Genome-wide Analyses of Chromatin State in Human Mast Cells Reveal Molecular Drivers and Mediators of Allergic and Inflammatory Diseases.

Mast cells (MCs) are versatile immune cells capable of rapidly responding to a diverse range of extracellular cues. Here, we mapped the genomic and transcriptomic changes in human MCs upon diverse stimuli. Our analyses revealed broad H3K4me3 domains and enhancers associated with activation. Notably, the rise of intracellular calcium concentration upon immunoglobulin E (IgE)-mediated crosslinking of the high-affinity IgE receptor (FcεRI) resulted in genome-wide reorganization of the chromatin landscape and was associated with a specific chromatin signature, which we term Ca2+-dependent open chromatin (COC) domains. Examination of differentially expressed genes revealed potential effectors of MC function, and we provide evidence for fibrinogen-like protein 2 (FGL2) as an MC mediator with potential relevance in chronic spontaneous urticaria. Disease-associated single-nucleotide polymorphisms mapped onto cis-regulatory regions of human MCs suggest that MC function may impact a broad range of pathologies. The datasets presented here constitute a resource for the further study of MC function.

TLR7 gain-of-function genetic variation causes human lupus.

Although circumstantial evidence supports enhanced Toll-like receptor 7 (TLR7) signalling as a mechanism of human systemic autoimmune disease1-7, evidence of lupus-causing TLR7 gene variants is lacking. Here we describe human systemic lupus erythematosus caused by a TLR7 gain-of-function variant. TLR7 is a sensor of viral RNA8,9 and binds to guanosine10-12. We identified a de novo, previously undescribed missense TLR7Y264H variant in a child with severe lupus and additional variants in other patients with lupus. The TLR7Y264H variant selectively increased sensing of guanosine and 2',3'-cGMP10-12, and was sufficient to cause lupus when introduced into mice. We show that enhanced TLR7 signalling drives aberrant survival of B cell receptor (BCR)-activated B cells, and in a cell-intrinsic manner, accumulation of CD11c+ age-associated B cells and germinal centre B cells. Follicular and extrafollicular helper T cells were also increased but these phenotypes were cell-extrinsic. Deficiency of MyD88 (an adaptor protein downstream of TLR7) rescued autoimmunity, aberrant B cell survival, and all cellular and serological phenotypes. Despite prominent spontaneous germinal-centre formation in Tlr7Y264H mice, autoimmunity was not ameliorated by germinal-centre deficiency, suggesting an extrafollicular origin of pathogenic B cells. We establish the importance of TLR7 and guanosine-containing self-ligands for human lupus pathogenesis, which paves the way for therapeutic TLR7 or MyD88 inhibition.

Messing with ßc: A unique receptor with many goals.

Our understanding of the biological role of the ßc family of cytokines has evolved enormously since their initial identification as bone marrow colony stimulating factors in the 1960's. It has become abundantly clear over the intervening decades that this family of cytokines has truly astonishing pleiotropic capacity, capable of regulating not only hematopoiesis but also many other normal and pathological processes such as development, inflammation, allergy and cancer. As noted in the current pandemic, ßc cytokines contribute to the cytokine storm seen in acutely ill COVID-19 patients. Ongoing studies to discover how these cytokines activate their receptor are revealing insights into the fundamental mechanisms that give rise to cytokine pleiotropy and are providing tantalizing glimpses of how discrete signaling pathways may be dissected for activation with novel ligands for therapeutic benefit.

Dual inhibition of airway inflammation and fibrosis by common ß cytokine receptor blockade.

