<0.05] (Fig. 7). Elevation of the mechanical threshold by rosiglitazone after CFA injection was reversed by naloxone. The injection of naloxone itself did not change the mechanical threshold.
Naloxone-reversed elevation of mechanical threshold by rosiglitazone (rosi). Withdrawal threshold to mechanical stimuli were examined. * P < 0.05. Each bar represents the mean ± SEM (n = 7 for each group). CFA, complete Freund’s adjuvant.
4 Discussion
We demonstrated that local administration of PPARγ agonist rosiglitazone attenuated CFA-induced mechanical hyperalgesia and promoted a phenotypic shift of infiltrating macrophages from M1 to M2 at the inflamed sites, with increased gene induction of endogenous opioid proenkephalin, through an HO-1-dependent mechanism (Fig. 8).
Mechanistic summary of peroxisome proliferator-activated receptor (PPAR)γ -mediated analgesia.
Mechanical allodynia was attenuated 5days after CFA injection, consistent with our previous data that rosiglitazone had late onset of analgesic effects in neuropathic pain induced by PSNL [33]. In a previous study, the mechanism by which rosiglitazone exerted its analgesic effects in the late phase was not clarified. It has been reported that macrophage differentiation is mediated by HO-1 [35]. We observed that HO-1 expression reached a maximum level at 2days after CFA injection (Fig. 2B), suggesting that the delayed analgesic effects of rosiglitazone might be explained by the time required for an increase in M2 macrophages after CFA injection followed by HO-1 induction. In support of this hypothesis, it was reported that an influx of M2 macrophages into sites of injured sciatic nerves was preceded by an influx of M1 macrophages [22]. Although gene expression of HO-1 was increased 4.1-fold in the vehicle-injected hind paw on day 2 (Fig. 2B), HO-1 was less evident in macrophages of vehicle-injected sites (Fig. 2C). This might be partly explained by rapid HO-1 protein turnover regulated by the ubiquitin–proteasome system [16].
It was also reported that CD206+ M2 macrophages expressing HO-1 had antioxidant and antiinflammatory properties [2,8]. In addition, HO-1 promotes macrophage polarization toward an M2 phenotype [35] and is antinociceptive against inflammatory pain induced by formalin injection in mouse hind paws [9,28]. These data support our idea that rosiglitazone might ameliorate hyperalgesia by regulating the macrophage phenotype towards M2 through HO-1. In contrast, it was demonstrated that mice lacking C-C chemokine receptor 2, a marker for M1 phenotype and the receptor for macrophage-specific chemoattractant macrophage chemoattractant protein-1, showed impaired inflammatory pain development and decreased macrophage infiltration to inflamed sites [1]. This suggests that infiltration of M1 macrophages exacerbates the development of hyperalgesia. M1 macrophages produce nociceptive mediators such as iNOS, interleukin-1β, and tumor necrosis factor-α [30]. Therefore, the balance between M1 and M2 populations might define the pain threshold followed by inflammation. However, as reported previously using the CFA model [5], macrophage depletion by administration of clodronate liposome did not alter pain hypersensitivity compared with that of empty control liposomes with vehicle in CFA-induced inflammation (Fig. 3C). In contrast, clodronate liposomes significantly reduced the nociceptive threshold compared with empty control liposomes at rosiglitazone-injected sites. Induction of apoptosis by clodronate liposome is a time-dependent process that requires at least 12hours [29], thus, infiltrating macrophages might be effectively depleted at late stages. Taken together with the results of immunostaining for M1/M2 markers (Fig. 4), we speculate that depletion of increased M2 macrophages infiltrating at late stages by clodronate liposome reverses the analgesic effects of rosiglitazone. We previously reported that transplantation of rosiglitazone-treated peritoneal macrophages into injured sites is sufficient to ameliorate pain hypersensitivity after PSNL and incision [11,33]. These data suggest that M2 macrophages mainly contribute to the analgesic effects in CFA-induced inflammatory pain.
