Author response: TRPV1 drugs alter core body temperature via central projections of primary afferent sensory neurons
Year: 2022
Type: peer-review
Abstract: Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract TRPV1, a capsaicin- and heat-activated ion channel, is expressed by peripheral nociceptors and has been implicated in various inflammatory and neuropathic pain conditions. Although pharmacological modulation of TRPV1 has attracted therapeutic interest, many TRPV1 agonists and antagonists produce thermomodulatory side effects in animal models and human clinical trials, limiting their utility. These on-target effects may result from the perturbation of TRPV1 receptors on nociceptors, which transduce signals to central thermoregulatory circuits and release proinflammatory factors from their peripheral terminals, most notably the potent vasodilative neuropeptide, calcitonin gene-related peptide (CGRP). Alternatively, these body temperature effects may originate from the modulation of TRPV1 on vascular smooth muscle cells (vSMCs), where channel activation promotes arteriole constriction. Here, we ask which of these pathways is most responsible for the body temperature perturbations elicited by TRPV1 drugs in vivo. We address this question by selectively eliminating TRPV1 expression in sensory neurons or vSMCs and show that only the former abrogates agonist-induced hypothermia and antagonist-induced hyperthermia. Furthermore, lesioning the central projections of TRPV1-positive sensory nerve fibers also abrogates drug-mediated thermomodulation, whereas eliminating CGRP has no effect. Thus, TRPV1 drugs alter core body temperature by modulating sensory input to the central nervous system, rather than through peripheral actions on the vasculature. These findings suggest how mechanistically distinct TRPV1 antagonists may diminish inflammatory pain without affecting core body temperature. Editor's evaluation This study establishes that the activity of TRPV1 channels in nociceptive neurons and their synaptic inputs to the central nervous system are required for the effects on body temperature of TRPV1-acting agonists and antagonists, whereas the activity of TRPV1 channels in the vasculature is dispensable, as is the release of neuropeptides from the peripheral nociceptive terminals. This conclusion is achieved by combining genetic tools to selectively ablate channel expression from either nociceptors or the vasculature, behavioral assays, pharmacology, and calcium imaging. The results have relevant implications for the physiology of pain, thermoregulation, and inflammatory processes, as well as for the clinical development of TRPV1-targeted analgesics. https://doi.org/10.7554/eLife.80139.sa0 Decision letter Reviews on Sciety eLife's review process Introduction The detection of painful stimuli begins with the activation of peripheral sensory neurons known as nociceptors. The capsaicin (vanilloid) receptor, TRPV1, is a nonselective cation channel expressed by a subset of small diameter unmyelinated (C) and medium diameter myelinated (Aδ) nociceptors (Basbaum et al., 2009). TRPV1 is activated by heat (Cao et al., 2013; Caterina et al., 1997) and is modulated by inflammatory mediators, including extracellular protons (Jordt et al., 2000; Tominaga et al., 1998) and bioactive lipids (Cao et al., 2013), making it one of the key polymodal receptors for detecting noxious stimuli. Many studies have found that TRPV1 contributes not only to acute pain, but also to persistent pain conditions, particularly those associated with inflammation (Julius, 2013). As such, TRPV1 has become an actively pursued drug target for analgesic therapy. Unfortunately, this pursuit has been stymied by the fact that many TRPV1 modulators produce core body temperature changes. Injection or consumption of capsaicin, the active component of chili peppers and a specific TRPV1 agonist, drives a range of thermoregulatory processes, including vasodilation, sweating, and panting, all of which promote a decrease in core body temperature (Jancsó-Gábor et al., 1970a; Szolcsányi, 2015). Conversely, many TRPV1 antagonists, including drug candidates, induce hyperthermia both in animal models and in human