BACKGROUND: Patients with severe asthma can present with eosinophilic type 2 (T2), neutrophilic, or mixed inflammation that drives airway remodeling and exacerbations and represents a major treatment challenge. The common ß (ßc) receptor signals for 3 cytokines, GM-CSF, IL-5, and IL-3, which collectively mediate T2 and neutrophilic inflammation. OBJECTIVE: To determine the pathogenesis of ßc receptor-mediated inflammation and remodeling in severe asthma and to investigate ßc antagonism as a therapeutic strategy for mixed granulocytic airway disease. METHODS: ßc gene expression was analyzed in bronchial biopsy specimens from patients with mild-to-moderate and severe asthma. House dust mite extract and Aspergillus fumigatus extract (ASP) models were used to establish asthma-like pathology and airway remodeling in human ßc transgenic mice. Lung tissue gene expression was analyzed by RNA sequencing. The mAb CSL311 targeting the shared cytokine binding site of ßc was used to block ßc signaling. RESULTS: ßc gene expression was increased in patients with severe asthma. CSL311 potently reduced lung neutrophils, eosinophils, and interstitial macrophages and improved airway pathology and lung function in the acute steroid-resistant house dust mite extract model. Chronic intranasal ASP exposure induced airway inflammation and fibrosis and impaired lung function that was inhibited by CSL311. CSL311 normalized the ASP-induced fibrosis-associated extracellular matrix gene expression network and strongly reduced signatures of cellular inflammation in the lung. CONCLUSIONS: ßc cytokines drive steroid-resistant mixed myeloid cell airway inflammation and fibrosis. The anti-ßc antibody CSL311 effectively inhibits mixed T2/neutrophilic inflammation and severe asthma-like pathology and reverses fibrosis gene signatures induced by exposure to commonly encountered environmental allergens.

Antipolar 2D Metallicity with Tunable Valence Wx+ (5 ≤ x ≤ 5.6) in the Layered Monophosphate Tungsten Bronzes [Ba(PO4)2]WmO3m-3.

The newly discovered series of layered monophosphate tungsten bronzes (L-MPTB) [Ba(PO4)2]WmO3m-3 consist of m-layer-thick slabs of WO6 octahedra separated by barium-phosphate spacers. They display a 2D metallic behavior confined in the central part of the perovskite slabs. Here, we report the missing m = 2 member of this series, containing the rather uncommon W5+ oxidation state. We have analyzed its structure-property relationships in relation to the other members of the L-MPTB family. In particular, we have determined its crystal structure by means of single-crystal X-ray and electron diffraction and investigated its physical properties from resistivity, Seebeck-coefficient and heat-capacity measurements combined with first-principles calculations. All the L-MPTB compounds show metallic behavior down to 1.8 K without any clear charge-density-wave (CDW) order. The m = 2 member, however, displays an increased influence of the spacer that translates into anisotropic negative thermal expansion, reversed thermopower and reversed crystal-field splitting of the tungsten t2g orbitals. Our analysis of the full [Ba(PO4)2]WmO3m-3 series reveals a systematic and significant W off-centering in their octahedral coordination. We identify the resulting anti-polar character of these W displacements as the crucial aspect behind the 2D metallicity of these systems: It leads to the presence of bound charges whose screening determines the distribution of mobile charges, tending to accumulate at the center of the [WmO3-m] block. We argue that this mechanism is analogous to enhanced conductivity observed for charged domain walls in ferroelectrics, thus providing a general design rule to promote 2D metallicity in layered systems.

Interleukin-3 production by basal-like breast cancer cells is associated with poor prognosis.

Breast cancer represents a collection of pathologies with different molecular subtypes, histopathology, risk factors, clinical behavior, and responses to treatment. "Basal-like" breast cancers predominantly lack the receptors for estrogen and progesterone (ER/PR), lack amplification of human epidermal growth factor receptor 2 (HER2) but account for 10-15% of all breast cancers, are largely insensitive to targeted treatment and represent a disproportionate number of metastatic cases and deaths. Analysis of interleukin (IL)-3 and the IL-3 receptor subunits (IL-3RA + CSF2RB) reveals elevated expression in predominantly the basal-like group. Further analysis suggests that IL-3 itself, but not the IL-3 receptor subunits, associates with poor patient outcome. Histology on patient-derived xenografts supports the notion that breast cancer cells are a significant source of IL-3 that may promote disease progression. Taken together, these observations suggest that IL-3 may be a useful marker in solid tumors, particularly triple negative breast cancer, and warrants further investigation into its contribution to disease pathogenesis.

