Elevated markers of gut leakage and inflammasome activation in COVID-19 patients with cardiac involvement.

BACKGROUND: A high proportion of COVID-19 patients have cardiac involvement, even those without known cardiac disease. Downregulation of angiotensin converting enzyme 2 (ACE2), a receptor for SARS-CoV-2 and the renin-angiotensin system, as well as inflammatory mechanisms have been suggested to play a role. ACE2 is abundant in the gut and associated with gut microbiota composition. We hypothesized that gut leakage of microbial products, and subsequent inflammasome activation could contribute to cardiac involvement in COVID-19 patients. METHODS: Plasma levels of a gut leakage marker (LPS-binding protein, LBP), a marker of enterocyte damage (intestinal fatty acid binding protein, IFABP), a gut homing marker (CCL25, ligand for chemokine receptor CCR9) and markers of inflammasome activation (IL-1ß, IL-18 and their regulatory proteins) were measured at three time points (day 1, 3-5 and 7-10) in 39 hospitalized COVID-19 patients and related to cardiac involvement. RESULTS: Compared to controls, COVID-19 patients had elevated plasma levels of LBP and CCL25 but not IFABP, suggesting impaired gut barrier function and accentuated gut homing of T cells without excessive enterocyte damage. Levels of LBP were twice as high at baseline in patients with elevated cardiac markers compared with those without and remained elevated during hospitalization. Also, markers of inflammasome activation were moderately elevated in patients with cardiac involvement. LBP was associated with higher NT-pro-BNP levels, whereas IL-18, IL-18BP and IL-1Ra were associated with higher troponin levels. CONCLUSION: Patients with cardiac involvement had elevated markers of gut leakage and inflammasome activation, suggestive of a potential gut-heart axis in COVID-19.

Profiling of immune-related gene expression in children with familial hypercholesterolaemia.

BACKGROUND: Innate and adaptive immune responses are pivotal in atherosclerosis, but their association with early-stage atherosclerosis in humans is incompletely understood. In this regard, untreated children with familial hypercholesterolaemia may serve as a human model to investigate the effect of elevated low-density lipoprotein (LDL)-cholesterol. OBJECTIVES: We aimed to study the immunological and inflammatory pathways involved in early atherosclerosis by examining mRNA molecules in peripheral blood mononuclear cells (PBMCs) from children with FH. METHODS: We analysed the level of 587 immune-related mRNA molecules using state-of-the-art Nanostring technology in PBMCs from children with (n = 30) and without (n = 21) FH, and from FH children before and after statin therapy (n = 10). RESULTS: 176 genes (30%) were differentially expressed between the FH and healthy children at P < 0.05. Compared to healthy children, the dysregulated pathways in FH children included the following: T cells (18/19); B cells (5/6); tumour necrosis factor super family (TNFSF) (6/8); cell growth, proliferation and differentiation (5/7); interleukins (5/9); toll-like receptors (2/5); apoptosis (3/7) and antigen presentation (1/7), where the ratio denotes higher expressed genes to total number of genes. Statin therapy reversed expression of thirteen of these mRNAs in FH children. CONCLUSION: FH children display higher PBMC expression of immune-related genes mapped to several pathways, including T and B cells, and TNFSF than healthy children. Our results suggest that LDL-C plays an important role in modulating expression of different immune-related genes, and novel data on the involvement of these pathways in the early atherosclerosis may represent future therapeutic targets for prevention of atherosclerotic progression.

Prognostic biomarkers for immunotherapy with ipilimumab in metastatic melanoma.

