Regulatory T-cells and IL-5 mediate pain outcomes in a preclinical model of chronic muscle pain.

Fibromyalgia (FM) is a chronic musculoskeletal pain disorder primarily diagnosed in women. Historically, clinical literature focusing on cytokines and immune cells has been inconsistent. However, recent key studies show several layers of immune system dysfunction in FM. Preclinically, studies of the immune system have focused on monocytes with little focus on other immune cells. Importantly, T-cells are implicated in the development and resolution of chronic pain states, particularly in females. Our previous work showed that monocytes from women with FM produced more interleukin 5 (IL-5) and systemic treatment of IL-5 reversed mechanical hypersensitivity in a preclinical model of FM. Typically, IL-5 is produced by TH2-cells, so in this study we assessed T-cell populations and cytokine production in female mice using the acid-induced chronic muscle pain model of FM before and after treatment with IL-5. Two unilateral injections of pH4.0 saline, five days apart, into the gastrocnemius muscle induce long-lasting widespread pain. We found that peripheral (blood) regulatory Thelper-cells (CD4+ FOXP3+) are downregulated in pH4.0-injected mice, with no differences in tissue (lymph nodes) or CD8+ T-cell populations. We tested the analgesic properties of IL-5 using a battery of spontaneous and evoked pain measures. Interestingly, IL-5 treatment induced place preference in mice previously injected with pH4.0 saline. Mice treated with IL-5 show limited changes in T-cell populations compared to controls, with a rescue in regulatory T-cells which positively correlates with improved mechanical hypersensitivity. The experiments in this study provide novel evidence that downregulation of regulatory T-cells play a role in chronic muscle pain pathology in the acidic saline model of FM and that IL-5 signaling is a promising target for future development of therapeutics.

Sensory Neuron TLR4 mediates the development of nerve-injury induced mechanical hypersensitivity in female mice.

Recent studies have brought to light the necessity to discern sex-specific differences in various pain states and different cell-types that mediate these differences. These studies have uncovered the role of neuroimmune interactions to mediate pain states in a sex-specific fashion. While investigating immune function in pain development, we discovered that females utilize immune components of sensory neurons to mediate neuropathic pain development. We utilized two novel transgenic mouse models that eitherrestore expression of toll-like receptor (TLR) 4 inNav1.8 nociceptors on a TLR4-null background (TLR4LoxTB) or remove TLR4 specifically from Nav1.8 nociceptors (TLR4fl/fl). After spared nerve injury (SNI), a model of neuropathic injury, we observed a robust female-specific onset of mechanical hypersensitivity in our transgenic animals. Female Nav1.8-TLR4fl/fl knockout animals were less mechanically sensitive than cre-negative TLR4fl/fl littermates. Conversely, female Nav1.8-TLR4LoxTB reactivated animals were as mechanically sensitive as their wild-type counterparts. These sex and cell-specific effects were not recapitulated in male animals of either strain. Additionally, we find the danger associated molecular pattern, high mobility group box-1 (HGMB1), a potent TLR4 agonist, localization and ATF3 expression in females is dependent on TLR4 expression in dorsal root ganglia (DRG) populations following SNI. These experiments provide novel evidence toward sensory neuron specific modulation of pain in a sex-dependent manner.

Cannabinoid Receptor Type 1 and Its Role as an Analgesic: An Opioid Alternative?