In Fig. 4, F4/80+CD206+ macrophages were increased on day 7, however, CD206 mRNA (Mrc1) was increased on day 2 (Fig. 5). We speculate that rosiglitazone promoted the survival of F4/80+CD206+ macrophages, rather than increased the expression of CD206 or infiltration of newly polarized CD206+ cells to the inflamed sites on day 7. The increased number of M2 macrophages by rosiglitazone (Fig. 4C) was associated with an increase in gene induction of proenkephalin in the inflamed hind paw (Fig. 5). It was demonstrated that the antinociceptive effects of endogenous PPARγ agonist, 15-deoxy-PGJ2 (15d-PGJ2), were enhanced by increased macrophage infiltration in paw tissue after local injection of thioglycollate in a prostaglandin E2-induced and carrageenan-induced pain model, which was inhibited by naloxone [23]. In addition, the antinociceptive action of 15d-PGJ2 mainly depends on activation of δ opioid receptors [27], whose main endogenous ligands are Leu- and Met-enkephalin. However, a link between the signaling of PPARγ and opioid receptor activation remains to be elucidated. It was reported that Met-enkephalin induces monocyte polarization to M1 but not M2 phenotype [7]. Although this report implies a negative feedback mechanism by Met-enkephalin, it also indicates that peripheral endogenous opioids, in turn, regulate macrophage polarization. Taken together with our data, rosiglitazone may increase the production of proenkephalin by altering infiltrated macrophage polarization to M2 phenotype, resulting in attenuation of inflammatory pain development. Although it is still unclear whether M2 macrophages predominantly produce endogenous opioids, the involvement of peripheral opioids produced from various types of immune cells in the development of hyperalgesia has been indicated [5,14]. Nadeau et al. reported that 30–40% of accumulated CD45+ immune cells at the site of chronic constriction injury contained endogenous opioids such as β-endorphin, Met-enkephalin, and dynorphin A, and the analgesic effects on neuropathic pain were mediated by these opioids expressed by leukocytes. Furthermore, the number of opioid-containing leukocytes (CD45+3E7+) at the injured nerve was higher in the early stages of neuropathy (2days) than the late stage (14days). Brack et al. demonstrated that the majority of opioid-containing leukocytes were ED1+ monocytes/macrophages at 48hours (71% of total CD45+ leukocytes) and 96hours (77% of total CD45+ leukocytes) after CFA injection to the hind paw. The influx of M2 macrophages was preceded by influx of M1 macrophages as reported previously [22]. Therefore, M2 macrophages might have a role in the production of peripheral opioids and endogenous opioid-dependent analgesia. The expression of Pomc and Pdyn was not different between vehicle- and rosiglitazone-treated sites, and those were not detected in peritoneal macrophages in our study, suggesting that the PPARγ signal in macrophages might attenuate the development of inflammatory hyperalgesia, in part, by regulating the production of proenkephalin.
Although it has been reported that intracerebroventricular or i.p. injection of rosiglitazone attenuates hyperalgesia to heat stimuli in a plantar carrageenan model of inflammatory pain or spinal cord injury model [19,25], rosiglitazone had little effect on thermal hyperalgesia in CFA-induced inflammation (Fig. 1). It was recently reported that rosiglitazone inhibits heat-related transient receptor potential (TRP) channel, TRPM3, but stimulates TRPC5 without activating PPARγ receptor [18]. Therefore, PPARγ-mediated analgesic effects of rosiglitazone on heat stimuli might be modulated by a PPARγ-independent mechanism. In addition, a more recent report by Morgenweck et al. has demonstrated that the blood–brain barrier permeant PPARγ agonist pioglitazone reduced spinal glial activation and that PPARγ activation blocks the development of and reduces established neuropathic pain [20]. Therefore, PPARγ signaling might be involved in the activation and polarization of spinal microglia, derived from circulating monocytes.
In summary, we demonstrated that administration of rosiglitazone markedly alleviated pain hypersensitivity by regulating macrophage polarity in CFA-induced acute inflammation. PPARγ signaling can ameliorate inflammatory responses by switching macrophage polarity to be tissue protective, which may result in reduced pain hypersensitivity. We propose that PPARγ signaling in macrophages might be a potential therapeutic target for the treatment of acute pain development.
Conflict of interest statement
There are no conflicting financial interests in this work.
Acknowledgements
This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (to M.H.-M.) and The Nakatomi Foundation.
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Keywords:
Peroxisome proliferator-activated receptor γ; Rosiglitazone; Heme oxygenase-1; Macrophage polarity; Inflammatory pain
© 2013 Lippincott Williams & Wilkins, Inc.