A noncanonical role for the engulfment gene ELMO1 in neutrophils that promotes inflammatory arthritis.

Rheumatoid arthritis is characterized by progressive joint inflammation and affects ~1% of the human population. We noted single-nucleotide polymorphisms (SNPs) in the apoptotic cell-engulfment genes ELMO1, DOCK2, and RAC1 linked to rheumatoid arthritis. As ELMO1 promotes cytoskeletal reorganization during engulfment, we hypothesized that ELMO1 loss would worsen inflammatory arthritis. Surprisingly, Elmo1-deficient mice showed reduced joint inflammation in acute and chronic arthritis models. Genetic and cell-biology studies revealed that ELMO1 associates with receptors linked to neutrophil function in arthritis and regulates activation and early neutrophil recruitment to the joints, without general inhibition of inflammatory responses. Further, neutrophils from the peripheral blood of human donors that carry the SNP in ELMO1 associated with arthritis display increased migratory capacity, whereas ELMO1 knockdown reduces human neutrophil migration to chemokines linked to arthritis. These data identify 'noncanonical' roles for ELMO1 as an important cytoplasmic regulator of specific neutrophil receptors and promoter of arthritis.

Pannexin 1 channels facilitate communication between T cells to restrict the severity of airway inflammation.

Allergic airway inflammation is driven by type-2 CD4+ T cell inflammatory responses. We uncover an immunoregulatory role for the nucleotide release channel, Panx1, in T cell crosstalk during airway disease. Inverse correlations between Panx1 and asthmatics and our mouse models revealed the necessity, specificity, and sufficiency of Panx1 in T cells to restrict inflammation. Global Panx1-/- mice experienced exacerbated airway inflammation, and T-cell-specific deletion phenocopied Panx1-/- mice. A transgenic designed to re-express Panx1 in T cells reversed disease severity in global Panx1-/- mice. Panx1 activation occurred in pro-inflammatory T effector (Teff) and inhibitory T regulatory (Treg) cells and mediated the extracellular-nucleotide-based Treg-Teff crosstalk required for suppression of Teff cell proliferation. Mechanistic studies identified a Salt-inducible kinase-dependent phosphorylation of Panx1 serine 205 important for channel activation. A genetically targeted mouse expressing non-phosphorylatable Panx1S205A phenocopied the exacerbated inflammation in Panx1-/- mice. These data identify Panx1-dependent Treg:Teff cell communication in restricting airway disease.

ARAP3 protects from excessive formylated peptide-induced microvascular leakage by acting on endothelial cells and neutrophils.

Vascular permeability is temporarily heightened during inflammation, but excessive inflammation-associated microvascular leakage can be detrimental, as evidenced in the inflamed lung. Formylated peptides regulate vascular leakage indirectly via formylated peptide receptor-1 (FPR1)-mediated recruitment and activation of neutrophils. Here we identify how the GTPase-activating protein ARAP3 protects against formylated peptide-induced microvascular permeability via endothelial cells and neutrophils. In vitro, Arap3-/- endothelial monolayers were characterised by enhanced formylated peptide-induced permeability due to upregulated endothelial FPR1 and enhanced vascular endothelial cadherin internalisation. In vivo, enhanced inflammation-associated microvascular leakage was observed in Arap3-/- mice. Leakage of plasma protein into the lungs of Arap3-/- mice increased within hours of formylated peptide administration. Adoptive transfer experiments indicated this was dependent upon ARAP3 deficiency in both immune and non-immune cells. Bronchoalveolar lavages of formylated peptide-challenged Arap3-/- mice contained neutrophil extracellular traps (NETs). Pharmacological inhibition of NET formation abrogated excessive microvascular leakage, indicating a critical function of NETs in this context. The observation that Arap3-/- mice developed more severe influenza suggests these findings are pertinent to pathological situations characterised by abundant formylated peptides. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Narrowing the diagnostic gap: Genomes, episignatures, long-read sequencing, and health economic analyses in an exome-negative intellectual disability cohort.