New therapies, including the anti-cytotoxic T lymphocyte antigen (CTLA)-4 antibody, ipilimumab, is approved for metastatic melanoma. Prognostic biomarkers need to be identified, because the treatment has serious side effects. Serum samples were obtained before and during treatment from 56 patients with metastatic or unresectable malignant melanoma, receiving treatment with ipilimumab in a national Phase IV study (NCT0268196). Expression of a panel of 17 inflammatory-related markers reflecting different pathways including extracellular matrix remodeling and fibrosis, vascular inflammation and monocyte/macrophage activation were measured at baseline and the second and/or third course of treatment with ipilimumab. Six candidate proteins [endostatin, osteoprotegerin (OPG), C-reactive protein (CRP), pulmonary and activation-regulated chemokine (PARC), growth differentiation factor 15 (GDF15) and galectin-3 binding-protein (Gal3BP)] were persistently higher in non-survivors. In particular, high Gal3BP and endostatin levels were also independently associated with poor 2-year survival after adjusting for lactate dehydrogenase, M-stage and number of organs affected. A 1 standard deviation increase in endostatin gave 1·74 times [95% confidence interval (CI) = 1·10-2·78, P = 0·019] and for Gal3BP 1·52 times (95% CI = 1·01-2·29, P = 0·047) higher risk of death in the adjusted model. Endostatin and Gal3BP may represent prognostic biomarkers for patients on ipilimumab treatment in metastatic melanoma and should be further evaluated. Owing to the non-placebo design, we could only relate our findings to prognosis during ipilimumab treatment.

Rifaximin alters gut microbiota profile, but does not affect systemic inflammation - a randomized controlled trial in common variable immunodeficiency.

Common variable immunodeficiency (CVID) patients have reduced gut microbial diversity compared to healthy controls. The reduced diversity is associated with gut leakage, increased systemic inflammation and ten "key" bacteria that capture the gut dysbiosis (dysbiosis index) in CVID. Rifaximin is a broad-spectrum non-absorbable antibiotic known to reduce gut leakage (lipopolysaccharides, LPS) in liver disease. In this study, we explored as a 'proof of concept' that altering gut microbial composition could reduce systemic inflammation, using CVID as a disease model. Forty adult CVID patients were randomized, (1:1) to twice-daily oral rifaximin 550 mg versus no treatment for 2 weeks in an open-label, single-centre study. Primary endpoints were reduction in plasma/serum levels of soluble (s) CD14, sCD25, sCD163, neopterin, CRP, TNF, LPS and selected cytokines measured at 0, 2 and 8 weeks. Secondary endpoint was changes in intra-individual bacterial diversity in stool samples. Rifaximin-use did not significantly change any of the inflammation or gut leakage markers, but decreased gut microbial diversity compared with no treatment (p = 0.002). Importantly, the gut bacteria in the CVID dysbiosis index were not changed by rifaximin. The results suggest that modulating gut microbiota by rifaximin is not the chosen intervention to affect systemic inflammation, at least not in CVID.

An association between YKL-40 and type 2 diabetes in psychotic disorders.

OBJECTIVE: This study examines if YKL-40 is increased in individuals with psychotic disorders and if elevated YKL-40 levels at baseline is associated with subsequent development of type 2 diabetes. METHOD: A total of 1383 patients with a diagnosis of schizophrenia or affective psychosis and 799 healthy controls were recruited in the period 2002-2015. Plasma YKL-40 and metabolic risk factors were measured and medication was recorded. Using national registry data, association between baseline risk factors and later development of type 2 diabetes was assessed using Cox proportional hazards models. RESULTS: Plasma YKL-40 was higher in patients vs. healthy controls also after adjusting for metabolic risk factors, with no difference between the schizophrenia and affective psychosis groups. Patients were diagnosed with type 2 diabetes at a significantly younger age. Multivariate Cox regression analyses showed that elevated YKL-40 (hazard ratio (HR) = 5.6, P = 0.001), elevated glucose (HR = 3.6, P = 0.001), and schizophrenia diagnosis (HR = 3.0, P = 0.014) at baseline were associated with subsequent development of type 2 diabetes. CONCLUSIONS: Patients with psychotic disorders have at baseline increased levels of YKL-40 beyond the effect of comorbid type 2 diabetes and metabolic risk factors. Elevated YKL-40 level at baseline is associated with later development of type 2 diabetes.

High physical fitness is associated with reduction in basal- and exercise-induced inflammation.

