Structural basis of ligand recognition and design of antihistamines targeting histamine H4 receptor.

The histamine H4 receptor (H4R) plays key role in immune cell function and is a highly valued target for treating allergic and inflammatory diseases. However, structural information of H4R remains elusive. Here, we report four cryo-EM structures of H4R/Gi complexes, with either histamine or synthetic agonists clobenpropit, VUF6884 and clozapine bound. Combined with mutagenesis, ligand binding and functional assays, the structural data reveal a distinct ligand binding mode where D943.32 and a π-π network determine the orientation of the positively charged group of ligands, while E1825.46, located at the opposite end of the ligand binding pocket, plays a key role in regulating receptor activity. The structural insight into H4R ligand binding allows us to identify mutants at E1825.46 for which the agonist clobenpropit acts as an inverse agonist and to correctly predict inverse agonism of a closely related analog with nanomolar potency. Together with the findings regarding receptor activation and Gi engagement, we establish a framework for understanding H4R signaling and provide a rational basis for designing novel antihistamines targeting H4R.

A virtual library of small molecules mimicking dipeptides.

Virtual combinatorial libraries are prevalent in drug discovery due to improvements in the prediction of synthetic reactions that can be performed. This has gone hand in hand with the development of virtual screening capabilities to effectively screen the large chemical spaces spanned by exhaustive enumeration of reaction products. In this study, we generated a small-molecule dipeptide mimic library to target proteins binding small peptides. The library was created based on the general idea of peptide synthesis, that is, amino acid mimics were reacted in silico to form the dipeptide mimics, yielding 2,036,819 unique compounds. After docking calculations, two compounds from the library were synthesized and tested against WD repeat-containing protein 5 (WDR5) and histamine receptors H1-H4 to evaluate whether these molecules are viable in assays. The compounds showed the highest potency at the histamine H3 receptor, with Ki values in the two-digit micromolar range.

Jiawei guomin decoction regulates the degranulation of mast cells in atopic dermatitis mice via the HIS/PAR-2 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Guomin decoction (GMD) is a traditional Chinese medicine commonly used in clinical practice. It has traditionally been used to treat all allergic diseases. Currently, Jiawei Guomin Decoction (JWGMD) is used to treat sensitive skin after initial therapy. Although it has a significant clinical therapeutic effect, the exact role of mast cell degranulation in treating atopic dermatitis (AD) is still unclear. AIM OF THE STUDY: GMD and JWGMD can both treat allergic diseases, while JWGMD focuses on skin allergies. This study aims to explore the potential effect of JWGMD on the degranulation of mast cells in an AD mouse model induced by 2,4-dinitrofluorobenzene (DNFB) and investigate the effectiveness of JWGMD in alleviating disease progression to further provide specific therapeutic targets for treating AD. MATERIALS AND METHODS: The scratching times and skin lesions of model mice induced by DNFB were observed, and skin tissues were collected for subsequent measurement. Histopathological changes in the back skin of mice were observed by haematoxylin eosin (H&E) staining, Toluidine blue staining was used to detect the degranulation of mouse skin mast cells, and the relationship between the expression of histamine (HIS), mast cell tryptase (MCT) and mast cell degranulation was analysed by enzyme-linked immunosorbent assay (ELISA). The expression of protease-activated receptor-2 (PAR-2), histamine 1 receptor (H1R), H2R, H4R and MCT proteins in AD mice was detected by Western blot (WB). Immunofluorescence assay (IFA) further confirmed the localization of PAR-2, H1R, H2R, H4R, and MCT proteins in the skin. Quantitative real-time PCR (qPCR) was used to determine PAR-2, H1R, H2R and H4R mRNA levels in skin lesions to further clarify the mechanism by which JWGMD amplifies mast cell degranulation in AD. In addition, a reliable ultrahigh-performance liquid chromatography-quadrupole electrostatic field orbitrap mass spectrometry (UPLC-QE-MS) nontargeted metabolomics analysis was performed to analyse the differences in metabolite abundance between GMD and JWGMD, and these results were used to identify the active components in JWGMD that may have antipruritic and anti-inflammatory properties and inhibit mast cell degranulation. RESULTS: After intermittent stimulation with DNFB, the skin lesions showed extensive desquamation, dryness, scabbing, skin thickening, and slight bleeding. Both treatments alleviated this phenomenon and reduced the number of scratches, with JWGMD being the most effective. JWGMD can significantly reduce inflammatory cell infiltration, oedema, and some capillary neogenesis in mice and reduce the degranulation of mast cells. The ELISA results showed that JWGMD can increase the levels of MCT and HIS proteins. The WB and IFA results demonstrated that JWGMD reduced the expression levels of PAR-2, H1R, H4R, and MCT proteins in skin lesions, with protein localization mainly in the epidermal layer, while H2R protein levels were increased and mainly localized in the dermis. In addition, JWGMD downregulates the mRNA expression of PAR-2, H1R, H2R, and H4R. Interestingly, through UPLC-QE-MS nontargeted metabolomic analysis, we detected the anti-inflammatory and antiallergy active substances in JWGMD, such as methyl eugenol, dictamnine and sinapine. CONCLUSIONS: JWGMD may alleviate itching through methyl syringol, dictamnine, sinapine and other substances, and its mechanism may be related to inhibiting the HIS/PAR-2 pathway in AD model mice and further regulating the self-amplification of mast cell degranulation. JWGMD is a potential drug for treating AD. Therefore, it deserves continuous attention and research.