PURPOSE: Genome sequencing (GS)-specific diagnostic rates in prospective tightly ascertained exome sequencing (ES)-negative intellectual disability (ID) cohorts have not been reported extensively. METHODS: ES, GS, epigenetic signatures, and long-read sequencing diagnoses were assessed in 74 trios with at least moderate ID. RESULTS: The ES diagnostic yield was 42 of 74 (57%). GS diagnoses were made in 9 of 32 (28%) ES-unresolved families. Repeated ES with a contemporary pipeline on the GS-diagnosed families identified 8 of 9 single-nucleotide variations/copy-number variations undetected in older ES, confirming a GS-unique diagnostic rate of 1 in 32 (3%). Episignatures contributed diagnostic information in 9% with GS corroboration in 1 of 32 (3%) and diagnostic clues in 2 of 32 (6%). A genetic etiology for ID was detected in 51 of 74 (69%) families. Twelve candidate disease genes were identified. Contemporary ES followed by GS cost US$4976 (95% CI: $3704; $6969) per diagnosis and first-line GS at a cost of $7062 (95% CI: $6210; $8475) per diagnosis. CONCLUSION: Performing GS only in ID trios would be cost equivalent to ES if GS were available at $2435, about a 60% reduction from current prices. This study demonstrates that first-line GS achieves higher diagnostic rate than contemporary ES but at a higher cost.

Tissue Proteomic Analysis Identifies Mechanisms and Stages of Immunopathology in Fatal COVID-19.

Immunopathology occurs in the lung and spleen in fatal coronavirus disease (COVID-19), involving monocytes/macrophages and plasma cells. Antiinflammatory therapy reduces mortality, but additional therapeutic targets are required. We aimed to gain mechanistic insight into COVID-19 immunopathology by targeted proteomic analysis of pulmonary and splenic tissues. Lung parenchymal and splenic tissue was obtained from 13 postmortem examinations of patients with fatal COVID-19. Control tissue was obtained from cancer resection samples (lung) and deceased organ donors (spleen). Protein was extracted from tissue by phenol extraction. Olink multiplex immunoassay panels were used for protein detection and quantification. Proteins with increased abundance in the lung included MCP-3, antiviral TRIM21, and prothrombotic TYMP. OSM and EN-RAGE/S100A12 abundance was correlated and associated with inflammation severity. Unsupervised clustering identified "early viral" and "late inflammatory" clusters with distinct protein abundance profiles, and differences in illness duration before death and presence of viral RNA. In the spleen, lymphocyte chemotactic factors and CD8A were decreased in abundance, and proapoptotic factors were increased. B-cell receptor signaling pathway components and macrophage colony stimulating factor (CSF-1) were also increased. Additional evidence for a subset of host factors (including DDX58, OSM, TYMP, IL-18, MCP-3, and CSF-1) was provided by overlap between 1) differential abundance in spleen and lung tissue; 2) meta-analysis of existing datasets; and 3) plasma proteomic data. This proteomic analysis of lung parenchymal and splenic tissue from fatal COVID-19 provides mechanistic insight into tissue antiviral responses, inflammation and disease stages, macrophage involvement, pulmonary thrombosis, splenic B-cell activation, and lymphocyte depletion.

DNA Origami Nanostructures Elicit Dose-Dependent Immunogenicity and Are Nontoxic up to High Doses In Vivo.

DNA origami (DO) nanotechnology enables the construction of precise nanostructures capable of functionalization with small molecule drugs, nucleic acids, and proteins, suggesting a promising platform for biomedical applications. Despite the potential for drug and vaccine delivery, the impact of DO vehicles on immunogenicity in vivo is not well understood. Here, two DO vehicles, a flat triangle and a nanorod, at varying concentrations are evaluated in vitro and with a repeated dosing regimen administered at a high dose in vivo to study early and late immunogenicity. The studies show normal CD11b+ myeloid cell populations preferentially internalize DO in vitro. DO structures distribute well systemically in vivo, elicit a modest pro-inflammatory immune response that diminishes over time and are nontoxic as shown by weight, histopathology, lack of cytokine storm, and a complete biochemistry panel at the day 10 end point. The results take critical steps to characterize the biological response to DO and suggest that DO vehicles represent a promising platform for drug delivery and vaccine development where immunogenicity should be a key consideration.

