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1.
Immunity ; 55(9): 1645-1662.e7, 2022 09 13.
Article in English | MEDLINE | ID: mdl-35882236

ABSTRACT

Healthy skin maintains a diverse microbiome and a potent immune system to fight off infections. Here, we discovered that the epithelial-cell-derived antimicrobial peptides defensins activated orphan G-protein-coupled receptors (GPCRs) Mrgpra2a/b on neutrophils. This signaling axis was required for effective neutrophil-mediated skin immunity and microbiome homeostasis. We generated mutant mouse lines lacking the entire Defensin (Def) gene cluster in keratinocytes or Mrgpra2a/b. Def and Mrgpra2 mutant animals both exhibited skin dysbiosis, with reduced microbial diversity and expansion of Staphylococcus species. Defensins and Mrgpra2 were critical for combating S. aureus infections and the formation of neutrophil abscesses, a hallmark of antibacterial immunity. Activation of Mrgpra2 by defensin triggered neutrophil release of IL-1ß and CXCL2 which are vital for proper amplification and propagation of the antibacterial immune response. This study demonstrated the importance of epithelial-neutrophil signaling via the defensin-Mrgpra2 axis in maintaining healthy skin ecology and promoting antibacterial host defense.


Subject(s)
Bacterial Infections , Neutrophils , Receptors, G-Protein-Coupled , Animals , Mice , Anti-Bacterial Agents , Carrier Proteins , Defensins/genetics , Dysbiosis , Keratinocytes , Receptors, G-Protein-Coupled/metabolism , Staphylococcus aureus
2.
Immunity ; 51(3): 426-428, 2019 09 17.
Article in English | MEDLINE | ID: mdl-31533055

ABSTRACT

The skin is densely innervated with nociceptive neurons specialized in detecting noxious and painful stimuli. In a recent issue of Cell, Cohen et al. report that activation of cutaneous nociceptive neurons leads to a nerve-reflex action that is sufficient to provide a danger signal that triggers regional immunity to fight a microbial challenge.


Subject(s)
Nociceptors , Pain , Humans , Neurons , Reflex , Skin , TRPV Cation Channels
3.
Immunity ; 50(5): 1163-1171.e5, 2019 05 21.
Article in English | MEDLINE | ID: mdl-31027996

ABSTRACT

Classical itch studies have focused on immunoglobulin E (IgE)-mediated mast cell activation and histamine release. Recently, members of the Mas-related G-protein-coupled receptor (Mrgpr) family have been identified as mast cell receptors, but their role in itch is unclear. Here, we report that mast cell activation via Mrgprb2 evoked non-histaminergic itch in mice independently of the IgE-Fc epsilon RI (FcεRI)-histamine axis. Compared with IgE-FcεRI stimulation, Mrgprb2 activation of mast cells was distinct in both released substances (histamine, serotonin, and tryptase) and the pattern of activated itch-sensory neurons. Mrgprb2 deficiency decreased itch in multiple preclinical models of allergic contact dermatitis (ACD), a pruritic inflammatory skin disorder, and both mast cell number and PAMP1-20 concentrations (agonist of the human Mrgprb2 homolog, MRGPRX2) were increased in human ACD skin. These findings suggest that this pathway may represent a therapeutic target for treating ACD and mast-cell-associated itch disorders in which antihistamines are ineffective.


Subject(s)
Mast Cells/immunology , Nerve Tissue Proteins/metabolism , Pruritus/pathology , Receptors, G-Protein-Coupled/metabolism , Receptors, IgE/metabolism , Receptors, Neuropeptide/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Female , Histamine/metabolism , Histamine Antagonists/therapeutic use , Humans , Immunoglobulin E/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Peptide Fragments/metabolism , Receptors, G-Protein-Coupled/genetics , Serotonin/metabolism , Skin/metabolism , Tryptases/metabolism , Young Adult
4.
Proc Natl Acad Sci U S A ; 121(6): e2309243121, 2024 Feb 06.
Article in English | MEDLINE | ID: mdl-38289950

ABSTRACT

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.