Understanding mast cell heterogeneity at single cell resolution.

Mast cells (MC)s are evolutionarily conserved, tissue-resident immune cells with diverse roles in allergy, cancer, and protection from infection by helminths and microorganisms. The significant diversity in MC development and tissue-specific functional characteristics has recently begun to be understood. Exciting developments in single-cell-based RNA, protein, and chromatin profiling technologies offer new opportunities to characterize MC heterogeneity and to uncover novel MC functions and subtypes; these developments might lead to new and clinically effective therapies for certain pathologies. In this review, we provide an overview of the current understanding of MC development and heterogeneity and discuss new insights gained from single-cell-based studies that may lead to future research directions and therapeutic opportunities.

Targeting human CALR-mutated MPN progenitors with a neoepitope-directed monoclonal antibody.

Calreticulin (CALR) is recurrently mutated in myelofibrosis via a frameshift that removes an endoplasmic reticulum retention signal, creating a neoepitope potentially targetable by immunotherapeutic approaches. We developed a specific rat monoclonal IgG2α antibody, 4D7, directed against the common sequence encoded by both insertion and deletion mutations. 4D7 selectively bound to cells co-expressing mutant CALR and thrombopoietin receptor (TpoR) and blocked JAK-STAT signalling, TPO-independent proliferation and megakaryocyte differentiation of mutant CALR myelofibrosis progenitors by disrupting the binding of CALR dimers to TpoR. Importantly, 4D7 inhibited proliferation of patient samples with both insertion and deletion CALR mutations but not JAK2 V617F and prolonged survival in xenografted bone marrow models of mutant CALR-dependent myeloproliferation. Together, our data demonstrate a novel therapeutic approach to target a problematic disease driven by a recurrent somatic mutation that would normally be considered undruggable.

Ferromagnetic Mn3+ Sawtooth Chains in A2(Mn2O)(SeO3)3 (A = K, Rb) Quaternary Selenites.

Two quaternary manganese selenites, A2(Mn2O)(SeO3)3 (A = K, Rb), have been synthesized by hydrothermal reactions. They both crystallize in a complex triclinic (P-1) structure built of Jahn-Teller (JT) distorted Mn3+O4+2 octahedra, connected into nearly isosceles [Mn3O14] triangles, themselves arranged into so-called "sawtooth (ST) chains". The K and Rb compounds show subtle variations in the orientations of the MnO4 planes inside the elementary triangles. The ST chains are structurally and magnetically isolated by SeO3 groups and alkali cations. In the ST chains, predominant ferromagnetic interactions were calculated and verified experimentally, which finally order antiferromagnetically between the chains around TN ≈ 22 K. The spin exchanges calculated by DFT + U and fitted by Monte Carlo simulations allow for the quantification of an effective "overall" model. The specific role of the µ3-O bridge on the ferromagnetic (FM) exchanges is discussed, together with spin reorientations observed in the ordered state. Magnetocrystalline anisotropy along the [110] direction stabilized by ∼50 meV per Mn by spin-orbit coupling (SOC) was found by DFT + U + SOC.

Elucidating the Mechanism of Metabolism of Cannabichromene by Human Cytochrome P450s.

Cannabichromene (CBC) is a nonpsychoactive phytocannabinoid well-known for its wide-ranging health advantages. However, there is limited knowledge regarding its human metabolism following CBC consumption. This research aimed to explore the metabolic pathways of CBC by various human liver cytochrome P450 (CYP) enzymes and support the outcomes using in vivo data from mice. The results unveiled two principal CBC metabolites generated by CYPs: 8'-hydroxy-CBC and 6',7'-epoxy-CBC, along with a minor quantity of 1″-hydroxy-CBC. Notably, among the examined CYPs, CYP2C9 demonstrated the highest efficiency in producing these metabolites. Moreover, through a molecular dynamics simulation spanning 1 µs, it was observed that CBC attains stability at the active site of CYP2J2 by forming hydrogen bonds with I487 and N379, facilitated by water molecules, which specifically promotes the hydroxy metabolite's formation. Additionally, the presence of cytochrome P450 reductase (CPR) amplified CBC's binding affinity to CYPs, particularly with CYP2C8 and CYP3A4. Furthermore, the metabolites derived from CBC reduced cytokine levels, such as IL6 and NO, by approximately 50% in microglia cells. This investigation offers valuable insights into the biotransformation of CBC, underscoring the physiological importance and the potential significance of these metabolites.

