Mrgprb2-dependent Mast Cell Activation Plays a Crucial Role in Acute Colitis.

BACKGROUND & AIMS: Mast cells (MCs) are typically found at mucosal surfaces, where their immunoglobulin E (IgE)-dependent activation plays a central role in allergic diseases. Over the past years, signaling through Mas-related G protein-coupled receptor b2 (Mrgprb2) in mice and MRGPRX2 in humans has gained a lot of interest as an alternative MC activation pathway with high therapeutic potential. The aim of this study was to explore the relevance of such IgE-independent, Mrgprb2-mediated signaling in colonic MCs in the healthy and acutely inflamed mouse colon. METHODS: Mrgprb2 expression and functionality was studied using a genetic labeling strategy combined with advanced microscopic imaging. Furthermore, Mrgprb2 knockout (Mrgprb2-/-) mice were used to determine the role of this pathway in a preclinical dextran sodium sulphate (DSS) colitis model. RESULTS: We found that Mrgprb2 acts as a novel MC degranulation pathway in a large subset of connective tissue MCs in the mouse distal colon. Acute DSS colitis induced a pronounced increase of Mrgprb2-expressing MCs, which were found in close association with Substance P-positive nerve fibers. Loss of Mrgprb2-mediated signaling impaired DSS-induced neutrophil influx and significantly impacted on acute colitis progression. CONCLUSIONS: Our findings uncover a novel, IgE-independent MC degranulation pathway in the mouse colon that plays a central role in acute colitis pathophysiology, mainly by safeguarding acute colitis progression and severity in mice. This pseudo allergic, Mrgprb2-induced signaling is part of a hitherto unconsidered colonic neuro-immune pathway and might have significant potential for the further development of effective therapeutic treatment strategies for gastrointestinal disorders, such as ulcerative colitis.

Visualizing the modulation of neurokinin 1 receptor-positive neurons in the superficial dorsal horn by spinal cord stimulation in vivo.

ABSTRACT: Spinal cord stimulation (SCS) is an effective modality for pain treatment, yet its underlying mechanisms remain elusive. Neurokinin 1 receptor-positive (NK1R+) neurons in spinal lamina I play a pivotal role in pain transmission. To enhance our mechanistic understanding of SCS-induced analgesia, we investigated how different SCS paradigms modulate the activation of NK1R+ neurons, by developing NK1R-Cre;GCaMP6s transgenic mice and using in vivo calcium imaging of superficial NK1R+ neurons under anesthesia (1.5% isoflurane). Neurokinin 1 receptor-positive neurons in the lumbar spinal cord (L4-5) showed a greater activation by electrical test stimulation (TS, 3.0 mA, 1 Hz) at the hindpaw at 2 weeks after tibia-sparing nerve injury (SNI-t) than in naïve mice. Spinal cord stimulation was then delivered through a bipolar plate electrode placed epidurally at L1-2 level. The short-term 50-Hz high-intensity SCS (80% motor threshold [MoT], 10 minutes) induced robust and prolonged inhibition of NK1R+ neuronal responses to TS in both naïve and SNI-t mice. The 30-minute 50-Hz and 900-Hz SCS applied at moderate intensity (50% MoT) also significantly inhibited neuronal responses in SNI-t mice. However, at low intensity (20% MoT), the 30-minute 900-Hz SCS only induced persistent neuronal inhibition in naïve mice, but not in SNI-t mice. In conclusion, both 10-minute high-intensity SCS and 30-minute SCS at moderate intensity inhibit the activation of superficial NK1R+ neurons, potentially attenuating spinal nociceptive transmission. Furthermore, in vivo calcium imaging of NK1R+ neurons provides a new approach for exploring the spinal neuronal mechanisms of pain inhibition by neuromodulation pain therapies.

Optotagging and characterization of GABAergic rostral ventromedial medulla (RVM) neurons.