Tissue-Specific Immunopathology in Fatal COVID-19.

Rationale: In life-threatening coronavirus disease (COVID-19), corticosteroids reduce mortality, suggesting that immune responses have a causal role in death. Whether this deleterious inflammation is primarily a direct reaction to the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or an independent immunopathologic process is unknown.Objectives: To determine SARS-CoV-2 organotropism and organ-specific inflammatory responses and the relationships among viral presence, inflammation, and organ injury.Methods: Tissue was acquired from 11 detailed postmortem examinations. SARS-CoV-2 organotropism was mapped by using multiplex PCR and sequencing, with cellular resolution achieved by in situ viral S (spike) protein detection. Histologic evidence of inflammation was quantified from 37 anatomic sites, and the pulmonary immune response was characterized by using multiplex immunofluorescence.Measurements and Main Results: Multiple aberrant immune responses in fatal COVID-19 were found, principally involving the lung and reticuloendothelial system, and these were not clearly topologically associated with the virus. Inflammation and organ dysfunction did not map to the tissue and cellular distribution of SARS-CoV-2 RNA and protein between or within tissues. An arteritis was identified in the lung, which was further characterized as a monocyte/myeloid-rich vasculitis, and occurred together with an influx of macrophage/monocyte-lineage cells into the pulmonary parenchyma. In addition, stereotyped abnormal reticuloendothelial responses, including excessive reactive plasmacytosis and iron-laden macrophages, were present and dissociated from viral presence in lymphoid tissues.Conclusions: Tissue-specific immunopathology occurs in COVID-19, implicating a significant component of the immune-mediated, virus-independent immunopathologic process as a primary mechanism in severe disease. Our data highlight novel immunopathologic mechanisms and validate ongoing and future efforts to therapeutically target aberrant macrophage and plasma-cell responses as well as promote pathogen tolerance in COVID-19.

Real-world effectiveness, its predictors and onset of action of cholinesterase inhibitors and memantine in dementia: retrospective health record study.

BACKGROUND: The efficacy of acetylcholinesterase inhibitors and memantine in the symptomatic treatment of Alzheimer's disease is well-established. Randomised trials have shown them to be associated with a reduction in the rate of cognitive decline. AIMS: To investigate the real-world effectiveness of acetylcholinesterase inhibitors and memantine for dementia-causing diseases in the largest UK observational secondary care service data-set to date. METHOD: We extracted mentions of relevant medications and cognitive testing (Mini-Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA) scores) from de-identified patient records from two National Health Service (NHS) trusts. The 10-year changes in cognitive performance were modelled using a combination of generalised additive and linear mixed-effects modelling. RESULTS: The initial decline in MMSE and MoCA scores occurs approximately 2 years before medication is initiated. Medication prescription stabilises cognitive performance for the ensuing 2-5 months. The effect is boosted in more cognitively impaired cases at the point of medication prescription and attenuated in those taking antipsychotics. Importantly, patients who are switched between agents at least once do not experience any beneficial cognitive effect from pharmacological treatment. CONCLUSIONS: This study presents one of the largest real-world examination of the efficacy of acetylcholinesterase inhibitors and memantine for symptomatic treatment of dementia. We found evidence that 68% of individuals respond to treatment with a period of cognitive stabilisation before continuing their decline at the pre-treatment rate.

Mcl-1 protects eosinophils from apoptosis and exacerbates allergic airway inflammation.

Eosinophils are key effector cells in allergic diseases. Here we investigated Mcl-1 (an anti-apoptotic protein) in experimental allergic airway inflammation using transgenic overexpressing human Mcl-1 mice (hMcl-1) and reducing Mcl-1 by a cyclin-dependent kinase inhibitor. Overexpression of Mcl-1 exacerbated allergic airway inflammation, with increased bronchoalveolar lavage fluid cellularity, eosinophil numbers and total protein, and an increase in airway mucus production. Eosinophil apoptosis was suppressed by Mcl-1 overexpression, with this resistance to apoptosis attenuated by cyclin-dependent kinase inhibition which also rescued Mcl-1-exacerbated allergic airway inflammation. We propose that targeting Mcl-1 may be beneficial in treatment of allergic airway disease.