Author Correction: A multi-cohort study of the immune factors associated with M. tuberculosis infection outcomes.

The spelling of author Qianting Yang was corrected; the affiliation of author Stephanus T. Malherbe was corrected; and graphs in Fig. 4b and c were corrected owing to reanalysis of the data into the correct timed intervals.

A multi-cohort study of the immune factors associated with M. tuberculosis infection outcomes.

Most infections with Mycobacterium tuberculosis (Mtb) manifest as a clinically asymptomatic, contained state, known as latent tuberculosis infection, that affects approximately one-quarter of the global population1. Although fewer than one in ten individuals eventually progress to active disease2, tuberculosis is a leading cause of death from infectious disease worldwide3. Despite intense efforts, immune factors that influence the infection outcomes remain poorly defined. Here we used integrated analyses of multiple cohorts to identify stage-specific host responses to Mtb infection. First, using high-dimensional mass cytometry analyses and functional assays of a cohort of South African adolescents, we show that latent tuberculosis is associated with enhanced cytotoxic responses, which are mostly mediated by CD16 (also known as FcγRIIIa) and natural killer cells, and continuous inflammation coupled with immune deviations in both T and B cell compartments. Next, using cell-type deconvolution of transcriptomic data from several cohorts of different ages, genetic backgrounds, geographical locations and infection stages, we show that although deviations in peripheral B and T cell compartments generally start at latency, they are heterogeneous across cohorts. However, an increase in the abundance of circulating natural killer cells in tuberculosis latency, with a corresponding decrease during active disease and a return to baseline levels upon clinical cure are features that are common to all cohorts. Furthermore, by analysing three longitudinal cohorts, we find that changes in peripheral levels of natural killer cells can inform disease progression and treatment responses, and inversely correlate with the inflammatory state of the lungs of patients with active tuberculosis. Together, our findings offer crucial insights into the underlying pathophysiology of tuberculosis latency, and identify factors that may influence infection outcomes.

Signatures of immune dysfunction in HIV and HCV infection share features with chronic inflammation in aging and persist after viral reduction or elimination.

Chronic inflammation is thought to be a major cause of morbidity and mortality in aging, but whether similar mechanisms underlie dysfunction in infection-associated chronic inflammation is unclear. Here, we profiled the immune proteome, and cellular composition and signaling states in a cohort of aging individuals versus a set of HIV patients on long-term antiretroviral therapy therapy or hepatitis C virus (HCV) patients before and after sofosbuvir treatment. We found shared alterations in aging-associated and infection-associated chronic inflammation including T cell memory inflation, up-regulation of intracellular signaling pathways of inflammation, and diminished sensitivity to cytokines in lymphocytes and myeloid cells. In the HIV cohort, these dysregulations were evident despite viral suppression for over 10 y. Viral clearance in the HCV cohort partially restored cellular sensitivity to interferon-α, but many immune system alterations persisted for at least 1 y posttreatment. Our findings indicate that in the HIV and HCV cohorts, a broad remodeling and degradation of the immune system can persist for a year or more, even after the removal or drastic reduction of the pathogen load and that this shares some features of chronic inflammation in aging.

Development of a Smartwatch with Gas and Environmental Sensors for Air Quality Monitoring.