The transmission of nociceptive and pruriceptive signals in the spinal cord is greatly influenced by descending modulation from brain areas such as the rostral ventromedial medulla (RVM). Within the RVM three classes of neurons have been discovered which are relevant to spinal pain modulation, the On, Off, and Neutral cells. These neurons were discovered due to their functional response to nociceptive stimulation. On cells are excited, Off cells are inhibited, and Neutral cells have no response to noxious stimulation. Since these neurons are identified by functional response characteristics it has been difficult to molecularly identify them. In the present study, we leverage our ability to perform optotagging within the RVM to determine whether RVM On, Off, and Neutral cells are GABAergic. We found that 27.27% of RVM On cells, 47.37% of RVM Off cells, and 42.6% of RVM Neutral cells were GABAergic. These results demonstrate that RVM On, Off, and Neutral cells represent a heterogeneous population of neurons and provide a reliable technique for the molecular identification of these neurons.

Human birth tissue products as a non-opioid medicine to inhibit post-surgical pain.

Pain after surgery causes significant suffering. Opioid analgesics cause severe side effects and accidental death. Therefore, there is an urgent need to develop non-opioid therapies for managing post-surgical pain. Local application of Clarix Flo (FLO), a human amniotic membrane (AM) product, attenuated established post-surgical pain hypersensitivity without exhibiting known side effects of opioid use in mice. This effect was achieved through direct inhibition of nociceptive dorsal root ganglion (DRG) neurons via CD44-dependent pathways. We further purified the major matrix component, the heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3) from human AM that has greater purity and water solubility than FLO. HC-HA/PTX3 replicated FLO-induced neuronal and pain inhibition. Mechanistically, HC-HA/PTX3 induced cytoskeleton rearrangements to inhibit sodium current and high-voltage activated calcium current on nociceptive neurons, suggesting it is a key bioactive component mediating pain relief. Collectively, our findings highlight the potential of naturally derived biologics from human birth tissues as an effective non-opioid treatment for post-surgical pain. Moreover, we unravel the underlying mechanisms of pain inhibition induced by FLO and HC-HA/PTX3.

Antibody-mediated autoimmunity in symptom-based disorders: position statement and proceedings from an international workshop.

A 2-day closed workshop was held in Liverpool, United Kingdom, to discuss the results of research concerning symptom-based disorders (SBDs) caused by autoantibodies, share technical knowledge, and consider future plans. Twenty-two speakers and 14 additional participants attended. This workshop set out to consolidate knowledge about the contribution of autoantibodies to SBDs. Persuasive evidence for a causative role of autoantibodies in disease often derives from experimental "passive transfer" approaches, as first established in neurological research. Here, serum immunoglobulin (IgM or IgG) is purified from donated blood and transferred to rodents, either systemically or intrathecally. Rodents are then assessed for the expression of phenotypes resembling the human condition; successful phenotype transfer is considered supportive of or proof for autoimmune pathology. Workshop participants discussed passive transfer models and wider evidence for autoantibody contribution to a range of SBDs. Clinical trials testing autoantibody reduction were presented. Cornerstones of both experimental approaches and clinical trial parameters in this field were distilled and presented in this article. Mounting evidence suggests that immunoglobulin transfer from patient donors often induces the respective SBD phenotype in rodents. Understanding antibody binding epitopes and downstream mechanisms will require substantial research efforts, but treatments to reduce antibody titres can already now be evaluated.

Molecular Display of the Animal Meta-Venome for Discovery of Novel Therapeutic Peptides.

Animal venoms, distinguished by their unique structural features and potent bioactivities, represent a vast and relatively untapped reservoir of therapeutic molecules. However, limitations associated with extracting or expressing large numbers of individual venoms and venom-like molecules have precluded their therapeutic evaluation via high throughput screening. Here, we developed an innovative computational approach to design a highly diverse library of animal venoms and "metavenoms". We employed programmable M13 hyperphage display to preserve critical disulfide-bonded structures for highly parallelized single-round biopanning with quantitation via high-throughput DNA sequencing. Our approach led to the discovery of Kunitz type domain containing proteins that target the human itch receptor Mas-related G protein-coupled receptor X4 (MRGPRX4), which plays a crucial role in itch perception. Deep learning-based structural homology mining identified two endogenous human homologs, tissue factor pathway inhibitor (TFPI) and serine peptidase inhibitor, Kunitz type 2 (SPINT2), which exhibit agonist-dependent potentiation of MRGPRX4. Highly multiplexed screening of animal venoms and metavenoms is therefore a promising approach to uncover new drug candidates.

