Angiotensin Signaling Contributes to Neuropathic Pain via Peripheral Macrophages
Angiotensin has been known for nearly 100 years as a hormone that controls blood pressure. But recently, the angiotensin II type 2 receptor (AT2R) has emerged as a promising target for therapeutics aimed at neuropathic pain. Now a pair of studies from D.P. Mohapatra, Washington University, St. Louis, US, and colleagues show that in mice and in human sensory neurons, angiotensin appears to induce neuropathic pain hypersensitivity by signaling through AT2R found not on sensory neurons but on peripheral macrophages that infiltrate the site of nerve injury.
The findings also suggest that peripheral macrophages contribute to nociceptive signaling in sensory neurons via reactive oxygen species (ROS) that activate the TRPA1 ion channel. Further, analgesic effects of blocking AT2R were observed in a mouse model of chronic neuropathic pain, but not in a chronic inflammatory pain model.
The studies, published August 8 in the Journal of Neuroscience and August 21 in PNAS (a third paper has also just been published October 12 in Anesthesia and Analgesia), concur with previous findings on the analgesic effectiveness of AT2R antagonists in rodent neuropathic pain models. But the work contradicts previous findings suggesting that Ang II acts directly at AT2R on rat and human sensory neurons, and that Ang II contributes to neuropathic pain by promoting peripheral nerve outgrowth and by modulating activity at the TRPV1 ion channel (Anand et al., 2013; Danser and Anand, 2014; Anand et al., 2015; and see PRF related news).
“We’ve been looking at AT2R as an intrinsic neuronal receptor, so these new papers present kind of a conundrum,” said Peter Smith, University of Kansas, Kansas City, US, who has studied Ang II in pain models but was not involved in the current studies. “They’re saying there’s no AT2R in sensory ganglia. So there are two camps—those who have found it and a small camp that says it isn’t there.”
However, Smith said, “The findings are not in complete conflict here. The scope of the work they did—it’s a nice body of work, a tour de force. There are a number of facets, and I think parts of the study are very strong, and others are less compelling.”
Translating back from the clinic
Interest in the angiotensin system for pain relief ramped up with the 2014 publication of a successful phase 2 clinical trial of an AT2R antagonist called EMA401 for post-herpetic neuralgia (Rice et al., 2014; also see PRF related comment by Peter Smith).
At that time, Mohapatra’s group was studying the effects of Ang II on dorsal root ganglia (DRG) neurons, but they did not see any evidence for direct action. Once the clinical trials happened, though, “that finding charged us up again—like, ‘Oh, it’s clinically active,’” said Mohapatra, senior author on the new papers. “So instead of the standard neurobiological approach in pain research of going from mechanism to translation, we thought, let’s use a back-translational approach to understand the mechanisms underlying neuropathic pain,” he said.
The clinical trial was conducted by Andrew Rice, Imperial College London, UK, and funded by Australian pharmaceutical company Spinifex. In 2015, the company was acquired by Novartis Pharmaceuticals, and two planned trials of EMA401 for post-herpetic neuralgia and painful diabetic neuropathy were suspended. According to clinicaltrials.gov, two new trials have since started up and are slated for completion in mid-2019.
Neurons are left out of the story
In the Journal of Neuroscience paper, the researchers began by showing that injection of angiotensin II (Ang II) in the hindpaw of mice caused mechanical but not thermal hypersensitivity. Mice that received an intrathecal injection did not develop mechanical hypersensitivity, suggesting that Ang II acted peripherally.
To determine which angiotensin receptor mediated the pain hypersensitivity, the investigators co-administered together with Ang II an antagonist of either the Ang II type 1 receptor (AT1R), which had no effect on the development of hypersensitivity, or an antagonist of AT2R, which blocked the Ang II-mediated pain. Mice lacking AT1R developed hypersensitivity following Ang II injection, but those lacking AT2R did not, confirming that AT2R was responsible for the effects of Ang II on pain sensitivity.
First author Andrew Shepherd and colleagues next investigated whether TRP channels played a role. Mice that received a systemic dose of the TRPA1 inhibitor AP-18 did not develop Ang II-induced mechanical hypersensitivity, nor did mice lacking TRPA1, suggesting that this receptor was involved in Ang II signaling. Ang II-induced hypersensitivity developed normally in mice lacking TRPV1 or TRPV4.