In recent years, there has been a growing interest in developing portable and personal devices for measuring air quality and surrounding pollutants, partly due to the need for ventilation in the aftermath of COVID-19 situation. Moreover, the monitoring of hazardous chemical agents is a focus for ensuring compliance with safety standards and is an indispensable component in safeguarding human welfare. Air quality measurement is conducted by public institutions with high precision but costly equipment, which requires constant calibration and maintenance by highly qualified personnel for its proper operation. Such devices, used as reference stations, have a low spatial resolution since, due to their high cost, they are usually located in a few fixed places in the city or region to be studied. However, they also have a low temporal resolution, providing few samples per hour. To overcome these drawbacks and to provide people with personalized and up-to-date air quality information, a personal device (smartwatch) based on MEMS gas sensors has been developed. The methodology followed to validate the performance of the prototype was as follows: firstly, the detection capability was tested by measuring carbon dioxide and methane at different concentrations, resulting in low detection limits; secondly, several experiments were performed to test the discrimination capability against gases such as toluene, xylene, and ethylbenzene. principal component analysis of the data showed good separation and discrimination between the gases measured.

Translating the biology of ß common receptor-engaging cytokines into clinical medicine.

The family of cytokines that comprises IL-3, IL-5, and GM-CSF was discovered over 30 years ago, and their biological activities and resulting impact in clinical medicine has continued to expand ever since. Originally identified as bone marrow growth factors capable of acting on hemopoietic progenitor cells to induce their proliferation and differentiation into mature blood cells, these cytokines are also recognized as key mediators of inflammation and the pathobiology of diverse immunologic diseases. This increased understanding of the functional repertoire of IL-3, IL-5, and GM-CSF has led to an explosion of interest in modulating their functions for clinical management. Key to the successful clinical translation of this knowledge is the recognition that these cytokines act by engaging distinct dimeric receptors and that they share a common signaling subunit called ß-common or ßc. The structural determination of how IL-3, IL-5, and GM-CSF interact with their receptors and linking this to their differential biological functions on effector cells has unveiled new paradigms of cell signaling. This knowledge has paved the way for novel mAbs and other molecules as selective or pan inhibitors for use in different clinical settings.

IgE receptor of mast cells signals mediator release and inflammation via adaptor protein 14-3-3ζ.

BACKGROUND: Mast cells (MCs) are tissue-resident immune cells that mediate IgE-dependent allergic responses. Downstream of FcεRI, an intricate network of receptor-specific signaling pathways and adaptor proteins govern MC function. The 14-3-3 family of serine-threonine phosphorylation-dependent adapter proteins are known to organize intracellular signaling. However, the role of 14-3-3 in IgE-dependent activation remains poorly defined. OBJECTIVE: We sought to determine whether 14-3-3 proteins are required for IgE-dependent MC activation and whether 14-3-3 is a viable target for the treatment of MC-mediated inflammatory diseases. METHODS: Genetic manipulation of 14-3-3ζ expression in human and mouse MCs was performed and IgE-dependent mediator release assessed. Pharmacologic inhibitors of 14-3-3 and 14-3-3ζ knockout mice were used to assess 14-3-3ζ function in a MC-dependent in vivo passive cutaneous anaphylaxis (PCA) model of allergic inflammation. Expression and function of 14-3-3ζ were assessed in human nasal polyp tissue MCs. RESULTS: IgE-dependent mediator release from human MCs was decreased by 14-3-3ζ knockdown and increased by 14-3-3ζ overexpression. Deletion of the 14-3-3ζ gene decreased IgE-dependent activation of mouse MCs in vitro and PCA responses in vivo. Furthermore, the 14-3-3 inhibitor, RB-11, which impairs dimerization of 14-3-3, inhibited cultured MC and polyp tissue MC activation and signaling downstream of the FcεRI receptor and dose-dependently attenuated PCA responses. CONCLUSION: IgE/FcεRI-mediated MC activation is positively regulated by 14-3-3ζ. We identify a critical role for this p-Ser/Thr-binding protein in the regulation of MC FcεRI signaling and IgE-dependent immune responses and show that this pathway may be amenable to pharmacologic targeting.