Understanding how the body regulates pain is fundamental to develop rational strategies to combat the growing prevalence of chronic pain states, opioid dependency, and the increased financial burden to the medical care system. Pain is the most prominent reason why Americans seek medical attention and extensive literature has identified the importance of the endocannabinoid pathway in controlling pain. Modulation of the endocannabinoid system offers new therapeutic opportunities for the selective control of excessive neuronal activity in several pain conditions (acute, inflammatory, chronic, and neuropathic). Cannabinoids have a long history of medicinal use and their analgesic properties are well documented; however, there are major impediments to understanding cannabinoid pain modulation. One major issue is the presence of psychotropic side effects associated with D9-tetrahydrocannabinol (THC) or synthetic derivatives, which puts an emphatic brake on their use. This dose-limiting effect prevents the appropriate degree of analgesia . Animal studies have shown that the psychotropic effects are mediated via brain cannabinoid type 1 (CB1) receptors, while analgesic activity in chronic pain states may be mediated via CB1R action in the spinal cord, brainstem, peripheral sensory neurons, or immune cells. The development of appropriate therapies is incumbent on our understanding of the role of peripheral versus central endocannabinoid-driven analgesia. Recent physiological, pharmacological, and anatomical studies provide evidence that one of the main roles of the endocannabinoid system is the regulation of gamma-aminobutyric acid (GABA) and/or glutamate release. This article will review this evidence in the context of its implications for pain. We first provide a brief overview of CB1R's role in the regulation of nociception, followed by a review of the evidence that the peripheral endocannabinoid system modulates nociception. We then look in detail at regulation of central-mediated analgesia, followed up with evidence that cannabinoidmediated modulation of pain involves modulation of GABAergic and glutamatergic neurotransmission in key brain regions. Finally, we discuss cannabinoid action on non-neuronal cells in the context of inflammation and direct modulation of neurons. This work stands to reveal long-standing controversies in the cannabinoid analgesia area that have had an impact on failed clinical trials and implementation of therapeutics targeting this system.

Indirect AMP-Activated Protein Kinase Activators Prevent Incision-Induced Hyperalgesia and Block Hyperalgesic Priming, Whereas Positive Allosteric Modulators Block Only Priming in Mice.

AMP-activated protein kinase (AMPK) is a multifunctional kinase that negatively regulates the mechanistic target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK) signaling, two signaling pathways linked to pain promotion after injury, such as surgical incision. AMPK can be activated directly using positive allosteric modulators, as well as indirectly through the upregulation of upstream kinases, such as liver kinase B1 (LKB1), which is a mechanism of action of metformin. Metformin's antihyperalgesic effects occur only in male mice, raising questions about how metformin regulates pain sensitivity. We used metformin and other structurally distinct AMPK activators narciclasine (NCLS), ZLN-024, and MK8722, to treat incision-induced mechanical hypersensitivity and hyperalgesic priming in male and female mice. Metformin was the only AMPK activator to have sex-specific effects. We also found that indirect AMPK activators metformin and NCLS were able to reduce mechanical hypersensitivity and block hyperalgesic priming, whereas direct AMPK activators ZLN-024 and MK8722 only blocked priming. Direct and indirect AMPK activators stimulated AMPK in dorsal root ganglion (DRG) neuron cultures to a similar degree; however, incision decreased phosphorylated AMPK (p-AMPK) in DRG. Because AMPK phosphorylation is required for kinase activity, we interpret our findings as evidence that indirect AMPK activators are more effective for treating pain hypersensitivity after incision because they can drive increased p-AMPK through upstream kinases like LKB1. These findings have important implications for the development of AMPK-targeting therapeutics for pain treatment. SIGNIFICANCE STATEMENT: Nonopioid treatments for postsurgical pain are needed. Our work focused on whether direct or indirect AMP-activated protein kinase (AMPK) activators would show greater efficacy for inhibiting incisional pain, and we also tested for potential sex differences. We conclude that indirect AMPK activators are likely to be more effective as potential therapeutics for postsurgical pain because they inhibit acute pain caused by incision and prevent the long-term neuronal plasticity that is involved in persistent postsurgical pain. Our work points to the natural product narciclasine, an indirect AMPK activator, as an excellent starting point for development of therapeutics.

The antidiabetic drug metformin prevents and reverses neuropathic pain and spinal cord microglial activation in male but not female mice.