Structural insights into the agonists binding and receptor selectivity of human histamine H4 receptor.

Histamine is a biogenic amine that participates in allergic and inflammatory processes by stimulating histamine receptors. The histamine H4 receptor (H4R) is a potential therapeutic target for chronic inflammatory diseases such as asthma and atopic dermatitis. Here, we show the cryo-electron microscopy structures of the H4R-Gq complex bound with an endogenous agonist histamine or the selective agonist imetit bound in the orthosteric binding pocket. The structures demonstrate binding mode of histamine agonists and that the subtype-selective agonist binding causes conformational changes in Phe3447.39, which, in turn, form the "aromatic slot". The results provide insights into the molecular underpinnings of the agonism of H4R and subtype selectivity of histamine receptors, and show that the H4R structures may be valuable in rational drug design of drugs targeting the H4R.

A single photoreceptor splits perception and entrainment by cotransmission.

Vision enables both image-forming perception, driven by a contrast-based pathway, and unconscious non-image-forming circadian photoentrainment, driven by an irradiance-based pathway1,2. Although two distinct photoreceptor populations are specialized for each visual task3-6, image-forming photoreceptors can additionally contribute to photoentrainment of the circadian clock in different species7-15. However, it is unknown how the image-forming photoreceptor pathway can functionally implement the segregation of irradiance signals required for circadian photoentrainment from contrast signals required for image perception. Here we report that the Drosophila R8 photoreceptor separates image-forming and irradiance signals by co-transmitting two neurotransmitters, histamine and acetylcholine. This segregation is further established postsynaptically by histamine-receptor-expressing unicolumnar retinotopic neurons and acetylcholine-receptor-expressing multicolumnar integration neurons. The acetylcholine transmission from R8 photoreceptors is sustained by an autocrine negative feedback of the cotransmitted histamine during the light phase of light-dark cycles. At the behavioural level, elimination of histamine and acetylcholine transmission impairs R8-driven motion detection and circadian photoentrainment, respectively. Thus, a single type of photoreceptor can achieve the dichotomy of visual perception and circadian photoentrainment as early as the first visual synapses, revealing a simple yet robust mechanism to segregate and translate distinct sensory features into different animal behaviours.

Recovery of the histamine H3 receptor activity lost in yeast cells through error-prone PCR and in vivo selection.

G protein-coupled receptors (GPCRs) are the largest protein family in humans and are important drug targets. Yeast, especially Saccharomyces cerevisiae, is a useful host for modifying the function and stability of GPCRs through protein engineering, which is advantageous for mammalian cells. When GPCRs are expressed in yeast, their function is often impaired. In this study, we performed random mutagenesis using error-prone PCR and then an in vivo screening to obtain mutants that recovered the activity of the human histamine H3 receptor (H3R), which loses its signaling function when expressed in yeast. Four mutations with recovered activity were identified after screening. Three of the mutations were identified near the DRY and NPxxY motifs of H3R, which are important for activation and are commonly found in class A GPCRs. The mutants responded exclusively to the yeast YB1 strain harboring Gi-chimera proteins, showing retention of G protein specificity. Analysis of one of the mutants with recovered activity, C415R, revealed that it maintained its ligand-binding characteristics. The strategy used in this study may enable the recovery of the activity of other GPCRs that do not function in S. cerevisiae and may be useful in creating GPCRs mutants stabilized in their active conformations.

