Endogenous Inflammatory Mediators Produced by Injury Activate TRPV1 and TRPA1 Nociceptors to Induce Sexually Dimorphic Cold Pain That Is Dependent on TRPM8 and GFRα3.

The detection of environmental temperatures is critical for survival, yet inappropriate responses to thermal stimuli can have a negative impact on overall health. The physiological effect of cold is distinct among somatosensory modalities in that it is soothing and analgesic, but also agonizing in the context of tissue damage. Inflammatory mediators produced during injury activate nociceptors to release neuropeptides, such as calcitonin gene-related peptide (CGRP) and substance P, inducing neurogenic inflammation, which further exasperates pain. Many inflammatory mediators induce sensitization to heat and mechanical stimuli but, conversely, inhibit cold responsiveness, and the identity of molecules inducing cold pain peripherally is enigmatic, as are the cellular and molecular mechanisms altering cold sensitivity. Here, we asked whether inflammatory mediators that induce neurogenic inflammation via the nociceptive ion channels TRPV1 (vanilloid subfamily of transient receptor potential channel) and TRPA1 (transient receptor potential ankyrin 1) lead to cold pain in mice. Specifically, we tested cold sensitivity in mice after intraplantar injection of lysophosphatidic acid or 4-hydroxy-2-nonenal, finding that each induces cold pain that is dependent on the cold-gated channel transient receptor potential melastatin 8 (TRPM8). Inhibition of CGRP, substance P, or toll-like receptor 4 (TLR4) signaling attenuates this phenotype, and each neuropeptide produces TRPM8-dependent cold pain directly. Further, the inhibition of CGRP or TLR4 signaling alleviates cold allodynia differentially by sex. Last, cold pain induced by both inflammatory mediators and neuropeptides requires TRPM8, as well as the neurotrophin artemin and its receptor GDNF receptor α3 (GFRα3). These results are consistent with artemin-induced cold allodynia requiring TRPM8, demonstrating that neurogenic inflammation alters cold sensitivity via localized artemin release that induces cold pain via GFRα3 and TRPM8.SIGNIFICANCE STATEMENT The cellular and molecular mechanisms that generate pain are complex with a diverse array of pain-producing molecules generated during injury that act to sensitize peripheral sensory neurons, thereby inducing pain. Here we identify a specific neuroinflammatory pathway involving the ion channel TRPM8 (transient receptor potential cation channel subfamily M member 8) and the neurotrophin receptor GFRα3 (GDNF receptor α3) that leads to cold pain, providing select targets for potential therapies for this pain modality.

Endogenous inflammatory mediators produced by injury activate TRPV1 and TRPA1 nociceptors to induce sexually dimorphic cold pain that is dependent on TRPM8 and GFRα3.

The detection of environmental temperatures is critical for survival, yet inappropriate responses to thermal stimuli can have a negative impact on overall health. The physiological effect of cold is distinct among somatosensory modalities in that it is soothing and analgesic, but also agonizing in the context of tissue damage. Inflammatory mediators produced during injury activate nociceptors to release neuropeptides, such as CGRP and substance P, inducing neurogenic inflammation which further exasperates pain. Many inflammatory mediators induce sensitization to heat and mechanical stimuli but, conversely, inhibit cold responsiveness, and the identity of molecules inducing cold pain peripherally is enigmatic, as are the cellular and molecular mechanisms altering cold sensitivity. Here, we asked if inflammatory mediators that induce neurogenic inflammation via the nociceptive ion channels TRPV1 and TRPA1 lead to cold pain in mice. Specifically, we tested cold sensitivity in mice after intraplantar injection of lysophosphatidic acid (LPA) or 4-hydroxy-2-nonenal (4HNE), finding each induces cold pain that is dependent on the cold-gated channel TRPM8. Inhibition of either CGRP, substance P, or toll-like receptor 4 (TLR4) signaling attenuates this phenotype, and each neuropeptide produces TRPM8-dependent cold pain directly. Further, the inhibition of CGRP or TLR4 signaling alleviates cold allodynia differentially by sex. Lastly, we find that cold pain induced by inflammatory mediators and neuropeptides requires the neurotrophin artemin and its receptor GFRα3. These results demonstrate that tissue damage alters cold sensitivity via neurogenic inflammation, likely leading to localized artemin release that induces cold pain via GFRα3 and TRPM8. Significance Statement: The cellular and molecular mechanisms that generate pain are complex with a diverse array of pain-producing molecules generated during injury that act to sensitize peripheral sensory neurons, thereby inducing pain. Here we identify a specific neuroinflammatory pathway involving the ion channel TRPM8 and the neurotrophin receptor GFRα3 that leads to cold pain, providing select targets for potential therapies for this pain modality.

