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Background: The lungs are the organ most likely to sustain serious injury from coronavirus disease 2019 (COVID-19). However, the mechanisms for long-term complications are not clear. Patients with severe COVID-19 have shorter telomere lengths and higher levels of cellular senescence, and we hypothesized that circulating levels of the telomere-associated senescence markers chitotriosidase, b-galactosidase, cathelicidin antimicrobial peptide and stathmin 1 (STMN1) were elevated in hospitalized COVID-19 patients compared to controls and could be associated with pulmonary sequelae following hospitalization. Methods: Ninety-seven hospitalized patients with COVID-19 who underwent assessment for pulmonary sequelae at threemonth follow-up were included in the study. b-Galactosidase and chitotriosidase were analysed by fluorescence; stathmin 1 and cathelicidin antimicrobial peptide were analysed by enzyme immuno-assay in plasma samples from the acute phase and after three-months. In addition, the classical senescence markers cyclin-dependent kinase inhibitor 1A and 2A were analysed by enzyme immuno-assay in peripheral blood mononuclear cell lysate after three months. Results: We found elevated plasma levels of the senescence markers chitotriosidase and stathmin 1 in patients three months after hospitalization with COVID-19, and these markers in addition to protein levels of cyclin-dependent kinase inhibitor 2A in cell lysate, were associated with pulmonary pathology. The elevated levels of these markers seem to reflect both age-dependent (chitotriosidase) and age-independent (stathmin 1, cyclin-dependent kinase inhibitor 2A) processes. Conclusions: We suggest that accelerated ageing or senescence could be important for long-term pulmonary complications of COVID-19, and our findings may be relevant for future research exploring the pathophysiology and management of these patients.

The global risk of cardiovascular disease, including ischemic disease such as stroke, remains high, and cardiovascular disease is the cause of one-third of all deaths worldwide. The main subjacent cause, atherosclerosis, is not fully understood. To improve early diagnosis and therapeutic strategies, it is crucial to unveil the key molecular mechanisms that lead to atherosclerosis development. The field of epitranscriptomics is blossoming and quickly advancing in fields like cancer research, nevertheless, poorly understood in the context of cardiovascular disease. Epitranscriptomic modifications are shown to regulate the metabolism and function of RNA molecules, which are important for cell functions such as cell proliferation, a key aspect in atherogenesis. As such, epitranscriptomic regulatory mechanisms can serve as novel checkpoints in gene expression during disease development. In this review, we describe examples of the latest research investigating epitranscriptomic modifications, in particular A-to-I editing and the covalent modification N 6 -methyladenosine and their regulatory proteins, in the context of cardiovascular disease. We additionally discuss the potential of these mechanisms as therapeutic targets and novel treatment options.

Purpose Triglycerides (TG) and their major transport lipoprotein in the circulation (VLDL) appear to be related to inflammation. Patients with common variable immunodeficiency (CVID) have inflammatory complications associated with gut microbial dysbiosis. We hypothesized that CVID patients have disturbed TG/VLDL profiles associated with these clinical characteristics. Methods We measured plasma concentrations of TGs, inflammatory markers, and lipopolysaccharide (LPS) in 95 CVID patients and 28 healthy controls. Additionally, in 40 CVID patients, we explored plasma lipoprotein profiling, fatty acid, gut microbial dysbiosis, and diet. Results TG levels were increased in CVID patients as compared to healthy controls (1.36 ± 0.53 mmol/l versus 1.08 ± 0.56 [mean, SD], respectively, P = 0.008), particularly in the clinical subgroup “Complications,” characterized by autoimmunity and organ-specific inflammation, compared to “Infection only” (1.41 mmol/l, 0.71[median, IQR] versus [1.02 mmol/l, 0.50], P = 0.021). Lipoprotein profile analyses showed increased levels of all sizes of VLDL particles in CVID patients compared to controls. TG levels correlated positively with CRP (rho = 0.256, P = 0.015), IL-6 (rho = 0.237, P = 0.021), IL-12 (rho = 0.265, P = 0.009), LPS (r = 0.654, P = 6.59 × 10−13), CVID-specific gut dysbiosis index (r = 0.315, P = 0.048), and inversely with a favorable fatty acid profile (docosahexaenoic acid [rho = − 0.369, P = 0.021] and linoleic acid [rho = − 0.375, P = 0.019]). TGs and VLDL lipids did not appear to be associated with diet and there were no differences in body mass index (BMI) between CVID patients and controls. Conclusion We found increased plasma levels of TGs and all sizes of VLDL particles, which were associated with systemic inflammation, LPS, and gut dysbiosis in CVID, but not diet or BMI.

Advanced science 10(5), 2203053 (2023). doi:10.1002/advs.202203053, Published by Wiley-VCH, Weinheim

Purpose Fatty acid (FA) abnormalities are found in various inflammatory disorders and have been related to disturbed gut microbiota. Patients with common variable immunodeficiency (CVID) have inflammatory complications associated with altered gut microbial composition. We hypothesized that there is an altered FA profile in CVID patients, related to gut microbial dysbiosis. Methods Plasma FAs were measured in 39 CVID patients and 30 healthy controls. Gut microbial profile, a food frequency questionnaire, and the effect of the oral antibiotic rifaximin were investigated in CVID patients. Results The n-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA) (1.4 [1.0–1.8] vs. 1.9 [1.2–2.5], median (IQR), P P n-6 PUFAs (34.3 ± 3.4 vs. 37.1 ± 2.8, mean ± SD, P P P n-6 PUFAs (r = 0.41, P r = 0.51, P n-3 PUFAs (P = 0.78). Moreover, a 2-week course of rifaximin significantly reduced the proportion of n-6 PUFAs (P = 0.04, UNIANOVA). Serum immunoglobulin G (IgG) levels correlated with plasma n-3 PUFAs (rho = 0.36, P = 0.03) and DHA (rho = 0.41, P = 0.009). Conclusion We found a potentially unfavorable FA profile in CVID, related to low IgG levels. High plasma n-6 PUFAs were related to increased gut microbial diversity and altered by rifaximin therapy.