The mRNA expression and secretion of granzyme B are up-regulated via the histamine H2 receptor in human CD4+ T cells.

INTRODUCTION: Granzyme B (GZMB), a serine protease with cytotoxic and immunomodulatory functions, shows elevated levels in blood plasma of patients with atopic dermatitis (AD). It has been observed that GZMB expression in CD4+ and CD8+ T cells is higher in lesional skin in AD than in healthy skin. Since histamine is present in high concentration in the skin of AD patients, we investigated the regulation of GZMB in human CD4+ T cells by histamine. METHODS: Naïve CD4+ T cells polarized into Th2 cells, total CD4+ T cells treated with IL-4 for 72 h and CD4+ T cells isolated from healthy donors and AD patients were investigated. The cells were stimulated with histamine or with different histamine-receptor agonists. Gene expression was evaluated by RNA-Seq. GZMB mRNA expression was detected by quantitative real time PCR, whereas GZMB secretion was measured by ELISpot and ELISA. T cell degranulation was evaluated by flow cytometry using CD107a surface expression as a degranulation marker. RESULTS: By RNA-Seq, we identified the up-regulation of various genes of the cytotoxic pathway, in particular of GZMB, by histamine in Th2-polarized CD4+ T cells. In Th2-polarized CD4+ T cells and in CD4+ T cells activated by IL-4 the mRNA expression of GZMB was significantly up-regulated by histamine and by histamine H2 receptor (H2R) agonists. The induction of GZMB secretion by histamine was significantly higher in CD4+ T cells from AD patients than in those from healthy donors. CD107a surface expression was up-regulated by trend in response to histamine in Th2-polarized CD4+ T cells. CONCLUSION: Our findings may help to elucidate novel mechanisms of the H2R and to achieve a better understanding of the role of GZMB in the pathogenesis of AD.

Histamine H1- and H4-receptor expression in human colon-derived cell lines.

In previous studies, we demonstrated the involvement of H4R in inflammatory bowel disease (IBD) and IBD-associated colon cancer in mice and could ascribe H4R-mediated histamine function to colon epithelial cells. The transferability of obtained data to humans is however lacking. Functional expression of H4R on colon epithelial cells is a prerequisite to pursue the hypothesis of involvement of H4R in carcinogenesis. Thus, we here compared the expression of histamine receptor subtypes in a series of cell lines. Out of these, three colon-derived cell lines displaying different combinations of H1R and H4R expression were submitted to functional analyses. Human hematopoietic HMC-1, HL-60, and U937, lung-derived A549 and Calu-3, and colorectal LoVo, SW 480, Caco-2, HT-29, and HCT116 cells were included in the study. mRNA expression was quantified by RT-qPCR. For functional analyses, Caco-2, HT-29, and HCT116 cells were treated by incubation with 1 - 10 µM histamine in the presence or absence of selective histamine receptor antagonists. Calcium mobilization, cAMP accumulation, and cell proliferation were measured by fluorimetry, mass spectrometry, and real-time bioimpedance measurements, respectively. Histamine receptor expression was heterogeneous in the cell lines tested. In most cell lines, we detected H1R mRNA while H4R mRNAs were found only occasionally. The colon-derived epithelial cell lines LoVo, SW480, and HT-29 expressed H1R mRNA exclusively, while in HCT116 cells H1R and H4R mRNAs and in CaCo-2 H2R mRNA were detectable. Subsequent functional analyses in HT29, Caco-2, and HCT116 cells, however, indicated that only HT-29 responded to histamine stimulation, by means of H1R. For a detailed analysis of histamine receptor function, esp. that of H1R and H4R, in human colon-derived cell lines, the cell lines tested here are not fully convenient unless genetically modified.

Insight into the Mode of Action of 8-Hydroxyquinoline-Based Blockers on the Histamine Receptor 2.

