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.

Formylated Peptide Receptor-1-Mediated Gut Inflammation as a Therapeutic Target in Inflammatory Bowel Disease.

Background: Formylated peptide receptor (FPR)-1 is a G-coupled receptor that senses foreign bacterial and host-derived mitochondrial formylated peptides (FPs), leading to innate immune system activation. Aim: We sought to investigate the role of FPR1-mediated inflammation and its potential as a therapeutic target in inflammatory bowel disease (IBD). Methods: We characterized FPR1 gene and protein expression in 8 human IBD (~1000 patients) datasets with analysis on disease subtype, mucosal inflammation, and drug response. We performed in vivo dextran-sulfate sodium (DSS) colitis in C57/BL6 FPR1 knockout mice. In ex vivo studies, we studied the role of mitochondrial FPs and pharmacological blockade of FPR1 using cyclosporin H in human peripheral blood neutrophils. Finally, we assess mitochondrial FPs as a potential mechanistic biomarker in the blood and stools of patients with IBD. Results: Detailed in silico analysis in human intestinal biopsies showed that FPR1 is highly expressed in IBD (n = 207 IBD vs 67 non-IBD controls, P < .001), and highly correlated with gut inflammation in ulcerative colitis (UC) and Crohn's disease (CD) (both P < .001). FPR1 receptor is predominantly expressed in leukocytes, and we showed significantly higher FPR1+ve neutrophils in inflamed gut tissue section in IBD (17 CD and 24 UC; both P < .001). Further analysis in 6 independent IBD (data available under Gene Expression Omnibus accession numbers GSE59071, GSE206285, GSE73661, GSE16879, GSE92415, and GSE235970) showed an association with active gut inflammation and treatment resistance to infliximab, ustekinumab, and vedolizumab. FPR1 gene deletion is protective in murine DSS colitis with lower gut neutrophil inflammation. In the human ex vivo neutrophil system, mitochondrial FP, nicotinamide adenine dinucleotide dehydrogenase subunit-6 (ND6) is a potent activator of neutrophils resulting in higher CD62L shedding, CD63 expression, reactive oxygen species production, and chemotactic capacity; these effects are inhibited by cyclosporin H. We screened for mitochondrial ND6 in IBD (n = 54) using ELISA and detected ND6 in stools with median values of 2.2 gg/mL (interquartile range [IQR] 0.0-4.99; range 0-53.3) but not in blood. Stool ND6 levels, however, were not significantly correlated with paired stool calprotectin, C-reactive protein, and clinical IBD activity. Conclusions: Our data suggest that FPR1-mediated neutrophilic inflammation is a tractable target in IBD; however, further work is required to clarify the clinical utility of mitochondrial FPs as a potential mechanistic marker for future stratification.

Analysis of AT7519 as a pro-resolution compound in an acetaminophen-induced mouse model of acute inflammation by UPLC-MS/MS.

BACKGROUND: Uncontrolled inflammation contributes to the progression of organ damage in acute conditions, such as acetaminophen-induced acute liver injury (APAP-ALI) and there are limited treatments for this condition. AT7519, a cyclic-dependent kinase inhibitor (CDKI), has been used successfully in several conditions, to resolve inflammation and return tissue homeostatic functions. AT7519 has not been assessed in APAP-ALI and its effect on APAP metabolism is unknown. Targeted chromatography and mass spectrometry can be used to assess multiple compounds simultaneously and this approach has not been applied yet to measure APAP and AT7519 in a mouse model. RESULTS: We show an optimised simple and sensitive LC-MS/MS method for determining concentrations of AT7519 and APAP in low volumes of mouse serum. Using positive ion mode electrospray ionisation, separation of AT7519 and APAP and their corresponding isotopically labelled internal standards [2H]8-AT16043M (d8-AT7519) and [2H]8-APAP (d4-APAP), was achieved on an Acquity UPLC BEH C18 column (100 × 2.1 mm; 1.7µm). A gradient mobile phase system of water and methanol was delivered at a flow rate of 0.5 mL/min with a run time of 9 min. Calibration curves were linear, intra-day and inter-day precision and accuracy were acceptable and the covariates of all standards and quality control replicates were less than 15%. The method was successfully applied to evaluate AT7519 and APAP levels 20 h post AT7519 (10 mg/mg) in C57Bl6J wild type mouse serum treated with either vehicle or APAP. Serum AT7519 was significantly higher in mice that had received APAP compared to control, but there was no correlation between APAP and AT7519 quantification. There was also no correlation of AT7519 and hepatic damage or proliferation markers. CONCLUSION: We optimised an LC-MS/MS method to quantify both AT7519 and APAP in mouse serum (50 µL), using labelled internal standards. Application of this method to a mouse model of APAP toxicity proved effective in accurately measuring APAP and AT7519 concentrations after i.p. dosing. AT7519 was significantly higher in mice with APAP toxicity, indicating hepatic metabolism of this CDKI, but there was no correlation with markers of hepatic damage or proliferation, demonstrating that this dose of AT7519 (10 mg/kg) does not contribute to hepatic damage or repair. This optimised method can be used for future investigations of AT7519 in APAP in mice.