Metformin is a widely prescribed drug used in the treatment of type II diabetes. While the drug has many mechanisms of action, most of these converge on AMP activated protein kinase (AMPK), which metformin activates. AMPK is a multifunctional kinase that is a negative regulator of mechanistic target of rapamycin (mTOR) and mitogen activated protein kinase (MAPK) signaling. Activation of AMPK decreases the excitability of dorsal root ganglion neurons and AMPK activators are effective in reducing chronic pain in inflammatory, post-surgical and neuropathic rodent models. We have previously shown that metformin leads to an enduring resolution of neuropathic pain in the spared nerve injury (SNI) model in male mice and rats. The precise mechanism underlying this long-lasting effect is not known. We conducted experiments to investigate the effects of metformin on SNI-induced microglial activation, a process implicated in the maintenance of neuropathic pain that has recently been shown to be sexually dimorphic. We find that metformin is effective at inhibiting development of neuropathic pain when treatment is given around the time of injury and that metformin is likewise effective at reversing neuropathic mechanical hypersensitivity when treatment is initiation weeks after injury. This effect is linked to decreased Iba-1 staining in the dorsal horn, a marker of microglial activation. Importantly, these positive behavioral and microglia effects of metformin were only observed in male mice. We conclude that the neuropathic pain modifying effects of metformin are sex-specific supporting a differential role for microglial activation in male and female mice.

The influence of sex on neuroimmune communication, pain, and physiology.

With the National Institutes of Health's mandate to consider sex as a biological variable (SABV), there has been a significant increase of studies utilizing both sexes. Historically, we have known that biological sex and hormones influence immunological processes and now studies focusing on interactions between the immune, endocrine, and nervous systems are revealing sex differences that influence pain behavior and various molecular and biochemical processes. Neuroendocrine-immune interactions represent a key integrative discipline that will reveal critical processes in each field as it pertains to novel mechanisms in sex differences and necessary therapeutics. Here we appraise preclinical and clinical literature to discuss these interactions and key pathways that drive cell- and sex-specific differences in immunity, pain, and physiology.

In the last decade, NIH-funded basic and clinical research studies have been mandated to include sex as a biological variable, and this has resulted in a boom of studies discovering sex differences. We know that females are more likely than males to develop chronic pain conditions and experience higher levels of pain for longer periods of time. Conditions that demonstrate this include migraine, arthritis, and peripheral neuropathy. Furthermore, these sex differences extend to surgical outcomes and are observable at the molecular, cellular, and systemic levels. Although pain perception pathways differ by sex, studies are also focusing on differences in the "conversation" between the immune system and the nervous system while addressing implications of sex hormones to gain a better understanding of the impact on pain, physiology, and behavior. Lifestyle factors such as diet and alcohol consumption also play a role in affecting the perception and impact of pain conditions. As this area of research continues to grow, the development and availability of sex-specific treatment options will grow and lead to improved patient outcomes and more effective pain management strategies.

Sex-specific differences in alcohol-induced pain sensitization.

Pain sensitization is a phenomenon that occurs to protect tissues from damage and recent studies have shown how a variety of non-noxious stimuli included in our everyday lives can lead to pain sensitization. Consumption of large amounts of alcohol over a long period of time invokes alcohol use disorder (AUD), a complex pathological state that has many manifestations, including alcohol peripheral neuropathy (neuropathic pain). We asked if 'non-pathological' alcohol consumption can cause pain sensitization in the absence of other pathology? Studies have pointed to glia and other immune cells and their role in pain sensitization that results in cell and sex-specific responses. Using a low-dose and short-term ethanol exposure model, we investigated whether this exposure would sensitize mice to a subthreshold dose of an inflammatory mediator that normally does not induce pain. We observed female mice exhibited specific mechanical and higher thermal sensitivity than males. We also observed an increase in CD68+ macrophages in the ipsilateral dorsal root ganglia (DRG) and Iba1+ microglia in the ipsilateral spinal dorsal horn of animals that were exposed to ethanol and injected with subthreshold inflammatory prostaglandin E2. Our findings suggest that short-term ethanol exposure stimulates peripheral and central, immune and glial activation, respectively to induce pain sensitization. This work begins to reveal a possible mechanism behind the development of alcoholic peripheral neuropathy.