Histamine receptor 2 (HRH2) blockers are used to treat peptic ulcers and gastric reflux. Chlorquinaldol and chloroxine, which contain an 8-hydroxyquinoline (8HQ) core, have recently been identified as blocking HRH2. To gain insight into the mode of action of 8HQ-based blockers, here, we leverage an HRH2-based sensor in yeast to evaluate the role of key residues in the HRH2 active site on histamine and 8HQ-based blocker binding. We find that the HRH2 mutations D98A, F254A, Y182A, and Y250A render the receptor inactive in the presence of histamine, while HRH2:D186A and HRH2:T190A retain residual activity. Based on molecular docking studies, this outcome correlates with the ability of the pharmacologically relevant histamine tautomers to interact with D98 via the charged amine. Docking studies also suggest that, unlike established HRH2 blockers that interact with both ends of the HRH2 binding site, 8HQ-based blockers interact with only one end, either the end framed by D98/Y250 or T190/D186. Experimentally, we find that chlorquinaldol and chloroxine still inactivate HRH2:D186A by shifting their engagement from D98 to Y250 in the case of chlorquinaldol and D186 to Y182 in the case of chloroxine. Importantly, the tyrosine interactions are supported by the intramolecular hydrogen bonding of the 8HQ-based blockers. The insight gained in this work will aid in the development of improved HRH2 therapeutics. More generally, this work demonstrates that Gprotein-coupled receptor (GPCR)-based sensors in yeast can help elucidate the mode of action of novel ligands for GPCRs, a family of receptors that bind 30% of FDA therapeutics.

Renal histaminergic system and acute effects of histamine receptor 2 blockade on renal damage in the Dahl salt-sensitive rat.

Histamine is involved in the regulation of immune response, vasodilation, neurotransmission, and gastric acid secretion. Although elevated histamine levels and increased expression of histamine metabolizing enzymes have been reported in renal disease, there is a gap in knowledge regarding the mechanisms of histamine-related pathways in the kidney. We report here that all four histamine receptors as well as enzymes responsible for the metabolism of histamine are expressed in human and rat kidney tissues. In this study, we hypothesized that the histaminergic system plays a role in salt-induced kidney damage in the Dahl salt-sensitive (DSS) rat, a model characterized with inflammation-driven renal lesions. To induce renal damage related to salt sensitivity, DSS rats were challenged with 21 days of a high-salt diet (4% NaCl); normal-salt diet (0.4% NaCl)-fed rats were used as a control. We observed lower histamine decarboxylase and higher histamine N-methyltransferase levels in high-salt diet-fed rats, indicative of a shift in histaminergic tone; metabolomics showed higher histamine and histidine levels in the kidneys of high-salt diet-fed rats, whereas plasma levels for both compounds were lower. Acute systemic inhibition of histamine receptor 2 in the DSS rat revealed that it lowered vasopressin receptor 2 in the kidney. In summary, we established here the existence of the local histaminergic system, revealed a shift in the renal histamine balance during salt-induced kidney damage, and provided evidence that blockage of histamine receptor 2 in the DSS rat affects water balance and urine concentrating mechanisms.NEW & NOTEWORTHY Histamine is a nitrogenous compound crucial for the inflammatory response. The knowledge regarding the renal effects of histamine is very limited. We showed that renal epithelia exhibit expression of the components of the histaminergic system. Furthermore, we revealed that there was a shift in the histaminergic tone in salt-sensitive rats when they were challenged with a high-salt diet. These data support the notion that histamine plays a role in renal epithelial physiological and pathophysiological functions.

Ergotamine Stimulates Human 5-HT4-Serotonin Receptors and Human H2-Histamine Receptors in the Heart.