Aims We recently reported five cases of vaccine-induced immune thrombotic thrombocytopenia (VITT) 7–10 days after receiving the first dose of the ChAdOx1 nCoV-19 adenoviral vector vaccine against corona virus disease 2019 (COVID-19). We aimed to investigate the pathogenic immunological responses operating in these patients. Methods and results We assessed circulating inflammatory markers by immune assays and immune cell phenotyping by flow cytometry analyses and performed immunoprecipitation with anti-platelet factor (PF)4 antibody in plasma samples followed by mass spectrometry from all five patients. A thrombus was retrieved from the sinus sagittal superior of one patient and analysed by immunohistochemistry and flow cytometry. Precipitated immune complexes revealed multiple innate immune pathway triggers for platelet and leucocyte activation. Plasma contained increased levels of innate immune response cytokines and markers of systemic inflammation, extensive degranulation of neutrophils, and tissue and endothelial damage. Blood analyses showed activation of neutrophils and increased levels of circulating H3Cit, dsDNA, and myeloperoxidase–DNA complex. The thrombus had extensive infiltration of neutrophils, formation of neutrophil extracellular traps (NETs), and IgG deposits. Conclusions The results show that anti-PF4/polyanion IgG-mediated thrombus formation in VITT patients is accompanied by a massive innate immune activation and particularly the fulminant activation of neutrophils including NETosis. These results provide novel data on the immune response in this rare adenoviral vector-induced VITT., Graphical Abstract Graphical Abstract

Background Prognostic markers for disease severity and identification of therapeutic targets in COVID‐19 are urgently needed. We have studied innate and adaptive immunity on protein and transcriptomic level in COVID‐19 patients with different disease severity at admission and longitudinally during hospitalization. Methods Peripheral blood mononuclear cells (PBMCs) were collected at three time points from 31 patients included in the Norwegian SARS‐CoV‐2 cohort study and analysed by flow cytometry and RNA sequencing. Patients were grouped as either mild/moderate (n = 14), severe (n = 11) or critical (n = 6) disease in accordance with WHO guidelines and compared with patients with SARS‐CoV‐2‐negative bacterial sepsis (n = 5) and healthy controls (n = 10). Results COVID‐19 severity was characterized by decreased interleukin 7 receptor alpha chain (CD127) expression in naïve CD4 and CD8 T cells. Activation (CD25 and HLA‐DR) and exhaustion (PD‐1) markers on T cells were increased compared with controls, but comparable between COVID‐19 severity groups. Non‐classical monocytes and monocytic HLA‐DR expression decreased whereas monocytic PD‐L1 and CD142 expression increased with COVID‐19 severity. RNA sequencing exhibited increased plasma B‐cell activity in critical COVID‐19 and yet predominantly reduced transcripts related to immune response pathways compared with milder disease. Conclusion Critical COVID‐19 seems to be characterized by an immune profile of activated and exhausted T cells and monocytes. This immune phenotype may influence the capacity to mount an efficient T‐cell immune response. Plasma B‐cell activity and calprotectin were higher in critical COVID‐19 while most transcripts related to immune functions were reduced, in particular affecting B cells. The potential of these cells as therapeutic targets in COVID‐19 should be further explored.

Background A substantial proportion of common variable immunodeficiency (CVID) patients has duodenal inflammation of largely unknown etiology. However, because of its histologic similarities with celiac disease, gluten sensitivity has been proposed as a potential mechanism. Objective We aimed to elucidate the role of the duodenal microenvironment in the pathogenesis of duodenal inflammation in CVID by investigating the transcriptional, proteomic, and microbial signatures of duodenal biopsy samples in CVID. Methods DNA, total RNA, and protein were isolated from snap-frozen pieces of duodenal biopsy samples from CVID (with and without duodenal inflammation), healthy controls, and patients with celiac disease (untreated). RNA sequencing, mass spectrometry–based proteomics, and 16S ribosomal DNA sequencing (bacteria) were then performed. Results CVID separated from controls in regulation of transcriptional response to lipopolysaccharide and cellular immune responses. These differences were independent of mucosal inflammation. Instead, CVID patients with duodenal inflammation displayed alterations in transcription of genes involved in response to viral infections. Four proteins were differently regulated between CVID patients and healthy controls—DBNL, TRMT11, GCHFR, and IGHA2—independent of duodenal inflammation. Despite similar histology, there were major differences in CVID with duodenal inflammation and celiac disease both at the RNA and protein level. No significant difference was observed in the bacterial gut microbial signature between CVID, celiac, and healthy controls. Conclusion Our findings suggest the existence of altered functions of the duodenal epithelium, particularly in response to lipopolysaccharide and viruses. The latter finding was related to duodenal inflammation, suggesting that viruses, not gluten sensitivity, could be related to duodenal inflammation in CVID. Key words CVID RNA sequencing proteomics microbiome gut microbiota microbiota gastrointestinal tract duodenum celiac disease Primary immunodeficiency IgA