clinical trials. For example, oral administration of AMG517 increases body temperature of some participants to 39–40°C for 1–4 days (Gavva et al., 2008). Other TRPV1 antagonists, for example, ABT-102 (Othman et al., 2013; Rowbotham et al., 2011), AZD1386 (Gomtsyan and Brederson, 2015; Quiding et al., 2013), and JNJ-39439335 (aka. Mavatrep) (Manitpisitkul et al., 2016), also produce varying degree of hyperthermia at therapeutic dosages. It is unclear what site and mechanism of action underlie these thermoregulatory effects of TRPV1-selective drugs. While nociceptors are the main site of TRPV1 expression, functional channels are also found on vascular smooth muscle cells (vSMCs) within a subset of terminal arterioles in peripheral (but not central) vasculature (Cavanaugh et al., 2011; Phan et al., 2020). The activation of vascular TRPV1 leads to vasoconstriction (Cavanaugh et al., 2011; Phan et al., 2022; Phan et al., 2020), which is generally thought to be associated with decreased blood flow and enhanced heat conservation. On the other hand, vasoconstriction at sites outside of thermoregulatory tissues may shunt blood flow into these areas and promote heat dissipation. Accordingly, it is unknown whether and how vascular TRPV1 participates in drug-mediated body temperature modulation, particularly in comparison with neuronal TRPV1. Also at issue is the relative contribution of the central versus peripheral consequences of the activation of primary afferents. Sensory neuron activation of central pathways involves spinal cord and trigeminal circuits that project to thermoregulatory centers in the brain, which can then mobilize adaptive physiological or behavioral programs, including the regulation of sympathetic outflow (Alawi et al., 2015) for control of thermogenesis, vascular tone, etc. Once activated, subsets of primary sensory neurons also release transmitters peripherally. The neuropeptide calcitonin gene-related peptide (CGRP) is most relevant here as it elicits pronounced local vasodilation following activation of TRPV1 on perivascular sensory nerves (Argunhan and Brain, 2022; Zygmunt et al., 1999). Here, we use genetic and pharmacological approaches to assess the relative contribution of TRPV1 on arterioles versus sensory neurons to agonist- or antagonist-evoked changes in core body temperature. We find that TRPV1 on sensory neurons predominates in mediating these actions, and furthermore, that this requires signal transmission to the central nervous system, but not peripheral release of CGRP. Results Neuronal TRPV1 is essential for drug-mediated changes in core body temperature To determine whether drug-evoked body temperature change is mediated by TRPV1 in peripheral nociceptors versus vSMCs, we generated mice lacking TRPV1 in each of these locales. First, Trpv1fl/fl mice were produced by inserting a pair of loxP sites flanking the second exon of the Trpv1 gene, which contains the initiator ATG codon (Figure 1A). Crossing this line with PirtCre (Anderson et al., 2018) or Tg(Myh11-CreERT2) (Wirth et al., 2008) mice allowed for specific elimination of TRPV1 expression in primary sensory neurons or vSMCs, respectively. We confirmed the selectivity of these genetic manipulations by immunohistochemical staining (Figure 1—figure supplement 1) as well as by functional analysis using calcium imaging, thereby demonstrating loss of TRPV1 expression and/or capsaicin sensitivity within dorsal root ganglion (DRG) neurons or ear skin arteriole segments harvested from these animals (Figure 1B, C). Both cell type-specific knockout lines exhibited normal basal core body temperature and showed no noticeable peculiarities in the circadian pattern of body temperature (Figure 1—figure supplement 2). These phenotypes resemble those observed in global TRPV1 knockout mice (Garami et al., 2011; Iida et al., 2005; Szelényi et al., 2004), likely reflecting redundant or compensatory actions of other thermosensitive receptors in these animals (Solinski and Hoon, 2019; Tan and Knight, 2018). Figure 1 with 2 supplements see all Download asset Open asset Tissue-specific knockout selectively abolishes functional expression of TRPV1 in sensory neurons or vascular smooth muscle