A novel dual NLRP1 and NLRP3 inflammasome inhibitor for the treatment of inflammatory diseases.

Objectives: Inflammasomes induce maturation of the inflammatory cytokines IL-1ß and IL-18, whose activity is associated with the pathophysiology of a wide range of infectious and inflammatory diseases. As validated therapeutic targets for the treatment of acute and chronic inflammatory diseases, there has been intense interest in developing small-molecule inhibitors to target inflammasome activity and reduce disease-associated inflammatory burden. Methods: We examined the therapeutic potential of a novel small-molecule inhibitor, and associated derivatives, termed ADS032 to target and reduce inflammasome-mediated inflammation in vivo. In vitro, we characterised ADS032 function, target engagement and specificity. Results: We describe ADS032 as the first dual NLRP1 and NLRP3 inhibitor. ADS032 is a rapid, reversible and stable inflammasome inhibitor that directly binds both NLRP1 and NLRP3, reducing secretion and maturation of IL-1ß in human-derived macrophages and bronchial epithelial cells in response to the activation of NLPR1 and NLRP3. ADS032 also reduced NLRP3-induced ASC speck formation, indicative of targeting inflammasome formation. In vivo, ADS032 reduced IL-1ß and TNF-α levels in the serum of mice challenged i.p. with LPS and reduced pulmonary inflammation in an acute model of lung silicosis. Critically, ADS032 protected mice from lethal influenza A virus challenge, displayed increased survival and reduced pulmonary inflammation. Conclusion: ADS032 is the first described dual inflammasome inhibitor and a potential therapeutic to treat both NLRP1- and NLRP3-associated inflammatory diseases and also constitutes a novel tool that allows examination of the role of NLRP1 in human disease.

Early Exacerbation Relapse is Increased in Patients with Asthma and Bronchiectasis (a Post hoc Analysis).

PURPOSE: Asthma is a common comorbidity in patients with bronchiectasis and has been shown to increase the risk of bronchiectasis exacerbations. This paper explores the impact of comorbid asthma on patients receiving intravenous antibiotic treatment for bronchiectasis exacerbations. METHODS: This was a post hoc analysis of the Meropenem randomised controlled trial of 90 patients that had intravenous antibiotic treatment for bronchiectasis exacerbations. The participants were split into two groups: group 1 (asthma and bronchiectasis) and group 2 (bronchiectasis). The authors assessed response to treatment and time to next exacerbation. RESULTS: There were 38 participants in group 1 and 34 participants in group 2. The groups were found to be comparable in terms of age, sex, and bronchiectasis severity (median (95% CI) group 1 and then group 2 data): age 64.0(59.3, 68.6) and 63.6(57.9, 69.4) years old, p = 0.8; 57.9% and 64.7% female, p = 0.6; Bronchiectasis Severity Index 11.1(9.8, 12.4) and 10.1(8.2, 12.0), p = 0.3. There was a similar response to treatment between the groups, but group 1 were found to relapse early by day 14, 31.6% in group 1 and 11.8% in group 2, p = 0.03. In the Cox proportional hazards model, asthma was the only independent risk factor for early relapse by day 14 (odds ratio (95% CI) 3.16 (1.02-9.79), p = 0.047). CONCLUSION: The clinical response to treatment was similar but patients with coexisting asthma were at increased risk of early relapse within 14 days of stopping intravenous antibiotic therapy. CLINICAL TRIAL REGISTRATION: NCT02047773.