Subject(s)
Dermatitis, Atopic , Eosinophilia , Staphylococcal Infections , Animals , Mice , Eosinophils/metabolism , Staphylococcus aureus/metabolism , Peptide Hydrolases/metabolism , Skin/metabolism , Dermatitis, Atopic/metabolism , Staphylococcal Infections/metabolism , Cellulitis/metabolism , Cellulitis/pathology , Inflammation/metabolism
5.
Proc Natl Acad Sci U S A ; 117(45): 28485-28495, 2020 11 10.
Article in English | MEDLINE | ID: mdl-33097666

ABSTRACT

The recent discovery of sensory (tastant and odorant) G protein-coupled receptors on the smooth muscle of human bronchi suggests unappreciated therapeutic targets in the management of obstructive lung diseases. Here we have characterized the effects of a wide range of volatile odorants on the contractile state of airway smooth muscle (ASM) and uncovered a complex mechanism of odorant-evoked signaling properties that regulate excitation-contraction (E-C) coupling in human ASM cells. Initial studies established multiple odorous molecules capable of increasing intracellular calcium ([Ca2+]i) in ASM cells, some of which were (paradoxically) associated with ASM relaxation. Subsequent studies showed a terpenoid molecule (nerol)-stimulated OR2W3 caused increases in [Ca2+]i and relaxation of ASM cells. Of note, OR2W3-evoked [Ca2+]i mobilization and ASM relaxation required Ca2+ flux through the store-operated calcium entry (SOCE) pathway and accompanied plasma membrane depolarization. This chemosensory odorant receptor response was not mediated by adenylyl cyclase (AC)/cyclic nucleotide-gated (CNG) channels or by protein kinase A (PKA) activity. Instead, ASM olfactory responses to the monoterpene nerol were predominated by the activity of Ca2+-activated chloride channels (TMEM16A), including the cystic fibrosis transmembrane conductance regulator (CFTR) expressed on endo(sarco)plasmic reticulum. These findings demonstrate compartmentalization of Ca2+ signals dictates the odorant receptor OR2W3-induced ASM relaxation and identify a previously unrecognized E-C coupling mechanism that could be exploited in the development of therapeutics to treat obstructive lung diseases.


Subject(s)
Anoctamin-1/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Muscle, Smooth/metabolism , Neoplasm Proteins/metabolism , Receptors, Odorant/metabolism , Adenylyl Cyclases/metabolism , Bronchi/metabolism , Calcium/metabolism , Cells, Cultured , Humans , Lung/metabolism , Muscle Contraction/physiology , Muscle Relaxation , Myocytes, Smooth Muscle/metabolism , Receptors, Odorant/genetics
6.
Proc Natl Acad Sci U S A ; 116(22): 10917-10926, 2019 05 28.
Article in English | MEDLINE | ID: mdl-31088972

ABSTRACT

T cell cytokines contribute to immunity against Staphylococcus aureus, but the predominant T cell subsets involved are unclear. In an S. aureus skin infection mouse model, we found that the IL-17 response was mediated by γδ T cells, which trafficked from lymph nodes to the infected skin to induce neutrophil recruitment, proinflammatory cytokines IL-1α, IL-1ß, and TNF, and host defense peptides. RNA-seq for TRG and TRD sequences in lymph nodes and skin revealed a single clonotypic expansion of the encoded complementarity-determining region 3 amino acid sequence, which could be generated by canonical nucleotide sequences of TRGV5 or TRGV6 and TRDV4 However, only TRGV6 and TRDV4 but not TRGV5 sequences expanded. Finally, Vγ6+ T cells were a predominant γδ T cell subset that produced IL-17A as well as IL-22, TNF, and IFNγ, indicating a broad and substantial role for clonal Vγ6+Vδ4+ T cells in immunity against S. aureus skin infections.


Subject(s)
Interleukin-17/physiology , Staphylococcal Infections/immunology , Staphylococcus aureus/pathogenicity , T-Lymphocyte Subsets/immunology , T-Lymphocytes/immunology , Animals , Disease Models, Animal , Humans , Lymph Nodes/immunology , Mice , Staphylococcal Infections/microbiology
7.
J Allergy Clin Immunol ; 148(2): 506-522.e8, 2021 08.
Article in English | MEDLINE | ID: mdl-33617860