cells. (A) Design of the Trpv1-floxed mouse line. Numbered gray boxes indicate exons. (B) Calcium responses of Fura2-AM-loaded dorsal root ganglion (DRG) neurons from sensory neuron-specific knockouts (PirtCre;Trpv1fl/fl), smooth muscle-specific knockouts (Tg(Myh11-CreERT2);Trpv1fl/fl), and their respective genotype controls following capsaicin (5 µM) application. In these and all following experiments, control animals received same tamoxifen treatment as knockouts. High (~50 mM) extracellular K+ was used to reveal all neurons in the imaging field. Left panels show representative images of 340/380 emission ratio in rainbow scale; summary data are in bar graph on right. Each datapoint represents one experimental preparation. Total numbers of K+ responders are indicated in brackets. Statistical analyses by unpaired, two-tailed Welch's t-test; ****: p ≤ 0.0001, ns: not significant. (C) Calcium responses of Fura2-AM-loaded ear arterioles of the indicated genotypes to capsaicin (10 µM) and high extracellular K+ application. Table on right shows the percentage of capsaicin-responsive segments among K+-responsive arteriole segments, with respective number of segments given in brackets. Source data for (B) can be found in Figure 1—source data 1. Figure 1—source data 1 Calcium imaging of DRG neurons from tissue-specific Trpv1 knockouts and controls (related to Figure 1B). https://cdn.elifesciences.org/articles/80139/elife-80139-fig1-data1-v1.xlsx Download elife-80139-fig1-data1-v1.xlsx To examine the acute effects of TRPV1 modulation in wild type or mutant mice, we administered an agonist (capsaicin, 9.8 µmol/kg, single dose, s.c.) and recorded core body temperature changes with telemetric probes implanted in the abdomen. Consistent with previous observations (Caterina et al., 2000), capsaicin-induced robust hypothermia of ~5°C at peak (~1 hr postinjection) in control (Trpv1fl/fl or Tg(Myh11-CreERT2)) mice. Strikingly, this capsaicin-evoked hypothermic response was completely abolished in sensory neuron-specific knockouts (PirtCre;Trpv1fl/fl) (see Figure 2A, upper left for genotype comparison and Figure 2—figure supplement 1 for vehicle vs. drug comparison). These results recapitulate the phenotype observed in global TRPV1 knockouts (Caterina et al., 2000). Unexpectedly, the smooth muscle-specific knockout (Tg(Myh11-CreERT2);Trpv1fl/fl) (Figure 2A, lower left) showed a robust hypothermic response that was even more pronounced in both amplitude and duration compared to controls. Together, these observations show that agonist-evoked hypothermia requires TRPV1 expressed by sensory nerve fibers, but the activation of channels on the vasculature has an opposing effect, likely related to vasoconstriction that limits radiant heat loss. Figure 2 with 1 supplement see all Download asset Open asset Neuronal, rather than vascular, TRPV1 mediates drug-evoked thermomodulatory effects. (A) Telemetric recordings of core body temperatures of PirtCre;Trpv1fl/fl mice (red), Tg(Myh11-CreERT2);Trpv1fl/fl mice (blue), and their respective genotype controls (black). Traces show responses to subcutaneous injection of capsaicin (9.8 µmol/kg) and intraperitoneal injection of AMG517 (250 nmol/kg) administered at time 0, after subtracting measurements from vehicle-alone trials. Baselines (mean temperature across the 1-hr window before drug administration) have been offset to zero for comparison. Responses to AMG517 were smoothened by taking running averages to better display trends. All traces are mean ± standard deviation (SD). Black bars above traces indicate the time windows over which statistical comparisons (mixed effects analyses) between genotypes were made. (B) Body temperature response of the above genotypes to changes in ambient temperature (dotted line, right axis). Statistical comparisons by mixed effects analyses. Source data are in Figure 2—source data 2. (Ci) Representative images (left) and quantification (right) of dorsal root ganglion (DRG) neurons from Tg(Avil-CreERT2);Trpv1fl/fl and control mice that are responsive to capsaicin (5 µM) and high extracellular K+. Source data are in Figure 2—source data 3. (Cii) Body temperature recordings as in (A) but from Tg(Avil-CreERT2);Trpv1fl/fl