Background and aims: Atherogenesis involves a complex interaction between immune cells and lipids, processes greatly influenced by the vascular smooth muscle cell (VSMC) phenotype. The DNA glycosylase NEIL3 has previously been shown to have a role in atherogenesis, though whether this is due to its ability to repair DNA damage or to other non-canonical functions is not yet clear. Hereby, we investigate the role of NEIL3 in atherogenesis, specifically in VSMC phenotypic modulation, which is critical in plaque formation and stability.Methods: Chow diet-fed atherosclerosis-prone Apoe(-/-) mice deficient in Neil3, and NEIL3-abrogated human primary aortic VSMCs were characterized by qPCR, and immunohistochemical and enzymatic-based assays; moreover, single-cell RNA sequencing, mRNA sequencing, and proteomics were used to map the molecular effects of Neil3/NEIL3 deficiency in the aortic VSMC phenotype. Furthermore, BrdU-based proliferation assays and Western blot were performed to elucidate the involvement of the Akt signaling pathway in the transdifferentiation of aortic VSMCs lacking Neil3/NEIL3.Results: We show that Neil3 deficiency increases atherosclerotic plaque development without affecting systemic lipids. This observation was associated with a shift in VSMC phenotype towards a proliferating, lipid-accumulating and secretory macrophage-like cell phenotype, without changes in DNA damage. VSMC transdifferentiation in Neil3-deficient mice encompassed increased activity of the Akt signaling pathway, supported by cell experiments showing Akt-dependent proliferation in NEIL3-abrogated human primary aortic VSMCs.Conclusions: Our findings show that Neil3 deficiency promotes atherosclerosis development through non-canonical mechanisms affecting VSMC phenotype involving activation of the Akt signaling pathway.

Background Reduction of the aortic valve area (AVA) may lead to aortic valve stenosis with considerable impact on morbidity and mortality if not identified and treated. Lipoprotein (a) [Lp(a)] and also inflammatory biomarkers, including platelet derived biomarkers, have been considered risk factor for aortic stenosis; however, the association between Lp(a), inflammatory biomarkers and AVA among patients with familial hypercholesterolemia (FH) is not clear. Objective We aimed to investigate the relation between concentration of Lp(a), measurements of the aortic valve including velocities and valve area and circulating inflammatory biomarkers in adult FH subjects and controls. Methods In this cross-sectional study aortic valve measures were examined by cardiac ultrasound and inflammatory markers were analyzed in non-fasting blood samples. The study participants were 64 FH subjects with high (n = 29) or low (n = 35) Lp(a), and 14 healthy controls. Results Aortic valve peak velocity was higher (p = 0.02), and AVA was lower (p = 0.04) in the FH patients compared to controls; however, when performing multivariable linear regression, there were no significant differences. Furthermore, there were no significant differences between the high and low FH Lp(a) groups regarding the aortic valve. FH subjects had higher levels of platelet-derived markers CD40L, PF4, NAP2 and RANTES compared to controls (0.003 ≤ P ≤ 0.03). This result persisted after multiple linear regression. Conclusions Middle-aged, intensively treated FH subjects have higher aortic valve velocity, lower AVA, and higher levels of the platelet-derived markers CD40L, PF4, NAP2 and RANTES compared to healthy control subjects. The aortic valve findings were not significant after multiple linear regression, whereas the higher levels of platelet-derived markers were maintained.

Background More than 170 post-transcriptional RNA modifications regulate the localization, processing and function of cellular RNAs, and aberrant RNA modifications have been linked to a range of human diseases. The RNA modification landscape in atherosclerosis, the main underlying cause of cardiovascular diseases, is still largely unknown. Methods We used mass spectrometry to analyse a selection of RNA-modifying enzymes and the N6-methyladenosine (m6A) in carotid atherosclerotic lesion samples representing early and advanced stages of atherosclerosis as compared to non-atherosclerotic arteries from healthy controls. Findings (i) the detection of different levels of several enzymes involved in methylations occurring in rRNA and mRNA; (ii) these findings included changes in the levels of methyltransferases (‘writers’), binding proteins (‘readers’) and demethylases (‘erasers’) during atherosclerosis as compared to non-atherosclerotic control arteries, with generally the most prominent differences in samples from early atherosclerotic lesions; and (iii) these changes were accompanied by a marked downregulation of m6A in rRNA, the most abundant and well-studied modification in mRNA with a wide range of effects on cell biology. Interpretation We show for the first time that RNA-modifying enzymes and the well-studied RNA modification m6A are differentially regulated in atherosclerotic lesions, which potentially could help creating new prognostic and treatment strategies.

Fatty liver diseases are a major health threat across the western world, leading to cirrhosis and premature morbidity and mortality. Recently, a correlation between the base excision repair enzyme SMUG1 and metabolic homeostasis was identified. As the molecular mechanisms remain unknown, we exploited a SMUG1-knockout mouse model to gain insights into this association by characterizing the liver phenotype in young vs old SMUG1-null mice. We observed increased weight and fat content in one-year old animals, with altered activity of enzymes important for fatty acids influx and uptake. Consistently, lipidomic profiling showed accumulation of free fatty acids and triglycerides in SMUG1-null livers. Old SMUG1-knockout mice also displayed increased hepatocyte senescence and DNA damage at telomeres. Interestingly, RNA sequencing revealed widespread changes in the expression of lipid metabolic genes already in three months old animals. In summary, SMUG1 modulates fat metabolism favouring net lipogenesis and resulting in development of a fatty liver phenotype.