ABSTRACT

BACKGROUND: Mas gene-related G protein-coupled receptors (MRGPRs) are a G protein-coupled receptor family responsive to various exogenous and endogenous agonists, playing a fundamental role in pain and itch sensation. The primate-specific family member MRGPRX2 and its murine orthologue MRGPRB2 are expressed by mast cells mediating IgE-independent signaling and pseudoallergic drug reactions. OBJECTIVES: Our aim was to increase knowledge about the function and regulation of MRGPRX2/MRGPRB2, which is of major importance in prevention of drug hypersensitivity reactions and drug-induced pruritus. METHODS: To identify novel MRGPR (ant)agonists, we screened a library of pharmacologically active compounds by utilizing a high-throughput calcium mobilization assay. The identified hit compounds were analyzed for their pseudoallergic and pruritogenic effects in mice and human. RESULTS: We found a class of commonly used drugs activating MRGPRX2 that, to a large extent, consists of antidepressants, antiallergic drugs, and antipsychotics. Three-dimensional pharmacophore modeling revealed structural similarities of the identified agonists, classifying them as cationic amphiphilic drugs. Mast cell activation was investigated by using the 3 representatively selected antidepressants clomipramine, paroxetine, and desipramine. Indeed, we were able to show a concentration-dependent activation and MRGPRX2-dependent degranulation of the human mast cell line LAD2 (Laboratory of Allergic Diseases-2). Furthermore, clomipramine, paroxetine, and desipramine were able to induce degranulation of human skin and murine peritoneal mast cells. These substances elicited dose-dependent scratching behavior following intradermal injection into C57BL/6 mice but less so in MRGPRB2-mutant mice, as well as wheal-and-flare reactions following intradermal injections in humans. CONCLUSION: Our results contribute to the characterization of structure-activity relationships and functionality of MRGPRX2 ligands and facilitate prediction of adverse reactions such as drug-induced pruritus to prevent severe drug hypersensitivity reactions.


Subject(s)
Antidepressive Agents/adverse effects , Behavior, Animal/drug effects , Cell Degranulation/drug effects , Drug Hypersensitivity/immunology , Mast Cells/immunology , Nerve Tissue Proteins/immunology , Receptors, G-Protein-Coupled/immunology , Receptors, Neuropeptide/immunology , Animals , Antidepressive Agents/pharmacology , Cell Line , Drug Hypersensitivity/pathology , Humans , Mast Cells/pathology , Mice , Nerve Tissue Proteins/agonists , Receptors, G-Protein-Coupled/agonists , Receptors, Neuropeptide/agonists
8.
PLoS Pathog ; 12(9): e1005840, 2016 09.
Article in English | MEDLINE | ID: mdl-27631986

ABSTRACT

Over 100 million women use progesterone therapies worldwide. Despite having immunomodulatory and repair properties, their effects on the outcome of viral diseases outside of the reproductive tract have not been evaluated. Administration of exogenous progesterone (at concentrations that mimic the luteal phase) to progesterone-depleted adult female mice conferred protection from both lethal and sublethal influenza A virus (IAV) infection. Progesterone treatment altered the inflammatory environment of the lungs, but had no effects on viral load. Progesterone treatment promoted faster recovery by increasing TGF-ß, IL-6, IL-22, numbers of regulatory Th17 cells expressing CD39, and cellular proliferation, reducing protein leakage into the airway, improving pulmonary function, and upregulating the epidermal growth factor amphiregulin (AREG) in the lungs. Administration of rAREG to progesterone-depleted females promoted pulmonary repair and improved the outcome of IAV infection. Progesterone-treatment of AREG-deficient females could not restore protection, indicating that progesterone-mediated induction of AREG caused repair in the lungs and accelerated recovery from IAV infection. Repair and production of AREG by damaged respiratory epithelial cell cultures in vitro was increased by progesterone. Our results illustrate that progesterone is a critical host factor mediating production of AREG by epithelial cells and pulmonary tissue repair following infection, which has important implications for women's health.


Subject(s)
Influenza A virus/immunology , Lung/immunology , Orthomyxoviridae Infections/immunology , Th17 Cells/immunology , Amphiregulin/genetics , Amphiregulin/immunology , Animals , Antigens, CD/genetics , Antigens, CD/immunology , Apyrase/genetics , Apyrase/immunology , Cytokines/genetics , Cytokines/immunology , Female , Lung/pathology , Mice , Mice, Knockout , Orthomyxoviridae Infections/genetics , Orthomyxoviridae Infections/pathology , Th17 Cells/pathology
9.
Proc Natl Acad Sci U S A ; 112(16): 5099-104, 2015 Apr 21.
Article in English | MEDLINE | ID: mdl-25840590