Neutrophils in the initiation and resolution of acute pulmonary inflammation: understanding biological function and therapeutic potential.

Acute respiratory distress syndrome (ARDS) is the often fatal sequelae of a broad range of precipitating conditions. Despite decades of intensive research and clinical trials there remain no therapies in routine clinical practice that target the dysregulated and overwhelming inflammatory response that characterises ARDS. Neutrophils play a central role in the initiation, propagation and resolution of this complex inflammatory environment by migrating into the lung and executing a variety of pro-inflammatory functions. These include degranulation with liberation of bactericidal proteins, release of cytokines and reactive oxygen species as well as production of neutrophil extracellular traps. Although these functions are advantageous in clearing bacterial infection, the consequence of associated tissue damage, the contribution to worsening acute inflammation and prolonged neutrophil lifespan at sites of inflammation are deleterious. In this review, the importance of the neutrophil will be considered, together with discussion of recent advances in understanding neutrophil function and the factors that influence them throughout the phases of inflammation in ARDS. From a better understanding of neutrophils in this context, potential therapeutic targets are identified and discussed. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Changes in cognitive flexibility and hypothesis search across human life history from childhood to adolescence to adulthood.

How was the evolution of our unique biological life history related to distinctive human developments in cognition and culture? We suggest that the extended human childhood and adolescence allows a balance between exploration and exploitation, between wider and narrower hypothesis search, and between innovation and imitation in cultural learning. In particular, different developmental periods may be associated with different learning strategies. This relation between biology and culture was probably coevolutionary and bidirectional: life-history changes allowed changes in learning, which in turn both allowed and rewarded extended life histories. In two studies, we test how easily people learn an unusual physical or social causal relation from a pattern of evidence. We track the development of this ability from early childhood through adolescence and adulthood. In the physical domain, preschoolers, counterintuitively, perform better than school-aged children, who in turn perform better than adolescents and adults. As they grow older learners are less flexible: they are less likely to adopt an initially unfamiliar hypothesis that is consistent with new evidence. Instead, learners prefer a familiar hypothesis that is less consistent with the evidence. In the social domain, both preschoolers and adolescents are actually the most flexible learners, adopting an unusual hypothesis more easily than either 6-y-olds or adults. There may be important developmental transitions in flexibility at the entry into middle childhood and in adolescence, which differ across domains.

Adsorption, surface relaxation and electrolyte structure at Pt(111) electrodes in non-aqueous and aqueous acetonitrile electrolytes.

In situ electrochemical surface X-ray diffraction was employed to investigate the atomic scale structure of the electrochemical double layer and the relaxation at the Pt(111) electrode surface in non-aqueous and aqueous acetonitrile electrolytes under potential control. The X-ray measurements provide insight into the potential-dependence of the interface structure by combining potentiodynamic measurements (X-ray voltammetry) with potentiostatic measurements (crystal truncation rod data) to probe both the metal and electrolyte sides of the interface. The crystal truncation rod measurements are consistent with the potential dependent reorientation of acetonitrile in the absence of water and a parallel arrangement in the presence of water. As acetonitrile concentration increases, the electron density closest to the electrode surface also increases. Finally, Pt surface relaxation in a range of aqueous and non-aqueous solvents is discussed in general with regards to the structure of the electrochemical double layer.

Causal Learning Across Culture and Socioeconomic Status.

Extensive research has explored the ability of young children to learn about the causal structure of the world from patterns of evidence. These studies, however, have been conducted with middle-class samples from North America and Europe. In the present study, low-income Peruvian 4- and 5-year-olds and adults, low-income U.S. 4- and 5-year-olds in Head Start programs, and middle-class children from the United States participated in a causal learning task (N = 435). Consistent with previous studies, children learned both specific causal relations and more abstract causal principles across culture and socioeconomic status (SES). The Peruvian children and adults generally performed like middle-class U.S. children and adults, but the low-SES U.S. children showed some differences.