Introduction: Use of high-dose androgens causes drastic changes in hormonal milieu and is associated with adverse medical, psychological, and cognitive effects. Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family of growth factors plays a critical role in neuroplasticity, with implications for cognitive function and mental health. The impact of long-term, high-dose androgen use on BDNF in a natural setting has not been investigated. This study examined the association between long-term androgen exposure and BDNF levels, and the links between BDNF, heavy resistance exercise, hormones, androgens, and mental health. Methods: We measured serum levels of BDNF and sex steroid hormones in male weightlifters (N = 141) with a history of current (n = 59), past (n = 29), or no (n = 52) androgen use. All participants completed questionnaires assessing maximum strength and measures of anxiety and depression. Group differences in BDNF were tested using general linear models adjusting for age and associations between BDNF and strength, anxiety, and depression using Pearson’s or Kendall’s correlations. Results: Both current (mean: 44.1 ng/mL [SD: 12.7]) and past (39.5 ng/mL [SD: 13.9]) androgen users showed lower serum BDNF levels compared to nonusing controls (51.5 [SD: 15.3], p < 0.001, ηp2 = 0.10). BDNF levels were negatively related to maximal strength, and with hormonal status in past androgen users, but no significant associations were found with measures of depression and anxiety. Conclusion: Lower circulating BDNF concentrations in current and past androgen users suggest that high-dose androgen exposure triggers persistent changes in BDNF expression. Further studies are needed to verify the relationship and its potential clinical implications.

Summary: Oxidation resistance gene 1 (OXR1) protects cells against oxidative stress. We find that male mice with brain-specific isoform A knockout (Oxr1A−/−) develop fatty liver. RNA sequencing of male Oxr1A−/− liver indicates decreased growth hormone (GH) signaling, which is known to affect liver metabolism. Indeed, Gh expression is reduced in male mice Oxr1A−/− pituitary gland and in rat Oxr1A−/− pituitary adenoma cell-line GH3. Oxr1A−/− male mice show reduced fasting-blood GH levels. Pull-down and proximity ligation assays reveal that OXR1A is associated with arginine methyl transferase PRMT5. OXR1A-depleted GH3 cells show reduced symmetrical dimethylation of histone H3 arginine 2 (H3R2me2s), a product of PRMT5 catalyzed methylation, and chromatin immunoprecipitation (ChIP) of H3R2me2s shows reduced Gh promoter enrichment. Finally, we demonstrate with purified proteins that OXR1A stimulates PRMT5/MEP50-catalyzed H3R2me2s. Our data suggest that OXR1A is a coactivator of PRMT5, regulating histone arginine methylation and thereby GH production within the pituitary gland. : Yang et al. show that OXR1A interacts with methyltransferase PRMT5 to promote arginine methylation of histone H3R2 and to regulate transcription of growth hormone in the pituitary gland. Male mice with OXR1A knockout display growth-hormone deficiency and develop fatty liver. Keywords: Oxidation resistance gene 1, OXR1, protein arginine methyltransferase, PRMT1, PRMT5, Arginine Methylation, H3R2me2s, pituitary gland, brain-liver axis, Growth hormone, Non-alcoholic fatty liver disease, NAFLD, neuroendocrine regulation, epigenetic regulation

Background and Purpose: Cardioembolic stroke due to paroxysmal atrial fibrillation (AF) may account for 1 out of 4 cryptogenic strokes (CS) and transient ischemic attacks (TIAs). The purpose of this pilot study was to search for biomarkers potentially predicting incident AF in patients with ischemic stroke or TIA. Methods: Plasma samples were collected from patients aged 18 years and older with ischemic stroke or TIA due to AF (n = 9) and large artery atherosclerosis (LAA) with ipsilateral carotid stenosis (n = 8) and age- and sex-matched controls (n = 10). Analyses were performed with the Olink technology simultaneously measuring 184 biomarkers of cardiovascular disease. For bioinformatics, acquired data were analyzed using gene set enrichment analysis (GSEA). Selected proteins were validated using ELISA. Individual receiver operating characteristic (ROC) curves and odds ratios from logistic regression were calculated. A randomForest (RF) model with out-of-bag estimate was applied for predictive modeling. Results: GSEA indicated enrichment of proteins related to inflammatory response in the AF group. Interleukin (IL)-6, growth differentiation factor (GDF)-15, and pentraxin-related protein PTX3 were the top biomarkers on the ranked list for the AF group compared to the LAA group and the control group. ELISA validated increased expression of all tested proteins (GDF-15, PTX3, and urokinase plasminogen activator surface receptor [U-PAR]), except for IL-6. 19 proteins had the area under the ROC curve (AUC) over 0.85 including all of the proteins with significant evolution in the logistic regression. AUCs were very discriminant in distinguishing patients with and without AF (LAA and control group together). GDF-15 alone reached AUC of 0.95. Based on RF model, all selected participants in the tested group were classified correctly, and the most important protein in the model was GDF-15. Conclusions: Our results demonstrate an association between inflammation and AF and that multiple proteins alone and in combination may potentially be used as indicators of AF in CS and TIA patients. However, further studies including larger samples sizes are needed to support these findings. In the ongoing NOR-FIB study, we plan further biomarker assessments in patients with CS and TIA undergoing long-term cardiac rhythm monitoring with insertable cardiac monitors.