Use of Integrated Optical Clearing and 2-Photon Imaging to Investigate Sex Differences in Neuroimmune Interactions After Peripheral Nerve Injury.

Peripheral nerve injury induces a myriad of immune-derived symptoms that negatively impacts pain, depression, and overall quality of life. Neuroimmune differences underlie sexual dimorphisms in various pain states. The innate immune system is a source of these sex differences, which promotes inflammation and pro-nociception through bidirectional signaling with the nervous system. Spatiotemporal interactions between leukocytes and sensory neurons could hold the key to explain ascribed differences between sexes. To date, studies have found it difficult to display these interactions. We are poised to answer important questions regarding the recruitment of peripheral leukocytes to key tissues of the pain system, the dorsal root ganglia (DRG) and sciatic nerve after nerve injury. We optically clear whole DRGs and sciatic nerves and concomitantly use multi-photon microscopy and transgenic reporter lines, to visualize leukocyte dynamics involved in neuropathic pain development following nerve injury. We observed robust sexual dimorphisms in leukocyte recruitment to the lumbar DRGs after nerve injury. We also assessed immune cell size and morphology to understand activation states in the context of nervous tissue inflammation. The altered mechanisms by which the male and female immune systems respond to nerve injury are still topics of further research, however; the continued use of next-generation imaging with advanced whole tissue image analysis remains an important tool in understanding the reciprocal interactions between neuronal and non-neuronal cells.

Neuroimmune Consequences of eIF4E Phosphorylation on Chemotherapy-Induced Peripheral Neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a major dose-limiting side effect that occurs in up to 63% of patients and has no known effective treatment. A majority of studies do not effectively assess sex differences in the onset and persistence of CIPN. Here we investigated the onset of CIPN, a point of therapeutic intervention where we may limit, or even prevent the development of CIPN. We hypothesized that cap-dependent translation mechanisms are important in early CIPN development and the bi-directional crosstalk between immune cells and nociceptors plays a complementary role to CIPN establishment and sex differences observed. In this study, we used wild type and eIF4E-mutant mice of both sexes to investigate the role of cap-dependent translation and the contribution of immune cells and nociceptors in the periphery and glia in the spinal cord during paclitaxel-induced peripheral neuropathy. We found that systemically administered paclitaxel induces pain-like behaviors in both sexes, increases helper T-lymphocytes, downregulates cytotoxic T-lymphocytes, and increases mitochondrial dysfunction in dorsal root ganglia neurons; all of which is eIF4E-dependent in both sexes. We identified a robust paclitaxel-induced, eIF4E-dependent increase in spinal astrocyte immunoreactivity in males, but not females. Taken together, our data reveals that cap-dependent translation may be a key pathway that presents relevant therapeutic targets during the early phase of CIPN. By targeting the eIF4E complex, we may reduce or reverse the negative effects associated with chemotherapeutic treatments.

Sex- and cell-dependent contribution of peripheral high mobility group box 1 and TLR4 in arthritis-induced pain.

ABSTRACT: Spinal high mobility group box 1 protein (HMGB1) plays crucial roles in arthritis-induced pain; however, the involvement of peripheral HMGB1 has not been examined previously. In this study, we addressed the role of peripheral HMGB1 and explored if sex contributes differentially to nociception in arthritis. We found Hmgb1 expression to be elevated in the ankle joints of male and female mice subjected to collagen antibody-induced arthritis. Blocking the action of peripheral HMGB1, however, only reversed collagen antibody-induced arthritis-mediated hypersensitivity in males. Intra-articular injection of the toll-like receptor (TLR)4-activating, partially reduced disulfide, but not the fully reduced all-thiol, HMGB1 evoked mechanical hypersensitivity in both sexes. A sex-dependent temporal profile in expression of inflammatory factors in the ankle joint was observed in response to intra-articular injection of disulfide HMGB1, with male mice showing a delayed, yet longer-lasting increase in mRNA levels for several of the investigated factors. Intra-articular HMGB1 did not induce cellular infiltration in the ankle joint suggesting its action on tissue resident cells. To further explore possible sex differences in cellular involvement, we used the macrophage inhibitor, minocycline, and mice with specific TLR4 depletion in myeloid cells or nociceptors. We found that inhibition of resident macrophages attenuated HMGB1-induced pain-like behavior only in male mice. Interestingly, although the contribution of TLR4 on myeloid cells to nociception was minimal in females compared to males, TLR4 on nociceptors are important for HMGB1-induced pain in both sexes. Collectively, our work highlights sex- and cellular location-dependent roles of HMGB1 and TLR4 in peripheral pain mechanisms.