Mer-mediated eosinophil efferocytosis regulates resolution of allergic airway inflammation.

BACKGROUND: Eosinophils play a central role in propagation of allergic diseases, including asthma. Both recruitment and retention of eosinophils regulate pulmonary eosinophilia, but the question of whether alterations in apoptotic cell clearance by phagocytes contributes directly to resolution of allergic airway inflammation remains unexplored. OBJECTIVES: In this study we investigated the role of the receptor tyrosine kinase Mer in mediating apoptotic eosinophil clearance and allergic airway inflammation resolution in vivo to establish whether apoptotic cell clearance directly affects the resolution of allergic airway inflammation. METHODS: Alveolar and bone marrow macrophages were used to study Mer-mediated phagocytosis of apoptotic eosinophils. Allergic airway inflammation resolution was modeled in mice by using ovalbumin. Fluorescently labeled apoptotic cells were administered intratracheally or eosinophil apoptosis was driven by administration of dexamethasone to determine apoptotic cell clearance in vivo. RESULTS: Inhibition or absence of Mer impaired phagocytosis of apoptotic human and mouse eosinophils by macrophages. Mer-deficient mice showed delayed resolution of ovalbumin-induced allergic airway inflammation, together with increased airway responsiveness to aerosolized methacholine, increased bronchoalveolar lavage fluid protein levels, altered cytokine production, and an excess of uncleared dying eosinophils after dexamethasone treatment. Alveolar macrophage phagocytosis was significantly Mer dependent, with the absence of Mer attenuating apoptotic cell clearance in vivo to enhance inflammation in response to apoptotic cells. CONCLUSIONS: We demonstrate that Mer-mediated apoptotic cell clearance by phagocytes contributes to resolution of allergic airway inflammation, suggesting that augmenting apoptotic cell clearance is a potential therapeutic strategy for treating allergic airway inflammation.

Delayed neutrophil apoptosis enhances NET formation in cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) lung disease is defined by large numbers of neutrophils and associated damaging products in the airway. Delayed neutrophil apoptosis is described in CF although it is unclear whether this is a primary neutrophil defect or a response to chronic inflammation. Increased levels of neutrophil extracellular traps (NETs) have been measured in CF and we aimed to investigate the causal relationship between these phenomena and their potential to serve as a driver of inflammation. We hypothesised that the delay in apoptosis in CF is a primary defect and preferentially allows CF neutrophils to form NETs, contributing to inflammation. METHODS: Blood neutrophils were isolated from patients with CF, CF pigs and appropriate controls. Neutrophils were also obtained from patients with CF before and after commencing ivacaftor. Apoptosis was assessed by morphology and flow cytometry. NET formation was determined by fluorescent microscopy and DNA release assays. NET interaction with macrophages was examined by measuring cytokine generation with ELISA and qRT-PCR. RESULTS: CF neutrophils live longer due to decreased apoptosis. This was observed in both cystic fibrosis transmembrane conductance regulator (CFTR) null piglets and patients with CF, and furthermore was reversed by ivacaftor (CFTR potentiator) in patients with gating (G551D) mutations. CF neutrophils formed more NETs and this was reversed by cyclin-dependent kinase inhibitor exposure. NETs provided a proinflammatory stimulus to macrophages, which was enhanced in CF. CONCLUSIONS: CF neutrophils have a prosurvival phenotype that is associated with an absence of CFTR function and allows increased NET production, which can in turn induce inflammation. Augmenting neutrophil apoptosis in CF may allow more appropriate neutrophil disposal, decreasing NET formation and thus inflammation.

Morphophysical dynamics of human endometrial cells during decidualization.