Ergotamine (2'-methyl-5'α-benzyl-12'-hydroxy-3',6',18-trioxoergotaman) is a tryptamine-related alkaloid from the fungus Claviceps purpurea. Ergotamine is used to treat migraine. Ergotamine can bind to and activate several types of 5-HT1-serotonin receptors. Based on the structural formula of ergotamine, we hypothesized that ergotamine might stimulate 5-HT4-serotonin receptors or H2-histamine receptors in the human heart. We observed that ergotamine exerted concentration- and time-dependent positive inotropic effects in isolated left atrial preparations in H2-TG (mouse which exhibits cardiac-specific overexpression of the human H2-histamine receptor). Similarly, ergotamine increased force of contraction in left atrial preparations from 5-HT4-TG (mouse which exhibits cardiac-specific overexpression of the human 5-HT4-serotonin receptor). An amount of 10 µM ergotamine increased the left ventricular force of contraction in isolated retrogradely perfused spontaneously beating heart preparations of both 5-HT4-TG and H2-TG. In the presence of the phosphodiesterase inhibitor cilostamide (1 µM), ergotamine 10 µM exerted positive inotropic effects in isolated electrically stimulated human right atrial preparations, obtained during cardiac surgery, that were attenuated by 10 µM of the H2-histamine receptor antagonist cimetidine, but not by 10 µM of the 5-HT4-serotonin receptor antagonist tropisetron. These data suggest that ergotamine is in principle an agonist at human 5-HT4-serotonin receptors as well at human H2-histamine receptors. Ergotamine acts as an agonist on H2-histamine receptors in the human atrium.

Anti-inflammatory effects of new human histamine H3 receptor ligands with flavonoid structure on BV-2 neuroinflammation.

OBJECTIVE: Microglia play an important role in the neuroinflammation developed in response to various pathologies. In this study, we examined the anti-inflammatory effect of the new human histamine H3 receptor (H3R) ligands with flavonoid structure in murine microglial BV-2 cells. MATERIAL AND METHODS: The affinity of flavonoids (E243 -flavone and IIIa-IIIc-chalcones) for human H3R was evaluated in the radioligand binding assay. The cytotoxicity on BV-2 cell viability was investigated with the MTS assay. Preliminary evaluation of anti-inflammatory properties was screened by the Griess assay in an in vitro neuroinflammation model of LPS-treated BV-2 cells. The expression and secretion of pro-inflammatory cytokines were evaluated by real-time qPCR and ELISA, respectively. The expression of microglial cell markers were determined by immunocytochemistry. RESULTS: Chalcone derivatives showed high affinity at human H3R with Ki values < 25 nM. At the highest nontoxic concentration (6.25 µM) compound IIIc was the most active in reducing the level of nitrite in Griess assay. Additionally, IIIc treatment attenuated inflammatory process in murine microglia cells by down-regulating pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α) at both the level of mRNA and protein level. Our immunocytochemistry studies revealed expression of microglial markers (Iba1, CD68, CD206) in BV-2 cell line. CONCLUSIONS: These results emphasize the importance of further research to accurately identify the anti-inflammatory mechanism of action of chalcones.

Expression pattern and prognostic potential of histamine receptors in epithelial ovarian cancer.

PURPOSE: Despite recent advances in the treatment of ovarian cancer (OC), long-term remissions remain scarce. For a targeted approach, prognostic markers are indispensable for predicting survival and treatment response. Given their association with multiple hallmarks of cancer, histamine receptors (HR) are emerging as promising candidates. Here, we investigate their expression pattern and prognostic value in OC. METHODS: Specimens of 156 epithelial OC patients were collected during cytoreductive surgery at the Department of Obstetrics and Gynecology, LMU, between 1990 and 2002 and combined in a tissue microarray. Immunohistochemical staining of the HR H1, H2, H3 and H4 was quantified by an immunoreactive score and linked with clinico-pathological data by Spearman's correlation. Via ROC curve analysis, optimal cut-off values for potential prognostic markers were defined. Overall survival (OS) was visualized in Kaplan-Maier curves and significances determined by log-rank testing. A Cox regression model was applied for multivariate analysis. RESULTS: HR H3 and H4 expression was restricted to the cytosol of OC cells, while H1 was also present in the nucleus. A significant association between HR H1, H3 and H4 expression with several clinico-pathological parameters was revealed. In addition, HR H1 and H3 expression correlated positively, HR H4 expression negatively with OS. In addition, HR H3 was identified as independent prognostic marker for OS. HR H2 expression had no prognostic value. CONCLUSIONS: HR H1, H3 and H4 could serve as potential predictors for OS of OC patients. Further research is warranted to elucidate their pathophysiologic role and their predictive and therapeutic potential in OC.