ABSTRACT

Telomere syndromes have their most common manifestation in lung disease that is recognized as idiopathic pulmonary fibrosis and emphysema. In both conditions, there is loss of alveolar integrity, but the underlying mechanisms are not known. We tested the capacity of alveolar epithelial and stromal cells from mice with short telomeres to support alveolar organoid colony formation and found that type 2 alveolar epithelial cells (AEC2s), the stem cell-containing population, were limiting. When telomere dysfunction was induced in adult AEC2s by conditional deletion of the shelterin component telomeric repeat-binding factor 2, cells survived but remained dormant and showed all the hallmarks of cellular senescence. Telomere dysfunction in AEC2s triggered an immune response, and this was associated with AEC2-derived up-regulation of cytokine signaling pathways that are known to provoke inflammation in the lung. Mice uniformly died after challenge with bleomycin, underscoring an essential role for telomere function in AEC2s for alveolar repair. Our data show that alveoloar progenitor senescence is sufficient to recapitulate the regenerative defects, inflammatory responses, and susceptibility to injury that are characteristic of telomere-mediated lung disease. They suggest alveolar stem cell failure is a driver of telomere-mediated lung disease and that efforts to reverse it may be clinically beneficial.


Subject(s)
Pulmonary Alveoli/pathology , Stem Cells/pathology , Telomere Shortening , Telomere/pathology , Aging/pathology , Animals , Cell Differentiation , Cell Proliferation , Epithelial Cells/metabolism , Gene Deletion , Immunity , Inflammation/pathology , Intercellular Signaling Peptides and Proteins , Mesoderm/pathology , Mice , Paracrine Communication , Peptides/metabolism , Pulmonary Alveoli/metabolism , Pulmonary Surfactant-Associated Protein C , Signal Transduction/immunology , Spheroids, Cellular/pathology , Stromal Cells/pathology , Telomeric Repeat Binding Protein 2/metabolism , Tumor Suppressor Protein p53/metabolism
10.
Am J Physiol Lung Cell Mol Physiol ; 309(7): L662-76, 2015 Oct 01.
Article in English | MEDLINE | ID: mdl-26232300

ABSTRACT

Emphysema, one of the major components of chronic obstructive pulmonary disease (COPD), is characterized by the progressive and irreversible loss of alveolar lung tissue. Even though >80% of COPD cases are associated with cigarette smoking, only a relatively small proportion of smokers develop emphysema, suggesting a potential role for genetic factors in determining individual susceptibility to emphysema. Although strain-dependent effects have been shown in animal models of emphysema, the molecular basis underlying this intrinsic susceptibility is not fully understood. In this present study, we investigated emphysema development using the elastase-induced experimental emphysema model in two commonly used mouse strains, C57BL/6J and BALB/cJ. The results demonstrate that mice with different genetic backgrounds show disparate susceptibility to the development of emphysema. BALB/cJ mice were found to be much more sensitive than C57BL/6J to elastase injury in both a dose-dependent and time-dependent manner, as measured by significantly higher mortality, greater body weight loss, greater decline in lung function, and a greater loss of alveolar tissue. The more susceptible BALB/cJ strain also showed the persistence of inflammatory cells in the lung, especially macrophages and lymphocytes. A comparative gene expression analysis following elastase-induced injury showed BALB/cJ mice had elevated levels of il17A mRNA and a number of classically (M1) and alternatively (M2) activated macrophage genes, whereas the C57BL/6J mice demonstrated augmented levels of interferon-γ. These findings suggest a possible role for these cellular and molecular mediators in modulating the severity of emphysema and highlight the possibility that they might contribute to the heterogeneity observed in clinical emphysema outcomes.


Subject(s)
Disease Models, Animal , Lymphocytes , Macrophages , Pancreatic Elastase/toxicity , Pulmonary Alveoli , Pulmonary Emphysema , Animals , Humans , Interferon-gamma/metabolism , Interleukin-17/metabolism , Lymphocytes/metabolism , Lymphocytes/pathology , Macrophages/metabolism , Macrophages/pathology , Mice , Mice, Inbred BALB C , Pulmonary Alveoli/metabolism , Pulmonary Alveoli/pathology , Pulmonary Emphysema/chemically induced , Pulmonary Emphysema/metabolism , Pulmonary Emphysema/pathology , RNA, Messenger/metabolism , Species Specificity
11.
Sci Transl Med ; 16(746): eadk8198, 2024 May 08.
Article in English | MEDLINE | ID: mdl-38718132

ABSTRACT

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.