During decidualization, human mesenchymal-like endometrial stromal cells undergo well characterized cellular and molecular transformations in preparation for accepting a developing embryo. Modulation of cellular biophysical properties during decidualization is likely to be important in receptivity and support of the embryo in the uterus. Here we assess the biophysical properties of human endometrial stromal cells including topography, roughness, adhesiveness and stiffness in cells undergoing in vitro decidualization. A significant reduction in cell stiffness and surface roughness was observed following decidualization. These morphodynamical changes have been shown to be associated with alterations in cellular behavior and homeostasis, suggesting that localized endometrial cell biophysical properties play a role in embryo implantation and pregnancy. This cell-cell communication process is thought to restrict trophoblast invasion beyond the endometrial stroma, be essential in the establishment of pregnancy, and demonstrate the altered endometrial dynamics affecting cell-cell contact and migration regimes at this crucial interface in human reproduction.

The cyclin-dependent kinase inhibitor AT7519 accelerates neutrophil apoptosis in sepsis-related acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a neutrophil-dominant disorder with no effective pharmacological therapies. While the cyclin-dependent kinase inhibitor AT7519 induces neutrophil apoptosis to promote inflammation resolution in preclinical models of lung inflammation, its potential efficacy in ARDS has not been examined. Untreated peripheral blood sepsis-related ARDS neutrophils demonstrated prolonged survival after 20 hours in vitro culture. AT7519 was able to override this phenotype to induce apoptosis in ARDS neutrophils with reduced expression of the pro-survival protein Mcl-1. We demonstrate the first pharmacological compound to induce neutrophil apoptosis in sepsis-related ARDS, highlighting cyclin-dependent kinase inhibitors as potential novel therapeutic agents.

Novel role for endogenous mitochondrial formylated peptide-driven formyl peptide receptor 1 signalling in acute respiratory distress syndrome.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is an often fatal neutrophil-dominant lung disease. Although influenced by multiple proinflammatory mediators, identification of suitable therapeutic candidates remains elusive. We aimed to delineate the presence of mitochondrial formylated peptides in ARDS and characterise the functional importance of formyl peptide receptor 1 (FPR1) signalling in sterile lung inflammation. METHODS: Mitochondrial formylated peptides were identified in bronchoalveolar lavage fluid (BALF) and serum of patients with ARDS by liquid chromatography-tandem mass spectrometry. In vitro, human neutrophils were stimulated with mitochondrial formylated peptides and their effects assessed by flow cytometry and chemotaxis assay. Mouse lung injury was induced by mitochondrial formylated peptides or hydrochloric acid. Bone marrow chimeras determined the contribution of myeloid and parenchymal FPR1 to sterile lung inflammation. RESULTS: Mitochondrial formylated peptides were elevated in BALF and serum from patients with ARDS. These peptides drove neutrophil activation and chemotaxis through FPR1-dependent mechanisms in vitro and in vivo. In mouse lung injury, inflammation was attenuated in Fpr1-/- mice, effects recapitulated by a pharmacological FPR1 antagonist even when administered after the onset of injury. FPR1 expression was present in alveolar epithelium and chimeric mice demonstrated that both myeloid and parenchymal FPR1 contributed to lung inflammation. CONCLUSIONS: We provide the first definitive evidence of mitochondrial formylated peptides in human disease and demonstrate them to be elevated in ARDS and important in a mouse model of lung injury. This work reveals mitochondrial formylated peptide FPR1 signalling as a key driver of sterile acute lung injury and a potential therapeutic target in ARDS.

Probing the charge distribution at the electrochemical interface.

The electrode/electrolyte interface is central to many electrochemical systems; however, gaining insight into the electronic structure at the interface is challenging. Due to its buried nature it is difficult to employ traditional techniques that provide spectroscopic information of localised atoms. To gain new insight into the charge distribution at the interface, we used resonant surface X-ray diffraction to select atoms at the interface via the diffraction conditions and obtained spectroscopic information simultaneously. Coupling the polarisation of the incident X-ray beam with the electron density at the interface allows direct probing of the charge transfer between the metal electrode and the adsorbing species in the electrolyte solution. Results for the adsorption of halide anions onto Cu and Au single crystal electrode surfaces reveal that there is significant modification of the charge distribution of both the surface and sub-surface atomic metal adlayers in the case of ionic bond formation. This has potential impact both in developing a theoretical understanding of the interface structure and in designing new materials for electrochemical applications.