mice (orange) and controls (black). Source data for (A) and (Cii) can be found in Figure 2—source data 1. For all statistical tests, *: p ≤ 0.05; ***: p ≤ 0.001; ns: not significant. Figure 2—source data 1 Core body temperature responses of tissue-specific Trpv1 knockouts and controls to Trpv1 drugs (related to Figure 2A and Cii). https://cdn.elifesciences.org/articles/80139/elife-80139-fig2-data1-v1.xlsx Download elife-80139-fig2-data1-v1.xlsx Figure 2—source data 2 Core body temperature responses of tissue-specific Trpv1 knockouts and controls to ambient temperature changes (related to Figure 2B). https://cdn.elifesciences.org/articles/80139/elife-80139-fig2-data2-v1.xlsx Download elife-80139-fig2-data2-v1.xlsx Figure 2—source data 3 Calcium imaging of DRG neurons from inducible, sensory neuron-specific Trpv1 knockouts and controls (related to Figure 2Ci). https://cdn.elifesciences.org/articles/80139/elife-80139-fig2-data3-v1.xlsx Download elife-80139-fig2-data3-v1.xlsx We next recorded core body temperature responses following administration of AMG517 (250 ng/kg, 3 doses, i.p.), which we selected as a representative TRPV1 antagonist because its hyperthermic effect has been verified to be on-target (i.e., absent in Trpv1−/− mice) and its dose–response relationship has been documented previously (Garami et al., 2010). Indeed, AMG517 elicited a characteristic hyperthermia in control animals (as in human clinical trials), reaching a peak of ~0.5°C within 2 hr after injection (cf. Garami et al., 2010). This response was abolished in PirtCre;Trpv1fl/fl mice but did not change significantly in Tg(Myh11-CreERT2);Trpv1fl/fl animals (Figure 2A, right column and Figure 2—figure supplement 1). Loss of TRPV1 in either cell type did not impact body temperature regulation in response to changes in ambient temperature (Figure 2B), suggesting that relevant thermosensory mechanisms (e.g., contribution from other thermosensitive TRP channels) and thermoeffector pathways (Tan and Knight, 2018) function normally in these animals and are unlikely to explain the differential pharmacological responses that we observed across genotypes. To rule out developmental factors that could account for the observed lack of capsaicin/AMG517-evoked responses in our neuronal TRPV1 knockout, we also studied an Tg(Avil-CreERT2) (Lau et al., 2011) line to ablate TRPV1 in primary sensory neurons after 8 weeks of age. Functional ablation was less effective and more variable in Tg(Avil-CreERT2);Trpv1fl/fl mice compared to PirtCre;Trpv1fl/fl animals (based on calcium imaging, 11.1 ± 6.0% of DRG neurons remained capsaicin responsive after tamoxifen treatment compared to 42.2 ± 10.2% in Trpv1fl/fl controls) (Figure 2Ci and Figure 1—figure supplement 1). Nonetheless, capsaicin/AMG517-induced body temperature changes were similarly abrogated in these inducible knockouts (Figure 2Cii). These data further support the conclusion that sensory TRPV1 predominates in mediating drug-evoked thermomodulatory responses. Body temperature perturbations require central transmission, but not peripheral CGRP release When activated, primary afferent nociceptors release transmitters from their central terminals to convey signals to the spinal cord and brain. However, these neurons are unique in that they also release transmitters peripherally to initiate neurogenic inflammatory processes, including CGRP-evoked vasodilation. Importantly, such peripheral release occurs independently of central transmission. We therefore asked whether peripheral or central transmission from TRPV1+ nociceptors is required for the capsaicin/AMG517-evoked responses. To address this question, we selectively ablated the central projections of TRPV1+ DRG neurons by intrathecal (IT) injection of capsaicin (10 µg/mouse; control mice received vehicle). Successful ablation of central TRPV1+ terminals was validated by decreased nocifensive behavior on a hotplate (Figure 3A), as previously described (Cavanaugh et al., 2009). This procedure leaves peripheral projections of TRPV1+ fibers intact and capable of releasing proinflammatory agents (Lin King et al., 2019). Indeed, intraplantar injection of the TRPA1 agonist, allyl isothiocyanate (AITC), which