Sex-dependent role of microglia in disulfide high mobility group box 1 protein-mediated mechanical hypersensitivity.

ABSTRACT: High mobility group box 1 protein (HMGB1) is increasingly regarded as an important player in the spinal regulation of chronic pain. Although it has been reported that HMGB1 induces spinal glial activation in a Toll-like receptor (TLR)4-dependent fashion, the aspect of sexual dimorphisms has not been thoroughly addressed. Here, we examined whether the action of TLR4-activating, partially reduced disulfide HMGB1 on microglia induces nociceptive behaviors in a sex-dependent manner. We found disulfide HMGB1 to equally increase microglial Iba1 immunoreactivity in lumbar spinal dorsal horn in male and female mice, but evoke higher cytokine and chemokine expression in primary microglial culture derived from males compared to females. Interestingly, TLR4 ablation in myeloid-derived cells, which include microglia, only protected male mice from developing HMGB1-induced mechanical hypersensitivity. Spinal administration of the glial inhibitor, minocycline, with disulfide HMGB1 also prevented pain-like behavior in male mice. To further explore sex difference, we examined the global spinal protein expression using liquid chromatography-mass spectrometry and found several antinociceptive and anti-inflammatory proteins to be upregulated in only male mice subjected to minocycline. One of the proteins elevated, alpha-1-antitrypsin, partially protected males but not females from developing HMGB1-induced pain. Targeting downstream proteins of alpha-1-antitrypsin failed to produce robust sex differences in pain-like behavior, suggesting that several proteins identified by liquid chromatography-mass spectrometry are required to modulate the effects. Taken together, the current study highlights the importance of mapping sex dimorphisms in pain mechanisms and point to processes potentially involved in the spinal antinociceptive effect of microglial inhibition in male mice.

Isolation, culture, and downstream characterization of primary microglia and astrocytes from adult rodent brain and spinal cord.

BACKGROUND: Neuroimmunologists aspire to understand the interactions between neurons, microglia, and astrocytes in the CNS. To study these cells, researchers work with either immortalized cell lines or primary cells acquired from animal tissue. Primary cells reflect in vivo characteristics and functionality compared to immortalized cells; however, they are challenging to acquire and maintain. NEW METHOD: Established protocols to harvest primary glia use neonatal rodents, here we provide a method for simultaneously isolating microglia and astrocytes from brain and/or spinal cord from adult rodents. We utilized a discontinuous percoll density gradient enabling easy discrimination of these cell populations without enzymatic digestion or complex sorting techniques. RESULTS: We found cells isolated from the percoll interface between 70 %-50 % were microglia, as they express ionizing calcium-binding adaptor molecule 1 (Iba1) in immunocytochemistry and CD11bhi and CD45lo using flow cytometry. Isolated cells from the 50 %-30 % interface were astrocytes as they express glial fibrillary acidic protein (GFAP) in immunocytochemistry and Glutamate aspartate transporter (GLAST)-1 using flow cytometry. Cultured microglia and astrocytes showed a functional increase in IL-6 production after treatment of lipopolysaccharide (LPS). COMPARISON WITH EXISTING METHODS: Our method allows for rapid isolation of both microglia and astrocytes in one protocol with relatively few resources, preserves cellular phenotype, and yields high cell numbers without magnetic or antibody sorting. CONCLUSION: Here we show a novel, single protocol to isolate microglia and astrocytes from brain and spinal cord tissue, allowing for culturing and other downstream applications from the cells of animals of various ages, which will be useful for researchers investigating these two major glial cell types from the brain or spinal cord of the same rodent.