MRGPRX4 mediates phospho-drug-associated pruritus in a humanized mouse model.

The phosphate modification of drugs is a common chemical strategy to increase solubility and allow for parenteral administration. Unfortunately, phosphate modifications often elicit treatment- or dose-limiting pruritus through an unknown mechanism. Using unbiased high-throughput drug screens, we identified the Mas-related G protein-coupled receptor X4 (MRGPRX4), a primate-specific, sensory neuron receptor previously implicated in itch, as a potential target for phosphate-modified compounds. Using both Gq-mediated calcium mobilization and G protein-independent GPCR assays, we found that phosphate-modified compounds potently activate MRGPRX4. Furthermore, a humanized mouse model expressing MRGPRX4 in sensory neurons exhibited robust phosphomonoester prodrug-evoked itch. To characterize and confirm this interaction, we further determined the structure of MRGPRX4 in complex with a phosphate-modified drug through single-particle cryo-electron microscopy (cryo-EM) and identified critical amino acid residues responsible for the binding of the phosphate group. Together, these findings explain how phosphorylated drugs can elicit treatment-limiting itch and identify MRGPRX4 as a potential therapeutic target to suppress itch and to guide future drug design.

Peripherally restricted cannabinoid and mu-opioid receptor agonists synergistically attenuate neuropathic mechanical hypersensitivity in mice.

ABSTRACT: Many medications commonly used to treat neuropathic pain are associated with significant, dose-limiting adverse effects, including sedation, dizziness, and fatigue. These adverse effects are due to the activity of these medications within the central nervous system. The objective of this work was to investigate the interactions between peripherally restricted cannabinoid receptor and mu-opioid receptor (MOR) agonists on ongoing and evoked neuropathic pain behaviors in mouse models. RNAscope analysis of cannabinoid receptor type 1 (CB1R) and MOR mRNA demonstrated that the mRNA of both receptors is colocalized in both mouse and human dorsal root ganglion. Single-cell RNAseq of dorsal root ganglion from chronic constriction injury mice showed that the mRNA of both receptors (Cnr1 and Oprm1) is coexpressed across different neuron clusters. Myc-CB1R and FLAG-MOR were cotransfected into immortalized HEK-293T cells and were found to interact at a subcellular level. We also find that CB-13 (a peripherally restricted dual CB1R and cannabinoid receptor type 2 agonist) and DALDA (a peripherally restricted MOR agonist) both attenuate mechanical hypersensitivity in a murine model of neuropathic pain. Using isobolographic analysis, we demonstrate that when coadministered, these agents synergistically attenuate mechanical hypersensitivity. Importantly, combination dosing of these agents does not cause any detectable preferential behaviors or motor impairment. However, repeated dosing of these agents is associated with the development of tolerance to these drugs. Collectively, these findings suggest that leveraging synergistic pain inhibition between cannabinoid receptor and MOR agonists in peripheral sensory neurons may be worth examining in patients with neuropathic pain.

The kainate receptor GluK2 mediates cold sensing in mice.

Thermosensors expressed in peripheral somatosensory neurons sense a wide range of environmental temperatures. While thermosensors detecting cool, warm and hot temperatures have all been extensively characterized, little is known about those sensing cold temperatures. Though several candidate cold sensors have been proposed, none has been demonstrated to mediate cold sensing in somatosensory neurons in vivo, leaving a knowledge gap in thermosensation. Here we characterized mice lacking the kainate-type glutamate receptor GluK2, a mammalian homolog of the Caenorhabditis elegans cold sensor GLR-3. While GluK2 knockout mice respond normally to heat and mechanical stimuli, they exhibit a specific deficit in sensing cold but not cool temperatures. Further analysis supports a key role for GluK2 in sensing cold temperatures in somatosensory DRG neurons in the periphery. Our results reveal that GluK2-a glutamate-sensing chemoreceptor mediating synaptic transmission in the central nervous system-is co-opted as a cold-sensing thermoreceptor in the periphery.

Development and characterization of a Gucy2d-cre mouse to selectively manipulate a subset of inhibitory spinal dorsal horn interneurons.