Characteristics of histamine H4 receptor agonist-induced allergic conjunctivitis model in Guinea pigs.

Histamine is strongly associated with the onset of allergic conjunctivitis. The most recent cloned histamine H4 receptor antagonist is highly expected as a new therapeutic drug candidate. As a model for a therapeutic drug targeting the histamine H4 receptor, a mouse model in which conjunctivitis symptoms are induced by instilling 4-methylhistamine, a histamine H4 receptor agonist, has been reported. However, the affinity of the H4 receptor for histamine varies in species, and it is known that the histamine binding affinity for the guinea pig H4 receptor is closer to that for human receptor than mice receptor. In this paper, we investigated a possibility that a guinea pig model would become a drug efficacy evaluation model with higher evaluation accuracy than the mouse model. As a result, hyperemia was observed in the conjunctivae and iris of guinea pigs after instillation of 4-methylhistamine and specifically suppressed by the histamine H4 receptor antagonist. Unlikely to the previously reported mouse model, however, none of edema, increased vascular permeability or scratching behavior was observed, suggesting that there may be differences between mice and guinea pigs not only in the binding affinity of histamine to the H4 receptor but also in the biological reaction to 4-methylhistamine. Although the symptoms of allergic conjunctivitis do not appear comprehensively in the guinea pig model, results of this study indicated a possibility that this model can be used as a simple screening model in the early stages of drug development.

Maprotiline restores ER homeostasis and rescues neurodegeneration via Histamine Receptor H1 inhibition in retinal ganglion cells.

When the protein or calcium homeostasis of the endoplasmic reticulum (ER) is adversely altered, cells experience ER stress that leads to various diseases including neurodegeneration. Genetic deletion of an ER stress downstream effector, CHOP, significantly protects neuron somata and axons. Here we report that three tricyclic compounds identified through a small-scale high throughput screening using a CHOP promoter-driven luciferase cell-based assay, effectively inhibit ER stress by antagonizing their common target, histamine receptor H1 (HRH1). We further demonstrated that systemic administration of one of these compounds, maprotiline, or CRISPR-mediated retinal ganglion cell (RGC)-specific HRH1 inhibition, delivers considerable neuroprotection of both RGC somata and axons and preservation of visual function in two mouse optic neuropathy models. Finally, we determine that maprotiline restores ER homeostasis by inhibiting HRH1-mediated Ca2+ release from ER. In this work we establish maprotiline as a candidate neuroprotectant and HRH1 as a potential therapeutic target for glaucoma.

Structural basis for recognition of antihistamine drug by human histamine receptor.

The histamine receptors belong to the G protein-coupled receptor (GPCR) superfamily, and play important roles in the regulation of histamine and other neurotransmitters in the central nervous system, as potential targets for the treatment of neurologic and psychiatric disorders. Here we report the crystal structure of human histamine receptor H3R bound to an antagonist PF-03654746 at 2.6 Å resolution. Combined with the computational and functional assays, our structure reveals binding modes of the antagonist and allosteric cholesterol. Molecular dynamic simulations and molecular docking of different antihistamines further elucidate the conserved ligand-binding modes. These findings are therefore expected to facilitate the structure-based design of novel antihistamines.

Recent advances in the application of microbial diamine oxidases and other histamine-oxidizing enzymes.

The consumption of foods fraught with histamine can lead to various allergy-like symptoms if the histamine is not sufficiently degraded in the human body. The degradation occurs primarily in the small intestine, naturally catalyzed by the human diamine oxidase (DAO). An inherent or acquired deficiency in human DAO function causes the accumulation of histamine and subsequent intrusion of histamine into the bloodstream. The histamine exerts its effects acting on different histamine receptors all over the body but also directly in the intestinal lumen. The inability to degrade sufficient amounts of dietary histamine is known as the 'histamine intolerance'. It would be preferable to solve this problem initially by the production of histamine-free or -reduced foods and by the oral supplementation of exogenous DAO supporting the human DAO in the small intestine. For the latter, DAOs from mammalian, herbal and microbial sources may be applicable. Microbial DAOs seem to be the most promising choice due to their possibility of an efficient biotechnological production in suitable microbial hosts. However, their biochemical properties, such as activity and stability under process conditions and substrate selectivity, play important roles for their successful application. This review deals with the advances and challenges of DAOs and other histamine-oxidizing enzymes for their potential application as processing aids for the production of histamine-reduced foods or as orally administered adjuvants to humans who have been eating food fraught with histamine.