Subject(s)
Disease Models, Animal , Pruritus , Receptors, G-Protein-Coupled , Animals , Pruritus/metabolism , Pruritus/chemically induced , Pruritus/pathology , Pruritus/drug therapy , Humans , Receptors, G-Protein-Coupled/metabolism , Mice , HEK293 Cells , Phosphorylation/drug effects , Phosphates/metabolism , Sensory Receptor Cells/metabolism , Sensory Receptor Cells/drug effects , Prodrugs/pharmacology , Cryoelectron Microscopy
12.
bioRxiv ; 2024 May 20.
Article in English | MEDLINE | ID: mdl-38826432

ABSTRACT

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 and, more importantly, preventing its transition to a chronic state. In a mouse model of 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. Importantly, preemptive drug treatment also inhibited the transition of post-surgical pain to a prolonged state. This effect was achieved through direct inhibition of nociceptive dorsal root ganglion (DRG) neurons via CD44-dependent pathways, and indirect pain relief by attenuating immune cell recruitment. 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 and unravel the underlying mechanisms.

13.
bioRxiv ; 2024 May 28.
Article in English | MEDLINE | ID: mdl-38854075

ABSTRACT

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.

14.
Cells ; 12(22)2023 11 14.
Article in English | MEDLINE | ID: mdl-37998359

ABSTRACT

The skin presents a multifaceted microbiome, a balanced coexistence of bacteria, fungi, and viruses. These resident microorganisms are fundamental in upholding skin health by both countering detrimental pathogens and working in tandem with the skin's immunity. Disruptions in this balance, known as dysbiosis, can lead to disorders like psoriasis and atopic dermatitis. Central to the skin's defense system are mast cells. These are strategically positioned within the skin layers, primed for rapid response to any potential foreign threats. Recent investigations have started to unravel the complex interplay between these mast cells and the diverse entities within the skin's microbiome. This relationship, especially during times of both balance and imbalance, is proving to be more integral to skin health than previously recognized. In this review, we illuminate the latest findings on the ties between mast cells and commensal skin microorganisms, shedding light on their combined effects on skin health and maladies.


Subject(s)
Dermatitis, Atopic , Microbiota , Psoriasis , Humans , Mast Cells , Skin , Psoriasis/microbiology
15.
Sci Rep ; 13(1): 12302, 2023 07 29.
Article in English | MEDLINE | ID: mdl-37516794

ABSTRACT

Migraine ranks among the most prevalent disorders worldwide, leading to disability and decreased quality of life in patients. Recently, neurogenic inflammation has been recognized as a potential underlying pathology contributing to the migraine pain pathway. Mast cells reside in the meninges and have been implicated in contributing to the pathophysiology of migraine. Here we report for the first time that the mouse Mas-Related G-protein-coupled Receptor B2 (MrgprB2), is expressed on meningeal connective tissue mast cells and contributes to Pituitary Adenylate Cyclase Activating Peptide (PACAP)-induced migraine-like pain behavior. We also found that PACAP was able to dose-dependently lead to enzyme release from human mast cells via activation of MRGPRX2; the human homolog of MrgprB2. Using a transgenic MRGPRX2 mouse, we observed significant increases in PACAP-induced migraine-like pain behavior in MRGPRX2+ mice vs mice lacking the receptor. These results reveal both MrgprB2 and MRGPRX2 as important contributors to neuropeptide-induced migraine pain.


Subject(s)
Migraine Disorders , Pituitary Adenylate Cyclase-Activating Polypeptide , Animals , Humans , Mice , Mast Cells , Meninges , Mice, Transgenic , Migraine Disorders/chemically induced , Nerve Tissue Proteins/genetics , Pain , Pituitary Adenylate Cyclase-Activating Polypeptide/genetics , Quality of Life , Receptors, G-Protein-Coupled/genetics , Receptors, Neuropeptide/genetics
16.
Pain ; 164(1): 98-110, 2023 01 01.
Article in English | MEDLINE | ID: mdl-35507377