Recent transcriptomic studies identified Gucy2d (encoding guanylate cyclase D) as a highly enriched gene within inhibitory dynorphin interneurons in the mouse spinal dorsal horn. To facilitate investigations into the role of the Gucy2d+ population in somatosensation, Gucy2d-cre transgenic mice were created to permit chemogenetic or optogenetic manipulation of this subset of spinal neurons. Gucy2d-cre mice created via CRISPR/Cas9 genomic knock-in were bred to mice expressing a cre-dependent reporter (either tdTomato or Sun1.GFP fusion protein), and the resulting offspring were characterized. Surprisingly, a much wider population of spinal neurons was labeled by cre-dependent reporter expression than previous mRNA-based studies would suggest. Although the cre-dependent reporter expression faithfully labeled ~75% of cells expressing Gucy2d mRNA in the adult dorsal horn, it also labeled a substantial number of additional inhibitory neurons in which no Gucy2d or Pdyn mRNA was detected. Moreover, cre-dependent reporter was also expressed in various regions of the brain, including the spinal trigeminal nucleus, cerebellum, thalamus, somatosensory cortex, and anterior cingulate cortex. Injection of AAV-CAG-FLEX-tdTomato viral vector into adult Gucy2d-cre mice produced a similar pattern of cre-dependent reporter expression in the spinal cord and brain, which excludes the possibility that the unexpected reporter-labeling of cells in the deep dorsal horn and brain was due to transient Gucy2d expression during early stages of development. Collectively, these results suggest that Gucy2d is expressed in a wider population of cells than previously thought, albeit at levels low enough to avoid detection with commonly used mRNA-based assays. Therefore, it is unlikely that these Gucy2d-cre mice will permit selective manipulation of inhibitory signaling mediated by spinal dynorphin interneurons, but this novel cre driver line may nevertheless be useful to target a broader population of inhibitory spinal dorsal horn neurons.

A GPCR-neuropeptide axis dampens hyperactive neutrophils by promoting an alternative-like polarization during bacterial infection.

The notion that neutrophils exist as a homogeneous population is being replaced with the knowledge that neutrophils adopt different functional states. Neutrophils can have a pro-inflammatory phenotype or an anti-inflammatory state, but how these states are regulated remains unclear. Here, we demonstrated that the neutrophil-expressed G-protein-coupled receptor (GPCR) Mrgpra1 is a negative regulator of neutrophil bactericidal functions. Mrgpra1-mediated signaling was driven by its ligand, neuropeptide FF (NPFF), which dictated the balance between pro- and anti-inflammatory programming. Specifically, the Mrgpra1-NPFF axis counter-regulated interferon (IFN) γ-mediated neutrophil polarization during acute lung infection by favoring an alternative-like polarization, suggesting that it may act to balance overzealous neutrophilic responses. Distinct, cross-regulated populations of neutrophils were the primary source of NPFF and IFNγ during infection. As a subset of neutrophils at steady state expressed NPFF, these findings could have broad implications in various infectious and inflammatory diseases. Therefore, a neutrophil-intrinsic pathway determines their cellular fate, function, and magnitude of infection.

Staphylococcus aureus proteases trigger eosinophil-mediated skin inflammation.

Staphylococcus aureus skin colonization and eosinophil infiltration are associated with many inflammatory skin disorders, including atopic dermatitis, bullous pemphigoid, Netherton's syndrome, and prurigo nodularis. However, whether there is a relationship between S. aureus and eosinophils and how this interaction influences skin inflammation is largely undefined. We show in a preclinical mouse model that S. aureus epicutaneous exposure induced eosinophil-recruiting chemokines and eosinophil infiltration into the skin. Remarkably, we found that eosinophils had a comparable contribution to the skin inflammation as T cells, in a manner dependent on eosinophil-derived IL-17A and IL-17F production. Importantly, IL-36R signaling induced CCL7-mediated eosinophil recruitment to the inflamed skin. Last, S. aureus proteases induced IL-36α expression in keratinocytes, which promoted infiltration of IL-17-producing eosinophils. Collectively, we uncovered a mechanism for S. aureus proteases to trigger eosinophil-mediated skin inflammation, which has implications in the pathogenesis of inflammatory skin diseases.