Interestingly, cultured mouse or human sensory neurons from the DRG did not respond to prolonged application of Ang II, nor did Ang II have any effect on TRPA1 or TRPV1 function. “Unfortunately, it failed to do anything on the neurons in vitro,” Mohapatra said. “It was surprising and frustrating, but it made us look further.” They wondered whether AT2R was actually expressed in DRG neurons.
In a line of mice (Agtr2GFP mice) expressing green fluorescent protein (GFP) driven by the Agtr2 promoter (which also drives AT2R expression), the animals showed no detectable GFP in the DRG. “It was not in the neurons or any other cells,” Mohapatra said. In a positive control experiment, AT2R-GFP neurons were present in the spinal cord.
Staining of the DRG with multiple AT2R antibodies was similar in wildtype mice and those lacking AT2R, suggesting that those antibodies lacked specificity. A reverse-transcriptase polymerase chain reaction (RT-PCR) analysis of RNA isolated from mouse and human DRGs did not detect any messenger RNA (mRNA) for Agtr2, the gene that encodes AT2R. Functionally, Ang II did not elicit calcium influx, electrophysiological excitation, or activation of intracellular signaling, all suggesting that Ang II did not activate DRG neurons. Finally, a search of published RNA sequencing data turned up no evidence of Agtr2 mRNA in mouse DRGs or AGTR2 mRNA in human DRGs, nor did deep sequencing, by multiple study authors, of total RNA from human DRGs from healthy controls and from patients with chronic pain.
“These are very divergent results that are difficult to reconcile,” said Peter Smith, but there are possible explanations. “There could be species differences. A lot of the previous work was done in rats, but there has not been much done in mice.”
Shepherd and colleagues examined DRG neurons from healthy controls and from patients with chronic pain, whereas “the human sensory neurons used by Anand et al. were brachial plexus avulsion-injured neurons,” wrote Maree Smith, University of Queensland, Brisbane, Australia, in an email to PRF; it was Smith’s work that sparked the clinical development of EMA401 but she was not involved in the current studies. “This is a point of difference that needs to be further investigated.”
“They’re basing their conclusions on the fact that they don’t pull up AT2R in sequencing,” Peter Smith said. “There is absence of evidence, but the question is, is there evidence of absence?”
In response, Mohapatra told PRF, “Of course we can’t be 100 percent positive that there is no AT2R in neurons under any circumstances. But we show absence of evidence of neuronal protein, of RNA and of effects on function of AT2R, all with both positive and negative controls at multiple levels.”
All eyes on macrophages
“With AT2R being absent from DRG neurons, we next wondered what other cells in the periphery could be contributing,” Mohapatra said. “We saw that a lot of immune cells infiltrated the site of nerve injury, and we found that the majority were macrophages.”
To get a picture of possible macrophage involvement in people with neuropathic pain, the researchers examined leg skin biopsies. “It was surprising that in patients with either diabetic or chemotherapy-induced peripheral neuropathy, the density of macrophages in the skin was way higher compared to healthy age-matched controls,” Mohapatra said.
In mice, staining for macrophages and to some extent for neutrophils was elevated in the hindpaw after Ang II injection. RNA amplification showed that mouse macrophages but not neutrophils contained Agtr1 and Agtr2 mRNA. In a functional assay, application of Ang II to mouse peritoneal macrophages led to increased phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2)—a readout of AT2R activation in cells—in wildtype mice but not those lacking Agtr2. In addition, an AT2R but not an AT1R blocker prevented Ang II-mediated phosphorylation in mouse and human macrophages.
“This is a way to verify that AT2R leads to functional activation of downstream G protein-coupled receptors. In the context of macrophage signaling in pain, we do not know what AT2R-mediated Erk1/2 phosphorylation does,” according to Mohapatra.
Using the Agtr2GFP reporter mice, the authors then showed that AT2R-expressing macrophages infiltrated the site of Ang II injection but not the DRG. The results pointed to peripheral macrophages as a primary player in Ang II-mediated pain sensitivity.
“The work showing the infiltration of AT2R-expressing macrophages into peripheral nerves in GFP mice appears to be compelling, wrote Maree Smith.
Next, Shepherd and colleagues chemogenetically depleted macrophages. Mice were genetically modified so that macrophages express a receptor that, when activated by a designer drug, triggers apoptotic cell death. After treatment, Mohapatra said, “Eighty to ninety percent of peripheral macrophages are dead. The beauty of this depletion method is that the designer drug doesn’t cross the blood-brain barrier, so we’re eliminating peripheral but not central macrophages, or microglia. It’s a powerful approach.”