Maria Belland Olsen,1,2 Camilla Huse,1,2 Mirta Mittelstedt Leal de Sousa,3,4 Sarah Louise Murphy,1,2 Antonio Sarno,3,5 Tobias Sebastian Obermann,3 Kuan Yang,1 Jan Cato Holter,2,6 Marte Jøntvedt Jørgensen,2,7 Erik Egeland Christensen,2,7 Wei Wang,3 Ping Ji,3 Lars Heggelund,8,9 Hedda Hoel,1,10 Anne Margarita Dyrhol-Riise,2,7 Ida Gregersen,1 Pål Aukrust,1,2,11 Magnar Bjørås,3,6 Bente Halvorsen,1,2 Tuva Børresdatter Dahl12 1Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway; 2Institute of Clinical Medicine, University of Oslo, Oslo, Norway; 3Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway; 4Proteomics and Modomics Experimental Core Facility (PROMEC), NTNU, Trondheim, Norway; 5Department of Fisheries and New Biomarine Industry, SINTEF Ocean, Trondheim, Norway; 6Department of Microbiology, Oslo University Hospital and University of Oslo, Oslo, Norway; 7Department of Infectious Diseases, Oslo University Hospital, Oslo, Norway; 8Department of Internal Medicine, Vestre Viken Hospital Trust, Drammen, Norway; 9Department of Clinical Science, Faculty of Medicine, University of Bergen, Bergen, Norway; 10Department of Medicine, Lovisenberg Diaconal Hospital, Oslo, Norway; 11Section of Clinical Immunology and Infectious Diseases, Oslo University Hospital, Oslo, Norway; 12Division of Critical Care and Emergencies, Oslo University Hospital, Oslo, NorwayCorrespondence: Tuva Børresdatter Dahl, Division of Critical Care and Emergencies and Research Institute of Internal Medicine, Oslo University Hospital, Sognsvannsveien 20, Oslo, Norway, Tel +4723072786, Email t.b.dahl@medisin.uio.noPurpose: Reactive oxygen species (ROS) are an important part of the inflammatory response during infection but can also promote DNA damage. Due to the sustained inflammation in severe Covid-19, we hypothesized that hospitalized Covid-19 patients would be characterized by increased levels of oxidative DNA damage and dysregulation of the DNA repair machinery.Patients and Methods: Levels of the oxidative DNA lesion 8-oxoG and levels of base excision repair (BER) proteins were measured in peripheral blood mononuclear cells (PBMC) from patients (8-oxoG, n = 22; BER, n = 17) and healthy controls (n = 10) (Cohort 1). Gene expression related to DNA repair was investigated in two independent cohorts of hospitalized Covid-19 patients (Cohort 1; 15 patents and 5 controls, Cohort 2; 15 patients and 6 controls), and by publicly available datasets.Results: Patients and healthy controls showed comparable amounts of oxidative DNA damage as assessed by 8-oxoG while levels of several BER proteins were increased in Covid-19 patients, indicating enhanced DNA repair in acute Covid-19 disease. Furthermore, gene expression analysis demonstrated regulation of genes involved in BER and double strand break repair (DSBR) in PBMC of Covid-19 patients and expression level of several DSBR genes correlated with the degree of respiratory failure. Finally, by re-analyzing publicly available data, we found that the pathway Hallmark DNA repair was significantly more regulated in circulating immune cells during Covid-19 compared to influenza virus infection, bacterial pneumonia or acute respiratory infection due to seasonal coronavirus.Conclusion: Although beneficial by protecting against DNA damage, long-term activation of the DNA repair machinery could also contribute to persistent inflammation, potentially through mechanisms such as the induction of cellular senescence. However, further studies that also include measurements of additional markers of DNA damage are required to determine the role and precise molecular mechanisms for DNA repair in SARS-CoV-2 infection.Keywords: Covid-19, oxidative stress, DNA damage, DNA repair, base excision repair, double strand break repair

While serum-circulating complement destroys invading pathogens, intracellularly active complement, termed the ‘complosome’, functions as a vital orchestrator of cell-metabolic events underlying T cell effector responses. Whether intracellular complement is also non-redundant for the activity of myeloid immune cells is currently unknown. Here, we show that monocytes and macrophages constitutively express complement component (C) 5 and generate autocrine C5a via formation of an intracellular C5 convertase. Cholesterol crystal-sensing by macrophages induced C5aR1 signaling on mitochondrial membranes, which shifted ATP production via reverse electron chain flux towards reactive oxygen species (ROS) generation and anaerobic glycolysis to favor IL-1β production, both at the transcriptional level and processing of pro-IL-1β. Consequently, atherosclerosis-prone mice lacking macrophage-specific C5ar1 had ameliorated cardiovascular disease on a high-cholesterol diet. Conversely, inflammatory gene signatures and IL-1β produced by cells in unstable atherosclerotic plaques of patients were normalized by a specific cell-permeable C5aR1 antagonist. Deficiency of the macrophage cell autonomous C5 system also protected mice from crystal nephropathy mediated by folic acid. These data demonstrate the unexpected intracellular formation of a C5 convertase and identify C5aR1 as a direct modulator of mitochondrial function and inflammatory output from myeloid cells. Together, these findings suggest that the complosome is a contributor to the biologic processes underlying sterile inflammation and indicate that targeting this system could be beneficial in macrophage-dependent diseases, such as atherosclerosis.

Background Soluble suppression of tumorigenesis-2 (sST2) and galectin (Gal)-3 are two biomarkers related to inflammation, metabolic disturbances and to myocardial fibrosis that characterize several cardiac pathological conditions. Increased circulating levels of these molecules have been associated with risk of cardiovascular death. Treatment with liraglutide, a glucagon-like peptide 1 analog, is associated with weight loss, improved glycemic control, and reduced cardiovascular risk. We wanted to assess (I) potential differences between subjects with prediabetes or type 2 diabetes mellitus (T2DM) and healthy controls in sST2 and Gal-3 circulating levels, and their relationship with glycemic control and markers of beta cell function and myocardial injury; (II) whether liraglutide treatment modulates these markers in subjects with prediabetes or early T2DM independently of weight loss; (III) whether baseline levels of any of these two molecules may predict the response to liraglutide treatment. Methods Forty metformin-treated obese subjects (BMI ≥ 30) with prediabetes [impaired fasting glucose (IFG) or impaired glucose tolerance (IGT) or both (n = 23)] or newly diagnosed T2DM (n = 17), were randomized to liraglutide or lifestyle counseling until achieving a comparable weight loss (7% of initial body weight). Thirteen subjects were enrolled as healthy controls for baseline sST2 and Gal-3 levels. Results Baseline sST2 levels were comparable between controls and obese patients (p = 0.79) whereas Gal-3 levels were significantly higher in patients as compared to controls (p Conclusions Liraglutide-induced reduction in sST2 and possibly hs-TnI suggests that in obese patients with prediabetes or early T2DM this drug may have a positive effect on (cardiac) fibrosis, whereas plasma level of Gal-3 before liraglutide initiation may predict response to the drug in terms of beta cell function improvement. Trial registration Eudract: 2013-001356-36