Fish Erythrocyte Extracellular Traps (FEETs) are an evolutionarily conserved cellular process triggered by different stimuli.

Fish erythrocytes remain nucleated, unlike mammalian erythrocytes that undergo enucleation during maturation. Besides oxygen transport, fish erythrocytes are capable of several immune defence processes and thus these cells are candidates for carrying out ETotic responses. ETosis is an evolutionarily conserved innate immune defence process found in both vertebrates and invertebrates, which involves the extrusion of DNA studded with antimicrobial effector proteins into the extracellular space that traps and kills microorganisms. In this present report, we demonstrate that erythrocytes from Danio rerio (zebrafish) produce ETotic-like responses when exposed to both chemical and physiological inducers of ETosis. Furthermore, erythrocytes from Salmo salar (Atlantic salmon) behaved in a similar way. We have termed these ET-like formations, as Fish Erythrocyte Extracellular Traps (FEETs). Several inducers of mammalian ETosis, such as the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin, induced FEETs. Moreover, we found that FEETs depend on the activation of PKC and generation of mitochondrial reactive oxygen species (mROS). This present report is the first demonstration that fish erythrocytes can exhibit ETotic-like responses, unveiling a previously unknown function, which sheds new light on the innate immune arsenal of these cells.

Metabolic regulation by prostaglandin E2 impairs lung group 2 innate lymphoid cell responses.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) play a critical role in asthma pathogenesis. Non-steroidal anti-inflammatory drug (NSAID)-exacerbated respiratory disease (NERD) is associated with reduced signaling via EP2, a receptor for prostaglandin E2 (PGE2 ). However, the respective roles for the PGE2 receptors EP2 and EP4 (both share same downstream signaling) in the regulation of lung ILC2 responses has yet been deciphered. METHODS: The roles of PGE2 receptors EP2 and EP4 on ILC2-mediated lung inflammation were investigated using genetically modified mouse lines and pharmacological approaches in IL-33-induced lung allergy model. The effects of PGE2 receptors and downstream signals on ILC2 metabolic activation and effector function were examined using in vitro cell cultures. RESULTS: Deficiency of EP2 rather than EP4 augments IL-33-induced mouse lung ILC2 responses and eosinophilic inflammation in vivo. In contrast, exogenous agonism of EP4 and EP2 or inhibition of phosphodiesterase markedly restricts IL-33-induced lung ILC2 responses. Mechanistically, PGE2 directly suppresses IL-33-dependent ILC2 activation through the EP2/EP4-cAMP pathway, which downregulates STAT5 and MYC pathway gene expression and ILC2 energy metabolism. Blocking glycolysis diminishes IL-33-dependent ILC2 responses in mice where endogenous PG synthesis or EP2 signaling is blocked but not in mice with intact PGE2 -EP2 signaling. CONCLUSION: We have defined a mechanism for optimal suppression of mouse lung ILC2 responses by endogenous PGE2 -EP2 signaling which underpins the clinical findings of defective EP2 signaling in patients with NERD. Our findings also indicate that exogenously targeting the PGE2 -EP4-cAMP and energy metabolic pathways may provide novel opportunities for treating the ILC2-initiated lung inflammation in asthma and NERD.

SILAC-based quantitative proteomics to investigate the eicosanoid associated inflammatory response in activated macrophages.