Mice treated with the designer drug did not develop mechanical hypersensitivity to Ang II as vehicle-treated mice did, though they did develop bradykinin-induced heat and mechanical hypersensitivity, indicating that macrophage depletion did not lead to gross deficits in pain sensitization.
The macrophage-ROS-TRPA1 link
How could macrophages elicit nociceptive signals from sensory neurons? Mouse macrophages did not show calcium responses to application of Ang II or to TRPA1 or TRPV1 agonists. However, macrophage exposure to Ang II led to increased production of reactive oxygen and nitrogen species (ROS/RNS), which was mimicked by an AT2R agonist and blocked by an AT2R antagonist or by a free radical scavenger. Exposure to Ang II did not result in ROS/RNS production in cultured mouse or human DRG neurons. In mice, an increase in ROS/RNS was detected in the hindpaw following Ang II injection, which was blocked by co-injection of an AT2R blocker or a free radical scavenger.
AT2R antagonism provides relief from neuropathic pain by blocking macrophage-to-sensory neuron oxidative stress signaling. Credit: D.P. Mohapatra.
Next, the team circled back to TRPA1, which they had shown was important to neuropathic pain development. Live-cell imaging revealed that mouse DRG neurons responded to Ang II with increased calcium influx only when co-cultured with mouse macrophages. The calcium influx was attenuated by either a free radical scavenger or an AT2R blocker, and it required that AT2R be expressed by macrophages but not by neurons. The group also found evidence for the same macrophage-to-neuron signaling via AT2R, ROS/RNS and TRPA1 in co-cultured human DRG neurons and macrophages. Finally, experiments in cultured human embryonic kidney (HEK) cells transfected with human TRPA1 showed that cysteine modification of the channel was required for the ROS/RNS-mediated signaling.
The authors suggest that several steps in the pathway could be targeted with drugs to alleviate neuropathic pain. And because the drugs would target a peripheral mechanism, that might avoid side effects in the central nervous system.
Mouse models of neuropathic and inflammatory pain
In the PNAS paper, the researchers investigated AT2R signaling in mouse models of neuropathic and inflammatory pain. Mice that received spared nerve injury (SNI) surgery developed mechanical and cold hypersensitivity that was reduced by systemic or local—but not intrathecal—delivery of an AT2R antagonist. After SNI, Ang II levels were elevated in the injured nerve but not in the spinal cord, bolstering the case for a peripheral action of Ang II, Mohapatra said.
Interestingly, inhibition of AT2R did not alleviate pain behaviors in mice with chronic hindpaw inflammation following injection of complete Freund’s adjuvant (CFA). “This preliminary finding suggests that Ang II may spur neuropathic but not inflammatory pain, but more rigorous experimentation needs to be conducted to understand the role of AT2R in inflammatory pain,” according to Mohapatra.
Systemic delivery of a TRPA1 antagonist attenuated both cold and mechanical hypersensitivity in SNI mice, lending further support to the idea that peripheral signaling between AT2R-containing macrophages and TRPA1 on sensory neurons contributes to neuropathic pain hypersensitivity.
Finally, after SNI, macrophages expressing AT2R had infiltrated the injured nerves. Using the chemogenetic strategy, the researchers showed that as peripheral macrophages were depleted, the cold and mechanical hypersensitivity were attenuated, but the hypersensitivity returned when peripheral macrophages were allowed to recover. Spinal microglia were not affected.
It’s complicated
Despite the discrepancy with previous studies of AT2R in neurons, the new work reveals a novel peripheral mechanism driven by Ang II. “DRG nociceptive signaling is very complex,” Peter Smith said. “There may be multiple convergent pathways here, and how they interact, whether they are sequential or parallel, is not entirely clear.”
Maree Smith wrote in an email, “the mechanism is complex, involving peripheral immune cells (T cells, macrophages), satellite glial cells and neurons. More work is required to further elucidate the mechanism.”
In the end, if a drug works, is it important to know how? “If we know the mechanism, we could identify multiple targets that could be utilized to develop new drugs.” Plus, Mohapatra said, “if we know where and how the drug works, we could potentially fix side effects,” should any arise. And, he added, “if a drug shows effectiveness in humans, it provides a valuable opportunity to understand the biology underlying the specific disease condition.”
Stephani Sutherland, PhD, is a neuroscientist, yogi, and freelance writer in Southern California.
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