Background We recently showed that interleukin (IL)-6 inhibition by tocilizumab improves myocardial salvage in ST-elevation myocardial infarction (STEMI). However, the mechanisms for this effect are not clear. Methods In this exploratory sub-study of the ASSAIL-MI trial, we examined leukocyte differential counts and their relation to myocardial salvage and peak troponin T (TnT) in STEMI patients randomised to tocilizumab (n = 101) or placebo (n = 98). We performed RNA-sequencing on whole blood (n = 40) and T cells (n = 20). B and T cell subpopulations were examined by flow cytometry (n = 69). Findings (i) STEMI patients had higher neutrophil counts at hospitalisation compared with stable angina patients. (ii) After percutaneous coronary intervention there was a gradual decline in neutrophils, which was significantly more pronounced in the tocilizumab group. (iii) The decrease in neutrophils in the tocilizumab group was associated with improved myocardial salvage and lower peak TnT. (iv) RNA-sequencing suggested that neutrophil function was also attenuated by tocilizumab. (v) B and T cell sub-populations changed only minimally after STEMI with minor effects of tocilizumab, supported as well by RNA-sequencing analyses of T cells. (vi) However, a low CD8+ count was associated with improved myocardial salvage in patients admitted to the hospital > 3 h after symptom onset. Interpretation Tocilizumab induced a rapid reduction in neutrophils and seemed to attenuate neutrophil function in STEMI patients potentially related to the beneficial effects of tocilizumab on myocardial salvage. Funding South-Eastern Norway Regional Health Authority (Nos. 2019067, 2017084), the Central Norway Regional Health Authority and Norwegian Research Council (No. 283867).

BackgroundThe functional status of lipoprotein particles contributes to atherogenesis. The tendency of plasma LDL particles to aggregate and the ability of HDL particles to induce and mediate reverse cholesterol transport associate with high and low risk for cardiovascular disease in adult patients, respectively. However, it is unknown whether children with familial hypercholesterolemia (FH) display lipoprotein function alterations.HypothesisWe hypothesized that FH children had disrupted lipoprotein function.MethodsWe analyzed LDL aggregation susceptibility and HDL-apoA-I exchange to apoA-I ratio (HAE/apoA-I ratio), and activity of four proteins that regulate lipoprotein metabolism (CETP, LCAT, PLTP and PON1) in plasma samples derived from children with FH (n = 47) and from healthy children (n = 56). Potential biological mechanisms behind any variation in lipoprotein functionalities were explored using an NMR-based metabolomics profiling approach.ResultsLDL aggregation was higher and HAE/apoA-I ratio was lower in FH children than in healthy children. LDL aggregation associated positively with LDL-C and negatively with triglycerides, and HAE/apoA-I ratio associated negatively with LDL-C. Generally, the metabolomic profile for LDL aggregation was a mirror image of that for HAE/apoA-I ratio.ConclusionsFH children displayed increased atherogenicity of LDL and disrupted HDL function. These newly observed functional alterations in LDL and HDL may increase the risk for atherosclerotic cardiovascular disease in FH children.

Background In cardiovascular diseases, atherosclerotic disorder are the most frequent and important with respect to morbidity and mortality. Inflammation mediated by immune cells is central in all parts of the atherosclerotic progress, and further understanding of the underlying mechanisms is needed. Growing evidence suggests that deamination of adenosine‐to‐inosine in RNA is crucial for a correct immune response; nevertheless, the role of adenosine‐to‐inosine RNA editing in atherogenesis has barely been studied. Several proteins have affinity for inosines in RNA, one being ENDOV (endonuclease V), which binds and cleaves RNA at inosines. Data on ENDOV in atherosclerosis are lacking. Methods and Results Quantitative polymerase chain reaction on ENDOV mRNA showed an increased level in human carotid atherosclerotic plaques compared with control veins. Inosine‐ribonuclease activity as measured by an enzyme activity assay is detected in immune cells relevant for the atherosclerotic process. Abolishing EndoV in atherogenic apolipoprotein E‐deficient ( ApoE −/− ) mice reduces the atherosclerotic plaque burden, both in size and lipid content. In addition, in a brain stroke model, mice without ENDOV suffer less damage than control mice. Finally, lack of EndoV reduces the recruitment of monocytes to atherosclerotic lesions in atherogenic ApoE −/− mice. Conclusions ENDOV is upregulated in human atherosclerotic lesions, and data from mice suggest that ENDOV promotes atherogenesis by enhancing the monocyte recruitment into the atherosclerotic lesion, potentially by increasing the effect of CCL2 activation on these cells.