BACKGROUND: Macrophages play a central role in inflammation by phagocytosing invading pathogens, apoptotic cells and debris, as well as mediating repair of tissues damaged by trauma. In order to do this, these dynamic cells generate a variety of inflammatory mediators including eicosanoids such as prostaglandins, leukotrienes and hydroxyeicosatraenoic acids (HETEs) that are formed through the cyclooxygenase, lipoxygenase and cytochrome P450 pathways. The ability to examine the effects of eicosanoid production at the protein level is therefore critical to understanding the mechanisms associated with macrophage activation. RESULTS: This study presents a stable isotope labelling with amino acids in cell culture (SILAC) -based proteomics strategy to quantify the changes in macrophage protein abundance following inflammatory stimulation with Kdo2-lipid A and ATP, with a focus on eicosanoid metabolism and regulation. Detailed gene ontology analysis, at the protein level, revealed several key pathways with a decrease in expression in response to macrophage activation, which included a promotion of macrophage polarisation and dynamic changes to energy requirements, transcription and translation. These findings suggest that, whilst there is evidence for the induction of a pro-inflammatory response in the form of prostaglandin secretion, there is also metabolic reprogramming along with a change in cell polarisation towards a reduced pro-inflammatory phenotype. CONCLUSIONS: Advanced quantitative proteomics in conjunction with functional pathway network analysis is a useful tool to investigate the molecular pathways involved in inflammation.

Galectin-3 inhibitor GB0139 protects against acute lung injury by inhibiting neutrophil recruitment and activation.

Rationale: Galectin-3 (Gal-3) drives fibrosis during chronic lung injury, however, its role in acute lung injury (ALI) remains unknown. Effective pharmacological therapies available for ALI are limited; identifying novel concepts in treatment is essential. GB0139 is a Gal-3 inhibitor currently under clinical investigation for the treatment of idiopathic pulmonary fibrosis. We investigate the role of Gal-3 in ALI and evaluate whether its inhibition with GB0139 offers a protective role. The effect of GB0139 on ALI was explored in vivo and in vitro. Methods: The pharmacokinetic profile of intra-tracheal (i.t.) GB0139 was investigated in C57BL/6 mice to support the daily dosing regimen. GB0139 (1-30 µg) was then assessed following acute i.t. lipopolysaccharide (LPS) and bleomycin administration. Histology, broncho-alveolar lavage fluid (BALf) analysis, and flow cytometric analysis of lung digests and BALf were performed. The impact of GB0139 on cell activation and apoptosis was determined in vitro using neutrophils and THP-1, A549 and Jurkat E6 cell lines. Results: GB0139 decreased inflammation severity via a reduction in neutrophil and macrophage recruitment and neutrophil activation. GB0139 reduced LPS-mediated increases in interleukin (IL)-6, tumor necrosis factor alpha (TNFα) and macrophage inflammatory protein-1-alpha. In vitro, GB0139 inhibited Gal-3-induced neutrophil activation, monocyte IL-8 secretion, T cell apoptosis and the upregulation of pro-inflammatory genes encoding for IL-8, TNFα, IL-6 in alveolar epithelial cells in response to mechanical stretch. Conclusion: These data indicate that Gal-3 adopts a pro-inflammatory role following the early stages of lung injury and supports the development of GB0139, as a potential treatment approach in ALI.

Transgenic zebrafish larvae as a non-rodent alternative model to assess pro-inflammatory (neutrophil) responses to nanomaterials.

Hazard studies for nanomaterials (NMs) commonly assess whether they activate an inflammatory response. Such assessments often rely on rodents, but alternative models are needed to support the implementation of the 3Rs principles. Zebrafish (Danio rerio) offer a viable alternative for screening NM toxicity by investigating inflammatory responses. Here, we used non-protected life stages of transgenic zebrafish (Tg(mpx:GFP)i114) with fluorescently-labeled neutrophils to assess inflammatory responses to silver (Ag) and zinc oxide (ZnO) NMs using two approaches. Zebrafish were exposed to NMs via water following a tail fin injury, or NMs were microinjected into the otic vesicle. Zebrafish were exposed to NMs at 3 days post-fertilization (dpf) and neutrophil accumulation at the injury or injection site was quantified at 0, 4, 6, 8, 24, and 48 h post-exposure. Zebrafish larvae were also exposed to fMLF, LTB4, CXCL-8, C5a, and LPS to identify a suitable positive control for inflammation induction. Aqueous exposure to Ag and ZnO NMs stimulated an enhanced and sustained neutrophilic inflammatory response in injured zebrafish larvae, with a greater response observed for Ag NMs. Following microinjection, Ag NMs stimulated a time-dependent neutrophil accumulation in the otic vesicle which peaked at 48 h. LTB4 was identified as a positive control for studies investigating inflammatory responses in injured zebrafish following aqueous exposure, and CXCL-8 for microinjection studies that assess responses in the otic vesicle. Our findings support the use of transgenic zebrafish to rapidly screen the pro-inflammatory effects of NMs, with potential for wider application in assessing chemical safety (e.g. pharmaceuticals).