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin.

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Temporal and sex differences in the role of BDNF/TrkB signaling in hyperalgesic priming in mice and rats.

Brain-derived neurotrophic factor (BDNF) signaling through its cognate receptor, TrkB, is a well-known promoter of synaptic plasticity at nociceptive synapses in the dorsal horn of the spinal cord. Existing evidence suggests that BDNF/TrkB signaling in neuropathic pain is sex dependent. We tested the hypothesis that the effects of BDNF/TrkB signaling in hyperalgesic priming might also be sexually dimorphic. Using the incision postsurgical pain model in male mice, we show that BDNF sequestration with TrkB-Fc administered at the time of surgery blocks the initiation and maintenance of hyperalgesic priming. However, when BDNF signaling was blocked prior to the precipitation of hyperalgesic priming with prostaglandin E2 (PGE2), priming was not reversed. This result is in contrast to our findings in male mice with interleukin-6 (IL6) as the priming stimulus where TrkB-Fc was effective in reversing the maintenance of hyperalgesic priming. Furthermore, in IL6-induced hyperalgesic priming, the BDNF sequestering agent, TrkB-fc, was effective in reversing the maintenance of hyperalgesic priming in male mice; however, when this experiment was conducted in female mice, we did not observe any effect of TrkB-fc. This markedly sexual dimorphic effect in mice is consistent with recent studies showing a similar effect in neuropathic pain models. We tested whether the sexual dimorphic role for BDNF was consistent across species. Importantly, we find that this sexual dimorphism does not occur in rats where TrkB-fc reverses hyperalgesic priming fully in both sexes. Finally, to determine the source of BDNF in hyperalgesic priming in mice, we used transgenic mice (Cx3cr1CreER  × Bdnfflx/flx mice) with BDNF eliminated from microglia. From these experiments we conclude that BDNF from microglia does not contribute to hyperalgesic priming and that the key source of BDNF for hyperalgesic priming is likely nociceptors in the dorsal root ganglion. These experiments demonstrate the importance of testing mechanistic hypotheses in both sexes in multiple species to gain insight into complex biology underlying chronic pain.

eIF4E phosphorylation regulates ongoing pain, independently of inflammation, and hyperalgesic priming in the mouse CFA model.

Mitogen activated protein kinase-interacting kinase (MNK)-mediated phosphorylation of the mRNA cap binding protein eIF4E controls the translation of a subset of mRNAs that are involved in neuronal and immune plasticity. MNK-eIF4E signaling plays a crucial role in the response of nociceptors to injury and/or inflammatory mediators. This signaling pathway controls changes in excitability that drive acute pain sensitization as well as the translation of mRNAs, such as brain-derived neurotrophic factor (BDNF), that enhance plasticity between dorsal root ganglion (DRG) nociceptors and second order neurons in the spinal dorsal horn. However, since MNK-eIF4E signaling also regulates immune responses, we sought to assess whether decreased pain responses are coupled to decreased inflammatory responses in mice lacking MNK-eIF4E signaling. Our results show that while inflammation resolves more quickly in mice lacking MNK-eIF4E signaling, peak inflammatory responses measured with infrared imaging are not altered in the absence of this signaling pathway even though pain responses are significantly decreased. We also find that inflammation fails to produce hyperalgesic priming, a model for the transition to a chronic pain state, in mice lacking MNK-eIF4E signaling. We conclude that MNK-eIF4E signaling is a critical signaling pathway for the generation of nociceptive plasticity leading to acute pain responses to inflammation and the development of hyperalgesic priming.