activates a subset of TRPV1+ nociceptors (Jordt et al., 2004), failed to induce nocifensive behaviors in animals treated intrathecally with capsaicin (Figure 3A). However, AITC elicited robust local edema in both Cap-IT and Veh-IT control mice (Figure 3A). Interestingly, capsaicin/AMG517-evoked body temperature responses were significantly reduced in mice with ablated central projections (Figure 3B), indicating that central transmission from TRPV1+ DRG fibers is required for drug actions. A small residual effect of these drugs was observed (Figure 3B), which could reflect contributions from incompletely ablated central projections, vagal or trigeminal afferents, or sustained peripheral release of the vasodilative neuropeptide, CGRP. To address this last possibility, we examined mice lacking CGRP (i.e., Calca−/− generated by crossing CalcaCreERT2 to homozygosity) (Song et al., 2012; Figure 3—figure supplement 1) and found that they showed normal body temperature responses to capsaicin or AMG517 (Figure 3C), suggesting that peripheral CGRP release from TRPV1+ nociceptors is largely dispensable in this drug-mediated thermomodulatory process, consistent with a centrally mediated mechanism. Figure 3 with 1 supplement see all Download asset Open asset Thermomodulatory effects of TRPV1-selective drugs require central transmission, but not peripheral calcitonin gene-related peptide (CGRP) release. (A) Comparison of nocifensive behaviors of mice injected intrathecally with capsaicin (Cap-IT, green, 10 µg/mouse, n = 7) versus vehicle controls (black, n = 5). Cap-IT mice were significantly slower (i.e., had higher latency) in showing the first nocifensive response in a hotplate test (left). Cap-IT mice also spent significantly less time in nocifensive behaviors (middle) after intraplantar AITC injection despite similar levels of local edema (right), suggesting successful selective ablation of TRPV1+ nociceptors' central projections. Unpaired, two-tailed Welch's t-test in all panels. See Figure 3—source data 1 for source data. (B) Core body temperature recordings showing diminished hypo- and hyperthermic responses to capsaicin and AMG517, respectively, in Cap-IT mice. (C) Normal hypo- and hyperthermic responses to capsaicin and AMG517, respectively, in Calca−/− mice. All traces are plotted and statistical tests carried out as in Figure 2. For all statistical tests, *: p ≤ 0.05; ****: p ≤ 0.0001; ns: not significant. Source data for (B) and (C) are in Figure 3—source data 2. Figure 3—source data 1 Nocifensive behaviors of Cap-IT and Veh-IT mice (related to Figure 3A). https://cdn.elifesciences.org/articles/80139/elife-80139-fig3-data1-v1.xlsx Download elife-80139-fig3-data1-v1.xlsx Figure 3—source data 2 Core body temperature responses to Trpv1 drugs of Cap-IT and Calca-/- mice as well as controls (related to Figure 3B and C). https://cdn.elifesciences.org/articles/80139/elife-80139-fig3-data2-v1.xlsx Download elife-80139-fig3-data2-v1.xlsx Discussion In the past two decades, TRPV1 antagonists have been actively pursued by the pharmaceutical industry to treat diverse persistent pain conditions. Despite analgesic efficacy in some indications, such as osteoarthritic knee pain, two main 'on-target' adverse side effects have limited further development of these drugs for clinical use: diminished sensitivity to potentially injurious heat, and increased core body temperature (Bamps et al., 2021; Garami et al., 2020; Gomtsyan and Brederson, 2015). Our current work addresses the mechanism underlying the latter adverse effect. Our findings show that the thermomodulatory responses of mice to acute systemic activation/inhibition of TRPV1 are mediated predominantly by the central projections of TRPV1-expressing nociceptors and thus likely involve output from thermoregulatory centers in the hypothalamus, which may recruit the sympathetic system, for example, with the engagement of adrenergic receptors (Alawi et al., 2015). On the other hand, the concurrent and robust agonist-evoked hypothermia in smooth muscle-specific knockouts reveals an opposing action by vascular TRPV1 that may help constrain hypothermic responses within a tolerable temperature or temporal window. Incidentally, Sharif-Naeini