ABSTRACT

ABSTRACT: Chronic pruritus is a prominent symptom of allergic contact dermatitis (ACD) and represents a huge unmet health problem. However, its underlying cellular and molecular mechanisms remain largely unexplored. TRPC3 is highly expressed in primary sensory neurons and has been implicated in peripheral sensitization induced by proinflammatory mediators. Yet, the role of TRPC3 in acute and chronic itch is still not well defined. Here, we show that, among mouse trigeminal ganglion (TG) neurons, Trpc3 mRNA is predominantly expressed in nonpeptidergic small diameter TG neurons of mice. Moreover, Trpc3 mRNA signal was present in most presumptively itch sensing neurons. TRPC3 agonism induced TG neuronal activation and acute nonhistaminergic itch-like and pain-like behaviors in naive mice. In addition, genetic deletion of Trpc3 attenuated acute itch evoked by certain common nonhistaminergic pruritogens, including endothelin-1 and SLIGRL-NH2. In a murine model of contact hypersensitivity (CHS), the Trpc3 mRNA expression level and function were upregulated in the TG after CHS. Pharmacological inhibition and global knockout of Trpc3 significantly alleviated spontaneous scratching behaviors without affecting concurrent cutaneous inflammation in the CHS model. Furthermore, conditional deletion of Trpc3 in primary sensory neurons but not in keratinocytes produced similar antipruritic effects in this model. These findings suggest that TRPC3 expressed in primary sensory neurons may contribute to acute and chronic itch through a histamine independent mechanism and that targeting neuronal TRPC3 might benefit the treatment of chronic itch associated with ACD and other inflammatory skin disorders.


Subject(s)
Dermatitis, Allergic Contact , Pruritus , Animals , Mice , Dermatitis, Allergic Contact/metabolism , Disease Models, Animal , Mice, Inbred C57BL , Pruritus/chemically induced , Pruritus/genetics , Pruritus/metabolism , Sensory Receptor Cells/metabolism , Skin/metabolism
17.
Bone Res ; 10(1): 24, 2022 Mar 02.
Article in English | MEDLINE | ID: mdl-35232979

ABSTRACT

The molecular control of osteoclast formation is still not clearly elucidated. Here, we show that a process of cell recognition mediated by Siglec15-TLR2 binding is indispensable and occurs prior to cell fusion in RANKL-mediated osteoclastogenesis. Siglec15 has been shown to regulate osteoclastic bone resorption. However, the receptor for Siglec15 has not been identified, and the signaling mechanism involving Siglec15 in osteoclast function remains unclear. We found that Siglec15 bound sialylated TLR2 as its receptor and that the binding of sialylated TLR2 to Siglec15 in macrophages committed to the osteoclast-lineage initiated cell fusion for osteoclast formation, in which sialic acid was transferred by the sialyltransferase ST3Gal1. Interestingly, the expression of Siglec15 in macrophages was activated by M-CSF, whereas ST3Gal1 expression was induced by RANKL. Both Siglec15-specific deletion in macrophages and intrafemoral injection of sialidase abrogated cell recognition and reduced subsequent cell fusion for the formation of osteoclasts, resulting in increased bone formation in mice. Thus, our results reveal that cell recognition mediated by the binding of sialylated TLR2 to Siglec15 initiates cell fusion for osteoclast formation.

18.
Front Immunol ; 13: 997389, 2022.
Article in English | MEDLINE | ID: mdl-36341461

ABSTRACT

Background: Allergic drug reaction or drug allergy is an immunologically mediated drug hypersensitivity reaction (DHR). G-protein coupled receptors (GPCRs) are common drug targets and communicate extracellular signals that initiate cellular responses. Recent evidence shows that GPCR MRGPRX2 is of major importance in IgE-independent pseudo-allergic DHRs based on the suspected interactions between many FDA-approved peptidergic compounds and MRGPRX2. Objective: Our aim was to uncover novel MRGPRX2-selective and -potent agonists as drug candidates responsible for clinical features of pseudo-allergic DHRs. Methods: We conducted a primary high-throughput screening (HTS), coupled with mutagenesis targeting the MRGPRX2 N62S mutation, on a panel of 3,456 library compounds. We discovered pharmacologically active hit compounds as agonists of the MRGPRX2 protein according to high degrees of potency evaluated by the calcium response and validated by the degranulation assay. Using the molecular tool Forge, we also characterized the structure-activity relationship shared by identified hit compounds. Results: The alternative allele of single nucleotide polymorphism rs10833049 (N62S) in MRGPRX2 demonstrated loss-of-function property in response to substance P and antineoplastic agent daunorubicin hydrochloride. We applied a unique assay system targeting the N62S mutation to the HTS and identified 84 MRGPRX2-selective active hit compounds representing diverse classes according to primary drug indications. The top five highly represented groups included fluoroquinolone and non-fluoroquinolone antibiotics; antidepressive/antipsychotic; antihistaminic and antineoplastic agents. We classified hit compounds into 14 clusters representing a variety of chemical and drug classes beyond those reported, such as opioids, neuromuscular blocking agents, and fluoroquinolones. We further demonstrated MRGPRX2-dependent degranulation in the human mast cell line LAD2 cells induced by three novel agonists representing the non-fluoroquinolone antibiotics (bacitracin A), anti-allergic agents (brompheniramine maleate) and tyrosine-kinase inhibitors (imatinib mesylate). Conclusion: Our findings could facilitate the development of interventions for personalized prevention and treatment of DHRs, as well as future pharmacogenetic investigations of MRGPRX2 in relevant disease cohorts.