Histamine Activates Human Eosinophils via H2R and H4R Predominantly in Atopic Dermatitis Patients.

Atopic dermatitis (AD) is maintained by a variety of cells and inflammatory mediators, including eosinophils and histamine. We recently reported that eosinophils from AD patients highly express the H4R. However, its immunomodulatory function in eosinophils is still largely unexplored. In this study, transcriptome analysis of blood eosinophils from AD patients stimulated with histamine and the H4R agonist ST-1006 revealed several regulated genes (e.g., IL-18R, IL-1RL1, PDE4B, CXCR4) involved in inflammation. Subsequently, the impact of histamine on one of the strongly regulated genes, the IL-18 receptor (IL-18Rα), was investigated in detail. Stimulation with histamine induced the upregulation of IL-18Rα at mRNA and at the protein level in human eosinophils, which was more pronounced in cells from AD patients than in cells from healthy controls. IL-18 was upregulated via histamine as well. After pre-incubation with histamine and IFN-γ, subsequent stimulation with IL-18 resulted in an increased ECP mRNA expression. The activation of eosinophils by histamine, in combination with IFN-γ and IL-5, was also accompanied by an upregulation of CD69. Thus, our results indicate a crucial role of histamine in the upregulation of the IL-18/IL-18R axis and in the activation of human eosinophils from AD patients.

Unraveling the venom chemistry with evidence for histamine as key regulator in the envenomation by caterpillar Automeris zaruma.

Over the past decades, envenomation by caterpillars of Automeris spp. became an increasing health problem in Latin America. Accidental contact with the stinging spines of these caterpillars cause acute local pain, itching, inflammation and skin rashes that persists for days. Even when the cause is obvious, the exact molecular mechanisms responsible for the observed symptoms are yet to be elucidated. Here, we describe for the first time, an active compound in the venom and the study of the bioactivity of the venom extracted from the spines of the caterpillar Automeris zaruma. Electrophysiological screening of a library of membrane proteins important for pain and itch enabled us to investigate and reveal the mode of action of the venom of A. zaruma. Further mass spectrometric analysis (Q-TOF-MS) made it possible to establish a link between the bioactivity and the components found in the venom. We show that the spine extract of A. zaruma contains histamine that potently activates the four types of the human histamine receptors (H1R, H2R, H3R and H4R) with a selectivity preference towards H3R and H4R. Furthermore, a modulation of the target MRGPRX2 was found. Together, these findings are the first to explain the symptomology of A. zaruma envenomation, enabling us a better understanding of caterpillar envenomation and predict that the hurdle of the scarce efficacy of the currently used antihistaminic drugs can be overcome by including H3R and H4R blockers in the clinical used medication. Such an approach might be used for other caterpillar envenomation in the world and represent a significant improvement for the well-being of the patient.

Nuclear localization of histamine receptor 2 in primary human lymphatic endothelial cells.

Histamine exerts its physiological functions through its four receptor subtypes. In this work, we report the subcellular localization of histamine receptor 2 (H2R), a G protein-coupled receptor (GPCR), which is expressed in a wide variety of cell and tissue types. A growing number of GPCRs have been shown to be localized in the nucleus and contribute toward transcriptional regulation. In this study, for the first time, we demonstrate the nuclear localization of H2R in lymphatic endothelial cells. In the presence of its ligand, we show significant upregulation of H2R nuclear translocation kinetics. Using fluorescently tagged histamine, we explored H2R-histamine binding interaction, which exhibits a critical role in this translocation event. Altogether, our results highlight the previously unrecognized nuclear localization pattern of H2R. At the same time, H2R as a GPCR imparts many unresolved questions, such as the functional relevance of this localization, and whether H2R can contribute directly to transcriptional regulation and can affect lymphatic specific gene expression. H2R blockers are commonly used medications that recently have shown significant side effects. Therefore, it is imperative to understand the precise molecular mechanism of H2R biology. In this aspect, our present data shed new light on the unexplored H2R signaling mechanisms. This article has an associated First Person interview with the first author of the paper.