et al., 2008 have implicated supraoptic hypothalamic expression of an N-terminal variant of TRPV1 in anticipatory vasopressin release during hyperthermia. Moreover, central injection of TRPV1 agonists have also been reported to trigger transient hypothermia (Hori, 1984; Jancsó-Gábor et al., 1970b; Muzzi et al., 2012) whereas intracerebroventricular injection of a TRPV1 antagonist prolonged hyperthermia induced by exposure to a 35°C environment (Yonghak et al., 2020). However, the precise site of action of such pharmacological manipulations has not been unambiguously demonstrated. Most importantly, our finding that ablation of the central projections of sensory neurons prevents the thermomodulatory side effects of pharmacological manipulation of TRPV1 argues that any hypothalamic expression is not sufficient to sustain these effects in vivo. In addition, our results suggest a rationale for developing TRPV1 antagonists that would selectively or preferentially inhibit peripheral release of proinflammatory transmitters, without compromising the central pathway and thus thermoregulation. This idea is related to our recent analysis of a scorpion toxin that stabilizes the TRPA1 channel in a unique open state with a low Ca2+ permeability (Lin King et al., 2019). Thus, the application of the toxin to nociceptors in vivo promotes neuronal transmission, but not the release of pain without neurogenic modulation of TRPV1 in produce by preferentially channel with high Ca2+ thereby neuropeptide release from peripheral terminals to diminish neurogenic Interestingly, the Ca2+ of TRPV1 current is the channel is activated by low rather than by capsaicin et al., the of Moreover, recent of TRPV1 antagonists that not channel activation less likely to alter core body temperature (Garami et al., (see et al., also for In one such has been on with knee pain and found not to be its analgesic effect was in this one et al., 2018). clinical studies determine whether such drugs are in inflammatory pain acute heat sensitivity and Materials and methods All animal were to and by the and at PirtCre mice were by at and CalcaCreERT2 mice were from the CalcaCreERT2 mice, in which the ATG initiator of the is by a were to to produce For Tg(Myh11-CreERT2) was with tamoxifen for 1 mice were at weeks of age. For Tg(Avil-CreERT2) mice were injected with 2 in for days at 8 weeks of and for days at 10 weeks of to effective were with to and In all experiments, we used mice that were and were because the is into the animals in most were used as genotype controls and were to the same tamoxifen in body were observed across genotypes. of Trpv1fl/fl mice a loxP sites were by the system across the second exon of the Trpv1 gene, where the ATG codon is two and were to at ratio in a at for and then to at The were mixed and with at a of both and at temperature for to the of A a from to of the two loxP sites was into The was by and and was as a to the at a of 10 The was through a to any and was into mouse the loxP were by on and confirmed by A of were used to genotype the Trpv1 Trpv1 The pair of Trpv1 and a for the whereas the pair of Trpv1 and a for the The Trpv1fl/fl mouse line is on to the Calcium imaging a were by by to For calcium imaging of sensory were harvested from a mouse into medium The tissues were with for 10 by and for 10 both at The were then in medium to the neurons and were with and mouse were in medium with and at When used for calcium imaging, neurons were with 2 2 10 and 10 with and with 10 1 in in at temperature in the for 1 neurons were in and and at a time of 2 with a were and with the in a to 10 from a 10 in was by to a of in the of as for and was to reveal neuronal from at two animals were examined for each For calcium imaging of arterioles were from in the following 1 10 and 1 with (Cavanaugh et al., were in and on and calcium imaging were in as that a of capsaicin was used to a of 10 in the a were and implanted each with a temperature
Source: eLife
Institution: University of California System
Language: English
Topic: Ion Channels and Receptors
Subfield: Sensory Systems
Field: Neuroscience
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,500
OpenAlex ID: W4291512912