The structure of the GM-CSF receptor complex reveals a distinct mode of cytokine receptor activation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that controls the production and function of blood cells, is deregulated in clinical conditions such as rheumatoid arthritis and leukemia, yet offers therapeutic value for other diseases. Its receptors are heterodimers consisting of a ligand-specific alpha subunit and a betac subunit that is shared with the interleukin (IL)-3 and IL-5 receptors. How signaling is initiated remains an enigma. We report here the crystal structure of the human GM-CSF/GM-CSF receptor ternary complex and its assembly into an unexpected dodecamer or higher-order complex. Importantly, mutagenesis of the GM-CSF receptor at the dodecamer interface and functional studies reveal that dodecamer formation is required for receptor activation and signaling. This unusual form of receptor assembly likely applies also to IL-3 and IL-5 receptors, providing a structural basis for understanding their mechanism of activation and for the development of therapeutics.

Kinetic monitoring of neuronal stress response to proteostasis dysfunction.

Proteostasis dysfunction and activation of the unfolded protein response (UPR) are characteristic of all major neurodegenerative diseases. Nevertheless, although the UPR and proteostasis dysfunction has been studied in great detail in model organisms like yeast and mammalian cell lines, it has not yet been examined in neurons. In this study, we applied a viral vector-mediated expression of a reporter protein based on a UPR transcription factor, ATF4, and time-lapse fluorescent microscopy to elucidate how mouse primary neurons respond to pharmacological and genetic perturbations to neuronal proteostasis. In in vitro models of endoplasmic reticulum (ER) stress and proteasome inhibition, we used the ATF4 reporter to reveal the time course of the neuronal stress response relative to neurite degeneration and asynchronous cell death. We showed how potential neurodegenerative disease co-factors, ER stress and mutant α-synuclein overexpression, impacted neuronal stress response and overall cellular health. This work therefore introduces a viral vector-based reporter that yields a quantifiable readout suitable for non-cell destructive kinetic monitoring of proteostasis dysfunction in neurons by harnessing ATF4 signaling as part of the UPR activation.

Layered Monophosphate Tungsten Bronzes [Ba(PO4 )2 ]Wm O3m-3 : 2D Metals with Locked Charge-Density-Wave Instabilities.

Phosphate tungsten and molybenum bronzes represent an outstanding class of materials displaying textbook examples of charge-density-wave (CDW) physics among other fundamental properties. Here we report on the existence of a novel structural branch with the general formula [Ba(PO4 )2 ][Wm O3m-3 ] (m=3, 4 and 5) denominated 'layered monophosphate tungsten bronzes' (L-MPTB). It results from thick [Ba(PO4 )2 ]4- spacer layers disrupting the cationic metal-oxide 2D units and enforcing an overall trigonal structure. Their symmetries are preserved down to 1.8 K and the compounds show metallic behaviour with no clear anomaly as a function of temperature. However, their electronic structure displays the characteristic Fermi surface of previous bronzes derived from 5d W states with hidden nesting properties. By analogy with previous bronzes, such a Fermi surface should result into CDW order. Evidence of CDW order was only indirectly observed in the low-temperature specific heat, giving an exotic context at the crossover between stable 2D metals and CDW order.

Synthesis of the Cannabimovone and Cannabifuran Class of Minor Phytocannabinoids and Their Anti-inflammatory Activity.

Despite centuries-long use of Cannabis in human culture and the now ubiquitous claims of its medicinal value, only a small handful of phytocannabinoids have been rigorously evaluated for pharmacological properties. While more than 100 distinct minor cannabinoids have been documented to date, a paucity of studies on their biological activities have been conducted due to a lack of routine access to sufficient quantities for testing. Herein, we report a strategy to prepare several structurally diverse minor cannabinoids deriving synthetically from readily available cannabidiol. Furthermore, we examined their ability to polarize activated microglia toward an anti-inflammatory phenotype using LPS-stimulated BV2 microglial cells. The minor cannabinoids studied, especially cannabielsoin, dehydrocannabielsoin, cannabimovone, and 3'-epicannabimovone, inhibited the production of prototypical pro-inflammatory biomarkers. This study represents the beginning of a systematic mapping of the roles minor cannabinoids may play in the medicinal properties of cannabis used for the treatment of pain and inflammation.