ObjectiveAlthough COVID-19 is primarily a respiratory infection, mounting evidence suggests that the GI tract is involved in the disease, with gut barrier dysfunction and gut microbiota alterations being related to disease severity. Whether these alterations persist and could be related to long-term respiratory dysfunction is unknown.DesignFrom the NOR-Solidarity trial (n=181), plasma was collected during hospital admission and after three months, and analyzed for markers of gut barrier dysfunction and inflammation. At the three-month follow-up, pulmonary function was assessed by measuring diffusing capacity of the lungs for carbon monoxide (DLCO), and rectal swabs for gut microbiota analyses were collected (n= 97) and analysed by sequencing of the 16S rRNA gene.ResultsGut microbiota diversity was reduced in COVID-19 patients with persistent respiratory dysfunction, defined as DLCO below lower limit of normal three months after hospitalization. These patients also had an altered global gut microbiota composition, with reduced abundance of Erysipelotrichaceae UCG-003 and increased abundance of Flavonifractor and Veillonella, the latter potentially being linked to fibrosis. During hospitalization, increased plasma levels of lipopolysaccharide-binding protein (LBP) were strongly associated with respiratory failure, defined as pO2/fiO2-(P/F-ratio)ConclusionPersistent respiratory dysfunction after COVID-19 is associated with reduced biodiversity and gut microbiota alterations, along with persistently elevated LBP levels. Our results point to a potential gut-lung axis that should be further investigated in relation to long-term pulmonary dysfunction and long COVID.Summary boxWhat is already known about this subject?Mounting evidence suggests that the gastrointestinal tract is involved in the pathogenesis of COVID-19, with the putative SARS-CoV-2 receptor ACE 2 ubiquitously expressed in the gut.In severe COVID-19, the gut-blood barrier is compromised, and leakage of microbial products, such as lipopolysaccharides (LPS), could affect the host’s response to COVID-19 infection.COVID-19 patients exhibit an altered gut microbiota composition, which has been related to disease severity. However, it is currently not known whether dysbiosis or gut barrier dysfunction persist long-term after hospitalization, or whether microbiota-related mechanisms could be related to persistent pulmonary dysfunction.What are the new findings?COVID-19 patients with persistent respiratory dysfunction after three months had a lower microbial diversity and an altered gut microbiota composition at the same time point.The microbiota alterations included reduced abundance of Erysipelotrichaceae UCG-003 and increased abundance of Veillonella and Flavonifractor.During hospitalization, increased plasma levels of LBP were strongly associated with respiratory failure.LBP levels remained elevated during and after hospitalization, and associated significantly with persistent respiratory dysfunction at three-month follow-up.How might it impact on clinical practice in the foreseeable future?Our findings point to a potential gut-lung axis in relation not only to respiratory failure during hospitalization, but also to long-term COVID-19 morbidity. Further studies on gut microbiota composition and gut barrier dysfunction as potential treatment targets and/or disease severity biomarkers in relation to long-term pulmonary dysfunction and long COVID are warranted.

Purpose: Fatty acid (FA) abnormalities have been found in various inflammatory disorders and have been related to disturbed gut microbiota. Patients with Common variable immunodeficiency (CVID) have inflammatory complications associated with altered gut microbial composition. We hypothesized that there is an altered FA profile in CVID patients, related to gut microbial dysbiosis. Methods: Plasma FAs were measured in 39 CVID patients and 30 healthy controls. Gut microbial profile, a food frequency questionnaire and the effect of the oral antibiotic rifaximin, were investigated in CVID patients. Results: The n-3 PUFAs, EPA (1.4 [1.0-1.8] vs 1.9 [1.2-2.5], median [IQR], Pn-6 PUFAs (34.3 ± 3.4, vs 37.1 ± 2.8, mean ± SD, P< 0.001) and linoleic acid (LA) (24.5 ± 3.3, vs 28.1 ± 2.7, P< 0.0001), and the FA anti-inflammatory index (98.9, [82.1-119.4], vs 117.0, [88.7-153.1], median [IQR]), Pn-6 PUFAs, (r=0.41, Pn-3 PUFAs (P= 0.78). Moreover, a 2-week course of rifaximin significantly reduced the proportion of n-6 PUFAs (P=0.04, UNIANOVA). Overall, the altered proportions of n-3 and n-6 PUFAs did not seem to be related to dietary intake, intestinal malabsorption or presence of CVID enteropathy.Conclusion: We found a potentially unfavourable FA profile in CVID, where plasma proportion of n-6 PUFAs was related to gut microbial diversity and altered by microbial therapy.

Angiogenesis is necessary for tumor growth and has been targeted in breast cancer; however, it is unclear which patients will respond and benefit from antiangiogenic therapy. We report noninvasive monitoring of patient response to neoadjuvant chemotherapy given alone or in combination with anti‐vascular endothelial growth factor (bevacizumab) in a randomized clinical trial. At four time points during neoadjuvant chemotherapy ± bevacizumab of receptor tyrosine‐protein kinase erbB‐2‐negative breast cancers, we measured metabolites and inflammation‐related markers in patient's serum. We report significant changes in the levels of several molecules induced by bevacizumab, the most prominent being an increase in pentraxin 3 (PTX3) and von Willebrand factor (VWF). Serum levels of AXL, VWF and pulmonary and activation‐regulated cytokine (PARC/CCL18) reflected response to chemotherapy alone or in combination with bevacizumab. We further analyzed serum cytokines in relation to tumor characteristics such as gene expression, tumor metabolites and tumor infiltrating leukocytes. We found that VWF and growth‐differentiation factor 15 tumor mRNA levels correlated with their respective serum protein levels suggesting that these cytokines may be produced by tumors and outflow to the bloodstream while influencing the tumor microenvironment locally. Finally, we used binomial logistic regression which allowed to predict patient's response using only 10 noninvasive biomarkers. Our study highlights the potential of monitoring circulating levels of cytokines and metabolites during breast cancer therapy © 2019 The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