Inhibition of Cyclin-Dependent Kinase 9 Downregulates Cytokine Production Without Detrimentally Affecting Human Monocyte-Derived Macrophage Viability.

Cyclin-dependent kinase (CDK) inhibitor drugs (CDKi), such as R-roscovitine and AT7519, induce neutrophil apoptosis in vitro and enhance the resolution of inflammation in a number of in vivo models. This class of compounds are potential novel therapeutic agents that could promote the resolution of acute and chronic inflammatory conditions where neutrophil activation contributes to tissue damage and aberrant tissue repair. In this study we investigated CDKi effects on macrophage pro-inflammatory mediator production and viability. Treatment of human monocyte-derived macrophages (MDMs) with the CDKi AT7519 and R-roscovitine at concentrations that induce neutrophil apoptosis had no significant effect on control or LPS-activated MDM apoptosis and viability, and did not detrimentally affect MDM efferocytosis of apoptotic cells. In addition, enhanced efferocytosis, induced by the glucocorticoid dexamethasone, was also unaffected after a short time treatment with R-roscovitine. Macrophage cytokine responses to inflammatory stimuli are also of importance during inflammation and resolution. As a key target of CDKi, CDK9, is involved in protein transcription via the RNA polymerase II complex, we investigated the effect of CDKi drugs on cytokine production. Our data show that treatment with AT7519 significantly downregulated expression and release of key MDM cytokines IL-6, TNF, IL-10 and IL-1ß, as well as markers of pro-inflammatory macrophage polarisation. R-Roscovitine was also able to downregulate inflammatory cytokine protein secretion from MDMs. Using siRNA transfection, we demonstrate that genetic knock-down of CDK9 replicates these findings, reducing expression and release of pro-inflammatory cytokines. Furthermore, overexpression of CDK9 in THP-1 cells can promote a pro-inflammatory phenotype in these cells, suggesting that CDK9 plays an important role in the inflammatory phenotype of macrophages. Overall, this study demonstrates that pharmacological and genetic targeting of CDK9 inhibits an inflammatory phenotype in human MDMs. As such these data indicate that CDK9 may be key to therapeutically targeting pro-inflammatory macrophage functions during chronic inflammation.

Macrophages trigger cardiomyocyte proliferation by increasing epicardial vegfaa expression during larval zebrafish heart regeneration.

Cardiac injury leads to the loss of cardiomyocytes, which are rapidly replaced by the proliferation of the surviving cells in zebrafish, but not in mammals. In both the regenerative zebrafish and non-regenerative mammals, cardiac injury induces a sustained macrophage response. Macrophages are required for cardiomyocyte proliferation during zebrafish cardiac regeneration, but the mechanisms whereby macrophages facilitate this crucial process are fundamentally unknown. Using heartbeat-synchronized live imaging, RNA sequencing, and macrophage-null genotypes in the larval zebrafish cardiac injury model, we characterize macrophage function and reveal that these cells activate the epicardium, inducing cardiomyocyte proliferation. Mechanistically, macrophages are specifically recruited to the epicardial-myocardial niche, triggering the expansion of the epicardium, which upregulates vegfaa expression to induce cardiomyocyte proliferation. Our data suggest that epicardial Vegfaa augments a developmental cardiac growth pathway via increased endocardial notch signaling. The identification of this macrophage-dependent mechanism of cardiac regeneration highlights immunomodulation as a potential strategy for enhancing mammalian cardiac repair.

Pannexin 1 drives efficient epithelial repair after tissue injury.