Immunoglobulin somatic hypermutation in a defined biochemical system recapitulates affinity maturation and permits antibody optimization.

We describe a purified biochemical system to produce monoclonal antibodies (Abs) in vitro using activation-induced deoxycytidine deaminase (AID) and DNA polymerase η (Polη) to diversify immunoglobulin variable gene (IgV) libraries within a phage display format. AID and Polη function during B-cell affinity maturation by catalyzing somatic hypermutation (SHM) of immunoglobulin variable genes (IgV) to generate high-affinity Abs. The IgV mutational motif specificities observed in vivo are conserved in vitro. IgV mutations occurred in antibody complementary determining regions (CDRs) and less frequently in framework (FW) regions. A unique feature of our system is the use of AID and Polη to perform repetitive affinity maturation on libraries reconstructed from a preceding selection step. We have obtained scFv Abs against human glucagon-like peptide-1 receptor (GLP-1R), a target in the treatment of type 2 diabetes, and VHH nanobodies targeting Fatty Acid Amide Hydrolase (FAAH), involved in chronic pain, and artemin, a neurotropic factor that regulates cold pain. A round of in vitro affinity maturation typically resulted in a 2- to 4-fold enhancement in Ab-Ag binding, demonstrating the utility of the system. We tested one of the affinity matured nanobodies and found that it reduced injury-induced cold pain in a mouse model.

Nociceptive afferent phenotyping reveals that transient receptor potential ankyrin 1 promotes cold pain through neurogenic inflammation upstream of the neurotrophic factor receptor GFRα3 and the menthol receptor transient receptor potential melastatin 8.

ABSTRACT: The proper detection and behavioral response to painfully cold temperatures is critical for avoiding potentially harmful tissue damage. Cold allodynia and hyperalgesia, pain associated with innocuous cooling and exaggerated pain with noxious cold, respectively, are common in patients with chronic pain. In peripheral somatosensory afferents, the ion channels transient receptor potential melastatin 8 (TRPM8) and transient receptor potential ankyrin 1 (TRPA1) are candidate receptors for innocuous and noxious cold temperatures, respectively. However, the role of TRPA1 as a cold sensor has remained controversial, and recent evidence suggests that TRPM8 channels and afferents mediate the detection of both pleasant and painful cold. To determine the role of TRPA1 afferents in cold-induced mouse behaviors in vivo, we used functional phenotyping by targeted nerve conduction block with the cell-impermeant lidocaine derivative QX-314. Surprisingly, we find that injection of QX-314 with TRPA1 agonists reduces cold-induced behaviors in mice, but does so in a TRPM8-dependent manner. Moreover, this effect is sexually dimorphic and requires the glial cell line-derived neurotrophic factor receptor GFRα3, as does cold hypersensitivity produced by the activation of TRPA1 channels. Taken together, these results suggest that under conditions of neurogenic inflammation, TRPA1 works upstream of GFRα3 and TRPM8 to produce cold hypersensitivity, providing novel insights into the role of TRPA1 channels in cold pain.

Dominant Role of the Gut Microbiota in Chemotherapy Induced Neuropathic Pain.

Chemotherapy induced peripheral neuropathy (CIPN), a toxic side effect of some cancer treatments, negatively impacts patient outcomes and drastically reduces survivor's quality of life (QOL). Uncovering the mechanisms driving chemotherapy-induced CIPN is urgently needed to facilitate the development of effective treatments, as currently there are none. Observing that C57BL/6 (B6) and 129SvEv (129) mice are respectively sensitive and resistant to Paclitaxel-induced pain, we investigated the involvement of the gut microbiota in this extreme phenotypic response. Reciprocal gut microbiota transfers between B6 and 129 mice as well as antibiotic depletion causally linked gut microbes to Paclitaxel-induced pain sensitivity and resistance. Microglia proliferated in the spinal cords of Paclitaxel treated mice harboring the pain-sensitive B6 microbiota but not the pain-resistant 129 microbiota, which exhibited a notable absence of infiltrating immune cells. Paclitaxel decreased the abundance of Akkermansia muciniphila, which could compromise barrier integrity resulting in systemic exposure to bacterial metabolites and products - that acting via the gut-immune-brain axis - could result in altered brain function. Other bacterial taxa that consistently associated with both bacteria and pain as well as microglia and pain were identified, lending support to our hypothesis that microglia are causally involved in CIPN, and that gut bacteria are drivers of this phenotype.