Subject(s)
Drug Hypersensitivity , Receptors, Neuropeptide , Humans , Receptors, Neuropeptide/metabolism , Cell Degranulation , Mast Cells , Nerve Tissue Proteins , Receptors, G-Protein-Coupled/metabolism , Anti-Bacterial Agents/pharmacology
19.
Neuron ; 110(2): 209-220.e6, 2022 01 19.
Article in English | MEDLINE | ID: mdl-34752775

ABSTRACT

Spontaneous pain refers to pain occurring without external stimuli. It is a primary complaint in chronic pain conditions and remains difficult to treat. Moreover, the mechanisms underlying spontaneous pain remain poorly understood. Here we employed in vivo imaging of dorsal root ganglion (DRG) neurons and discovered a distinct form of abnormal spontaneous activity following peripheral nerve injury: clusters of adjacent DRG neurons firing synchronously and sporadically. The level of cluster firing correlated directly with nerve injury-induced spontaneous pain behaviors. Furthermore, we demonstrated that cluster firing is triggered by activity of sympathetic nerves, which sprout into DRGs after injury, and identified norepinephrine as a key neurotransmitter mediating this unique firing. Chemogenetic and pharmacological manipulations of sympathetic activity and norepinephrine receptors suggest that they are necessary and sufficient for DRG cluster firing and spontaneous pain behavior. Therefore, blocking sympathetically mediated cluster firing may be a new paradigm for treating spontaneous pain.


Subject(s)
Ganglia, Spinal , Spinal Nerves , Ganglia, Spinal/physiology , Humans , Pain , Sensory Receptor Cells , Spinal Nerves/injuries , Sympathetic Nervous System/physiology
20.
Front Genet ; 12: 624561, 2021.
Article in English | MEDLINE | ID: mdl-33868365

ABSTRACT

Environmental exposures have been linked to increased asthma risk, particularly during pregnancy and in early life. Here we use a mouse model of allergic lung disease to examine the effects of pre- and perinatal house dust mite (HDM) allergen exposure on offspring phenotypic and transcriptional outcomes in three generations. We show that maternal HDM exposure (F0) acts synergistically with adult HDM exposure, leading to enhanced airway hyperresponsiveness (AHR) and lung inflammation when compared to mice exposed solely in adulthood. Additionally, a subset of F1 males were not challenged in adulthood, and used to generate F2 progeny, which was then used to generate F3 progeny. Upon adult challenge to HDM, F2, and F3 males generated from the maternal HDM (F0) exposure lineage displayed increased airway reactivity and inflammation when compared to mice exposed solely in adulthood. These findings indicate that maternal allergen exposure is capable of enhancing either susceptibly to or severity of allergic airway disease. To examine the role of epigenetic inheritance of asthma susceptibility induced by maternal HDM exposure, we utilized a genome-wide MeDIP-seq and hMeDIP-seq analysis to identify genes differentially methylated (DMG) and hydroxymethylated (DHG), and their association with the enhanced AHR. In addition, we validated the relationship between DNA methylation and mRNA expression of the DMGs and DHGs in the male sub-generations (F1-F3). We found the expression of Kchn1, Nron, and Spag17 to be differentially hydroxymethylated and upregulated in the F1 exposed to HDM both in early life and in adulthood when compared to F1 mice exposed solely in adulthood. Kcnh1 remained upregulated in the F2 and F3 from the maternal HDM (F0) exposure lineage, when compared to F1 mice exposed solely in adulthood. In summary, we demonstrated that maternal HDM exposure in early life can alter the gene expression and phenotype of offspring upon adult HDM exposure, resulting in more severe disease. These effects persist at least two generations past the initial insult, transmitted along the paternal line.

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