Epithelial tissues such as lung and skin are exposed to the environment and therefore particularly vulnerable to damage during injury or infection. Rapid repair is therefore essential to restore function and organ homeostasis. Dysregulated epithelial tissue repair occurs in several human disease states, yet how individual cell types communicate and interact to coordinate tissue regeneration is incompletely understood. Here, we show that pannexin 1 (Panx1), a cell membrane channel activated by caspases in dying cells, drives efficient epithelial regeneration after tissue injury by regulating injury-induced epithelial proliferation. Lung airway epithelial injury promotes the Panx1-dependent release of factors including ATP, from dying epithelial cells, which regulates macrophage phenotype after injury. This process, in turn, induces a reparative response in tissue macrophages that includes the induction of the soluble mitogen amphiregulin, which promotes injury-induced epithelial proliferation. Analysis of regenerating lung epithelium identified Panx1-dependent induction of Nras and Bcas2, both of which positively promoted epithelial proliferation and tissue regeneration in vivo. We also established that this role of Panx1 in boosting epithelial repair after injury is conserved between mouse lung and zebrafish tailfin. These data identify a Panx1-mediated communication circuit between epithelial cells and macrophages as a key step in promoting epithelial regeneration after injury.

Pulmonary macrophages and SARS-Cov2 infection.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the largest global pandemic in living memory, with between 4.5 and 15M deaths globally from coronavirus disease 2019 (COVID-19). This has led to an unparalleled global, collaborative effort to understand the pathogenesis of this devastating disease using state-of-the-art technologies. A consistent feature of severe COVID-19 is dysregulation of pulmonary macrophages, cells that under normal physiological conditions play vital roles in maintaining lung homeostasis and immunity. In this article, we will discuss a selection of the pivotal findings examining the role of monocytes and macrophages in SARS-CoV-2 infection and place this in context of recent advances made in understanding the fundamental immunobiology of these cells to try to understand how key homeostatic cells come to be a central pathogenic component of severe COVID-19 and key cells to target for therapeutic gain.

Selective Cdk9 inhibition resolves neutrophilic inflammation and enhances cardiac regeneration in larval zebrafish.

Sustained neutrophilic inflammation is detrimental for cardiac repair and associated with adverse outcomes following myocardial infarction (MI). An attractive therapeutic strategy to treat MI is to reduce or remove infiltrating neutrophils to promote downstream reparative mechanisms. CDK9 inhibitor compounds enhance the resolution of neutrophilic inflammation; however, their effects on cardiac repair/regeneration are unknown. We have devised a cardiac injury model to investigate inflammatory and regenerative responses in larval zebrafish using heartbeat-synchronised light-sheet fluorescence microscopy. We used this model to test two clinically approved CDK9 inhibitors, AT7519 and flavopiridol, examining their effects on neutrophils, macrophages and cardiomyocyte regeneration. We found that AT7519 and flavopiridol resolve neutrophil infiltration by inducing reverse migration from the cardiac lesion. Although continuous exposure to AT7519 or flavopiridol caused adverse phenotypes, transient treatment accelerated neutrophil resolution while avoiding these effects. Transient treatment with AT7519, but not flavopiridol, augmented wound-associated macrophage polarisation, which enhanced macrophage-dependent cardiomyocyte number expansion and the rate of myocardial wound closure. Using cdk9-/- knockout mutants, we showed that AT7519 is a selective CDK9 inhibitor, revealing the potential of such treatments to promote cardiac repair/regeneration.

Vitamin D insufficiency in COVID-19 and influenza A, and critical illness survivors: a cross-sectional study.