NAD+ is an essential cofactor in reduction-oxidation metabolism with impact on metabolic and inflammatory diseases. However, data elucidating the effects of NAD+ on the proinflammatory features of human primary monocytes are scarce. In this study, we explored how NAD+ affects TLR4 and NOD-like receptor with a PYD-domain 3 (NLRP3) inflammasome activation, two key innate immune responses. Human primary monocytes were isolated from buffy coats obtained from healthy individuals. Intracellular NAD+ was manipulated by nicotinamide riboside and the NAMPT inhibitor FK866. Cells were primed with LPS with or without subsequent NLRP3 activation with ATP or cholesterol crystals to analyze the effects of NAD+ levels on TLR4-mediated NF-κB activation and NLRP3 activity, respectively. Cytokine release was quantified, and the downstream signal pathway of TLR4 was investigated with Western blot and proteomic analysis. The impact of sirtuin and PARP inhibition was also explored. Our main findings were: 1) elevated NAD+ enhanced IL-1β release in LPS-primed human monocytes exposed to ATP in vitro, 2) both NLRP3-dependent and -independent inflammatory responses in LPS-exposed monocytes were inhibited by NAD+ depletion with FK866, 3) the inhibition was not caused by suppression of sirtuins or PARP1, and 4) phosphorylation of several proteins TLR4 signal pathway was inhibited by FK866-mediated NAD+ depletion, specifically TAK1, IKKβ, IkBα, MEK 1/2, ERK 1/2, and p38. Hence, we suggest a novel mechanism in which NAD+ affects TLR4 signal transduction. Furthermore, our data challenge previous reports of the interaction between NAD+ and inflammation and question the use of nicotinamide riboside in the therapy of inflammatory disorders.

Objectives. Exercise training has been proposed to have anti-inflammatory effects. We examined whether aerobic interval training (AIT) can attenuate the inflammatory response in ischemic heart failure (HF) as measured by serum biomarkers representing a broad spectrum of activated inflammatory pathways. Design. We conducted a controlled prospective trial recruiting 30 patients (19 in the AIT group and 11 in the control group) with ischemic HF and an implantable cardioverter defibrillator (ICD). This study is a sub study of the previously reported “Aerobic interval training in patients with heart failure and an ICD” (Eur J Prev Cardiol. 22 March 2015; 22:296–303). Patients in the AIT group exercised for 12-weeks completing a total of 36 AIT sessions. We analyzed serum levels of C-reactive protein, pentraxin-3, osteoprotegerin, brain natriuretic peptide, neopterin, and soluble tumor necrois factor type 1 and 2, all known to predict an adverse outcome in HF, at baseline and following the 12-week AIT intervention. Results. The AIT group significantly increased peak oxygen uptake and improved endothelial function compared to the sedentary control group. No statistically significant changes in serum levels of the biomarkers were detected from baseline following the AIT intervention and, there were no significant differences in changes of these mediators between the AIT and the control group. Conclusions. A 12-week AIT intervention, although improving exercise capacity and endothelial function, did not attenuate serum inflammatory biomarkers in stable ischemic HF patients with an ICD on optimal medical therapy. acceptedVersion

Elevated lipoprotein(a) (Lp(a)) is associated with CVD and is mainly genetically determined. Studies suggest a role of dietary fatty acids (FA) in the regulation of Lp(a); however, no studies have investigated the association between plasma Lp(a) concentration and n-6 FA. We aimed to investigate whether plasma Lp(a) concentration was associated with dietary n-6 FA intake and plasma levels of arachidonic acid (AA) in subjects with familial hypercholesterolaemia (FH). We included FH subjects with (n 68) and without (n 77) elevated Lp(a) defined as ≥75 nmol/l and healthy subjects (n 14). Total FA profile was analysed by GC–flame ionisation detector analysis, and the daily intake of macronutrients (including the sum of n-6 FA: 18 : 2n-6, 20 : 2n-6, 20 : 3n-6 and 20 : 4n-6) were computed from completed FFQ. FH subjects with elevated Lp(a) had higher plasma levels of AA compared with FH subjects without elevated Lp(a) (P = 0·03). Furthermore, both FH subjects with and without elevated Lp(a) had higher plasma levels of AA compared with controls (P < 0·001). The multivariable analyses showed associations between dietary n-6 FA intake and plasma levels of AA (P = 0·02) and between plasma levels of Lp(a) and AA (P = 0·006). Our data suggest a novel link between plasma Lp(a) concentration, dietary n-6 FA and plasma AA concentration, which may explain the small diet-induced increase in Lp(a) levels associated with lifestyle changes. Although the increase may not be clinically relevant, this association may be mechanistically interesting in understanding more of the role and regulation of Lp(a).

Highlights • Development of cardiovascular disease and inflammation are heavily intertwined, and inflammasome activation is thought play an important role in this interaction. • This review provides an overview of preclinical and clinical studies supporting inflammasomes as a therapeutic target in atherosclerosis and heart failure. • Future studies exploring direct inflammasome inhibition, either NLRP3 or the lesser-studied inflammasomes, are also discussed., Summary The pathogenesis of cardiovascular disease (CVD) is complex and multifactorial, and inflammation plays a central role. Inflammasomes are multimeric protein complexes that are activated in a 2-step manner in response to infection or tissue damage. Upon activation the proinflammatory cytokines, interleukins-1β and -18 are released. In the last decade, the evidence that inflammasome activation plays an important role in CVD development became stronger. We discuss the role of different inflammasomes in the pathogenesis of CVD, focusing on atherosclerosis and heart failure. This review also provides an overview of existing experimental studies and clinical trials on inflammasome inhibition as a therapeutic target in these disorders., Central Illustration