Single-Cell RNA Sequencing Reveals Dysregulated POSTN+WNT5A+ Fibroblast Subclusters in Prurigo Nodularis.

Prurigo nodularis (PN) is an intensely pruritic, inflammatory skin disease with a poorly understood pathogenesis. We performed single-cell transcriptomic profiling of 28,695 lesional and nonlesional PN cells. Lesional PN has increased dysregulated fibroblasts (FBs) and myofibroblasts. FBs in lesional PN were shifted toward a cancer-associated FB-like phenotype, with POSTN+WNT5A+ cancer-associated FBs increased in PN and similarly so in squamous cell carcinoma. A multicenter cohort study revealed an increased risk of squamous cell carcinoma and cancer-associated FB-associated malignancies (breast and colorectal) in patients with PN. Systemic fibroproliferative diseases (renal sclerosis and idiopathic pulmonary fibrosis) were upregulated in patients with PN. Ligand-receptor analyses demonstrated an FB neuronal axis with FB-derived WNT5A and periostin interactions with neuronal receptors melanoma cell adhesion molecule and ITGAV. These findings identify a pathogenic and targetable POSTN+WNT5A+ FB subpopulation that may predispose cancer-associated FB-associated malignancies in patients with PN.

The MRGPR family of receptors in immunity.

The discovery of Mas-related G protein-coupled receptors (Mrgprs) has opened a compelling chapter in our understanding of immunity and sensory biology. This family of receptors, with their unique expression and diverse ligands, has emerged as key players in inflammatory states and hold the potential to alleviate human diseases. This review will focus on the members of this receptor family expressed on immune cells and how they govern immune and neuro-immune pathways underlying various physiological and pathological states. Immune cell-specific Mrgprs have been shown to control a variety of manifestations, including adverse drug reactions, inflammatory conditions, bacterial immunity, and the sensing of environmental exposures like allergens and irritants.

IL-33 potentiates histaminergic itch.

BACKGROUND: Itch is a common symptom that can greatly diminish quality of life. Histamine is a potent endogenous pruritogen, and while antihistamines are often the first-line treatment for itch, in conditions like chronic spontaneous urticaria (CSU), many patients remain symptomatic while receiving maximal doses. Mechanisms that drive resistance to antihistamines are poorly defined. OBJECTIVES: Signaling of the alarmin cytokine IL-33 in sensory neurons is postulated to drive chronic itch by inducing neuronal sensitization to pruritogens. Thus, we sought to determine if IL-33 can augment histamine-induced (histaminergic) itch. METHODS: Itch behavior was assessed in response to histamine after IL-33 or saline administration. Various stimuli and conditional and global knockout mice were utilized to dissect cellular mechanisms. Multiple existing transcriptomic data sets were evaluated, including single-cell RNA sequencing of human and mouse skin, microarrays of isolated mouse mast cells at steady state and after stimulation with IL-33, and microarrays of skin biopsy samples from subjects with CSU and healthy controls. RESULTS: IL-33 amplifies histaminergic itch independent of IL-33 signaling in sensory neurons. Mast cells are the top expressors of the IL-33 receptor in both human and mouse skin. When stimulated by IL-33, mouse mast cells significantly increase IL-13 levels. Enhancement of histaminergic itch by IL-33 relies on a mast cell- and IL-13-dependent mechanism. IL-33 receptor expression is increased in lesional skin of subjects with CSU compared to healthy controls. CONCLUSIONS: Our findings suggest that IL-33 signaling may be a key driver of histaminergic itch in mast cell-associated pruritic conditions such as CSU.

Targeting sensory neuron GPCRs for peripheral neuropathic pain.

Despite the high prevalence of peripheral neuropathic pain (NP) conditions and significant progress in understanding its underlying mechanisms, the management of peripheral NP remains inadequate. Existing pharmacotherapies for NP act primarily on the central nervous system (CNS) and are often associated with CNS-related adverse effects, limiting their clinical effectiveness. Mounting preclinical evidence indicates that reducing the heightened activity in primary sensory neurons by targeting G-protein-coupled receptors (GPCRs), without activating these receptors in the CNS, relieves pain without central adverse effects. In this review, we focus on recent advancements in GPCR-mediated peripheral pain relief and discuss strategies to advance the development of more effective and safer therapies for peripheral NP by shifting from traditional CNS modulatory approaches toward selective targeting of GPCRs on primary sensory neurons.