OBJECTIVES: The steroid hormone vitamin D has roles in immunomodulation and bone health. Insufficiency is associated with susceptibility to respiratory infections. We report 25-hydroxy vitamin D (25(OH)D) measurements in hospitalised people with COVID-19 and influenza A and in survivors of critical illness to test the hypotheses that vitamin D insufficiency scales with illness severity and persists in survivors. DESIGN: Cross-sectional study. SETTING AND PARTICIPANTS: Plasma was obtained from 295 hospitalised people with COVID-19 (International Severe Acute Respiratory and emerging Infections Consortium (ISARIC)/WHO Clinical Characterization Protocol for Severe Emerging Infections UK study), 93 with influenza A (Mechanisms of Severe Acute Influenza Consortium (MOSAIC) study, during the 2009-2010 H1N1 pandemic) and 139 survivors of non-selected critical illness (prior to the COVID-19 pandemic). Total 25(OH)D was measured by liquid chromatography-tandem mass spectrometry. Free 25(OH)D was measured by ELISA in COVID-19 samples. OUTCOME MEASURES: Receipt of invasive mechanical ventilation (IMV) and in-hospital mortality. RESULTS: Vitamin D insufficiency (total 25(OH)D 25-50 nmol/L) and deficiency (<25 nmol/L) were prevalent in COVID-19 (29.3% and 44.4%, respectively), influenza A (47.3% and 37.6%) and critical illness survivors (30.2% and 56.8%). In COVID-19 and influenza A, total 25(OH)D measured early in illness was lower in patients who received IMV (19.6 vs 31.9 nmol/L (p<0.0001) and 22.9 vs 31.1 nmol/L (p=0.0009), respectively). In COVID-19, biologically active free 25(OH)D correlated with total 25(OH)D and was lower in patients who received IMV, but was not associated with selected circulating inflammatory mediators. CONCLUSIONS: Vitamin D deficiency/insufficiency was present in majority of hospitalised patients with COVID-19 or influenza A and correlated with severity and persisted in critical illness survivors at concentrations expected to disrupt bone metabolism. These findings support early supplementation trials to determine if insufficiency is causal in progression to severe disease, and investigation of longer-term bone health outcomes.

Prostaglandin E2 directly inhibits the conversion of inducible regulatory T cells through EP2 and EP4 receptors via antagonizing TGF-ß signalling.

Regulatory T (Treg) cells are essential for control of inflammatory processes by suppressing effector T-cell functions. The actions of PGE2 on the development and function of Treg cells, particularly under inflammatory conditions, are debated. In this study, we employed pharmacological and genetic approaches to examine whether PGE2  had a direct action on T cells to modulate de novo differentiation of Treg cells. We found that TGF-ß-induced Foxp3 expression and iTreg cell differentiation in vitro is markedly inhibited by PGE2 , which was mediated by the receptors EP2 and EP4. Mechanistically, PGE2 -EP2/EP4 signalling interrupts TGF-ß signalling during iTreg differentiation. Moreover, EP4 deficiency in T cells impaired iTreg cell differentiation in vivo. Thus, our results demonstrate that PGE2 negatively regulates iTreg cell differentiation through a direct action on T cells, highlighting the potential for selectively targeting the PGE2 -EP2/EP4 pathway to control T cell-mediated inflammation.

Release of chromatin extracellular traps by phagocytes of Atlantic salmon, Salmo salar (Linnaeus, 1758).

Neutrophils release chromatin extracellular traps (ETs) as part of the fish innate immune response to counter the threats posed by microbial pathogens. However, relatively little attention has been paid to this phenomenon in many commercially farmed species, despite the importance of understanding host-pathogen interactions and the potential to influence ET release to reduce disease outbreaks. The aim of this present study was to investigate the release of ETs by Atlantic salmon (Salmo salar L.) immune cells. Extracellular structures resembling ETs of different morphology were observed by fluorescence microscopy in neutrophil suspensions in vitro, as these structures stained positively with Sytox Green and were digestible with DNase I. Immunofluorescence studies confirmed the ET structures to be decorated with histones H1 and H2A and neutrophil elastase, which are characteristic for ETs in mammals and other organisms. Although the ETs were released spontaneously, release in neutrophil suspensions was stimulated most significantly with 5 µg/ml calcium ionophore (CaI) for 1 h, whilst the fish pathogenic bacterium Aeromonas salmonicida (isolates 30411 and Hooke) also exerted a stimulatory effect. Microscopic observations revealed bacteria in association with ETs, and fewer bacterial colonies of A. salmonicida Hooke were recovered at 3 h after co-incubation with neutrophils that had been induced to release ETs. Interestingly, spontaneous release of ETs was inversely associated with fish mass (p < 0.05), a surrogate for age. Moreover, suspensions enriched for macrophages and stimulated with 5 µg/ml CaI released ET-like structures that occasionally led to the formation of large clumps of cells. A deeper understanding for the roles and functions of ETs within innate immunity of fish hosts, and their interaction with microbial pathogens, may open new avenues towards protecting cultured stocks against infectious diseases.