Updates in SJS/TEN: collaboration, innovation, and community.

Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis (SJS/TEN) is a predominantly drug-induced disease, with a mortality rate of 15-20%, that engages the expertise of multiple disciplines: dermatology, allergy, immunology, clinical pharmacology, burn surgery, ophthalmology, urogynecology, and psychiatry. SJS/TEN has an incidence of 1-5/million persons per year in the United States, with even higher rates globally. One of the challenges of SJS/TEN has been developing the research infrastructure and coordination to answer questions capable of transforming clinical care and leading to improved patient outcomes. SJS/TEN 2021, the third research meeting of its kind, was held as a virtual meeting on August 28-29, 2021. The meeting brought together 428 international scientists, in addition to a community of 140 SJS/TEN survivors and family members. The goal of the meeting was to brainstorm strategies to support the continued growth of an international SJS/TEN research network, bridging science and the community. The community workshop section of the meeting focused on eight primary themes: mental health, eye care, SJS/TEN in children, non-drug induced SJS/TEN, long-term health complications, new advances in mechanisms and basic science, managing long-term scarring, considerations for skin of color, and COVID-19 vaccines. The meeting featured several important updates and identified areas of unmet research and clinical need that will be highlighted in this white paper.

Effects of Early Exposure to Isoflurane on Susceptibility to Chronic Pain Are Mediated by Increased Neural Activity Due to Actions of the Mammalian Target of the Rapamycin Pathway.

Patients who have undergone surgery in early life may be at elevated risk for suffering neuropathic pain in later life. The risk factors for this susceptibility are not fully understood. Here, we used a mouse chronic pain model to test the hypothesis that early exposure to the general anesthetic (GA) Isoflurane causes cellular and molecular alterations in dorsal spinal cord (DSC) and dorsal root ganglion (DRG) that produces a predisposition to neuropathic pain via an upregulation of the mammalian target of the rapamycin (mTOR) signaling pathway. Mice were exposed to isoflurane at postnatal day 7 (P7) and underwent spared nerve injury at P28 which causes chronic pain. Selected groups were treated with rapamycin, an mTOR inhibitor, for eight weeks. Behavioral tests showed that early isoflurane exposure enhanced susceptibility to chronic pain, and rapamycin treatment improved outcomes. Immunohistochemistry, Western blotting, and q-PCR indicated that isoflurane upregulated mTOR expression and neural activity in DSC and DRG. Accompanying upregulation of mTOR and rapamycin-reversible changes in chronic pain-associated markers, including N-cadherin, cAMP response element-binding protein (CREB), purinergic P2Y12 receptor, glial fibrillary acidic protein (GFAP) in DSC; and connexin 43, phospho-extracellular signal-regulated kinase (p-ERK), GFAP, Iba1 in DRG, were observed. We concluded that early GA exposure, at least with isoflurane, alters the development of pain circuits such that mice are subsequently more vulnerable to chronic neuropathic pain states.

A tactile twist: decoding the phenomena of mechanical itch and alloknesis.

Itch is a sensation in the skin which provokes the desire to scratch. In the past few decades there has been a significant elucidation of the immune and neural pathways which underly the sensation of itch. An interesting divergence in the itch pathway relates to the type of stimulation used to evoke an itchy sensation. Commonly, chemical mediators of itch such as histamine are injected into the skin where they activate their cognate receptors on sensory neurons. Another way to evoke itch, particularly in patients with chronic itch, is to use light mechanical stimulation. Investigation into these pathways utilizing the mouse model have shown that the neuronal pathways which underly chemical itch are distinct from those which mediate itch in response to mechanical stimulation. Specific populations of primary sensory neurons, spinal interneurons and transmission neurons have been identified which suggests a labeled line for itch transmission. Additionally, Piezo channels, which underly mechanosensation, were discovered to play an important role in the mechanical itch pathway. Given these novel findings relating to the mechanical itch pathway, the purpose of this review is to summarize the reports from human subjects and animal studies to highlight the advances in our understanding of mechanical itch and alloknesis.