C-reactive protein (CRP) increases after strenuous exercise. It has been a concern that prolonged strenuous exercise may be harmful and induce a deleterious inflammatory response. The purpose of this study was to (a) assess and quantify the magnitude of CRP response following an endurance cycling competition in healthy middle-aged recreational cyclists. (b) Identify important determinants of this response. (c) Identify the relationship between CRP, myocardial damage (cardiac Troponin I (cTnI)), and myocardial strain (B-type natriuretic peptide [BNP]). (d) Identify the relationship between CRP and clinical events, defined as utilization of healthcare services or self-reported unusual discomfort. Race time was used as a measure of physical fitness. A total of 97 individuals (43±10 years of age, 74 [76%] males) were assessed prior to and 0, 3, and 24 hours following the 91-km mountain bike race "Nordsjørittet" (Sandnes, Norway, June 2013). There was a highly significant increase in CRP from baseline to 24 hours (0.9 (0.5-1.8) mg/L vs. 11.6 (6.0-17.5) mg/L (median[IQR]), P<.001), with no correlation of CRP to cTnI and BNP at any time-point. CRP was strongly correlated to race time at baseline (r=.38, P<.001) and at 24 hours following the race (r=.43, P<.001), In multivariate models, race time was an independent predictor of CRP both at baseline and at 24 hours (P<.01). There was no relationship between CRP levels and clinical events. In conclusion, high physical fitness was associated with reduction in both basal- and exercise-induced CRP. No adverse relationship was found between high intensity physical exercise, CRP levels, and outcomes.

Persistent increase in TNF and IL-1 markers in severe mental disorders suggests trait-related inflammation: a one year follow-up study.

OBJECTIVE: We evaluated if plasma levels of inflammatory markers are persistently altered in severe mental disorders with psychotic symptoms or associated with state characteristics in a longitudinal study. METHODS: Soluble tumor necrosis factor receptor 1 (sTNF-R1), interleukin-1 receptor antagonist (IL-1Ra), von Willebrand factor (VWF), and osteoprotegerin (OPG) were measured in schizophrenia (n = 69) and affective (n = 55) spectrum patients at baseline and at one-year follow-up, and compared to healthy controls (HC) (n = 92) with analysis of covariance. Association between change in symptoms and inflammatory markers was analyzed with mixed-effects models. RESULTS: sTNF-R1 was higher in the schizophrenia (P < 0.0001) and affective disorders (P = 0.02) compared to HC, while IL-1Ra was higher in schizophrenia (P = 0.01) compared to HC at one year follow-up. There were no significant differences between schizophrenia and affective groups; however, levels in the affective group were in between schizophrenia and HC for sTNF-R1 and IL-1Ra. There were no significant associations between change in symptoms and inflammatory markers. CONCLUSION: Persistently increased sTNF-R1 and IL-1Ra after one year in patients with severe mental disorders primarily reflecting data from the schizophrenia group may suggest that inflammation is a trait phenomenon, and not only the result of stress-related mechanisms associated with acute episodes.

Serum osteoprotegrin (OPG) in subclinical atherosclerosis in systemic lupus erythematosus.

Introduction Osteoprotegerin (OPG) is a member of the tumor necrosis factor (TNF) receptor family. It has recently been demonstrated that OPG is produced by a variety of tissues, including the cardiovascular system (heart, arteries, veins), lung, kidney, immune tissues, and bone. The OPG-RANKL signaling pathway is strongly related to vascular calcification. We determined the association of this biomarker with subclinical atherosclerosis in systemic lupus erythematous (SLE). Methods We measured OPG and markers of subclinical atherosclerosis (coronary artery calcium (CAC), carotid intima-media thickness (cIMT) carotid plaque) in 166 SLE patients (91% female, 64% Caucasian, 31% African American, 5% others, mean age 45 years). Subgroups of patients with different levels of OPG level were compared with respect to average levels of CAC, cIMT, and with respect to presence of carotid plaque. Age was adjusted for using multiple regression. Results OPG was highly correlated with age ( p < 0.0001). Individuals with higher levels of OPG tended to have higher measures of CAC, cIMT, and more carotid plaque. However, after adjustment for age, these associations, while still positive, were no longer statistically significant. Conclusion In our study much of the association observed was due to confounding by age, and after adjusting for age, our findings do not rule out the possibility of a null association.

Rickettsia conorii is a potent complement activator in vivo and combined inhibition of complement and CD14 is required for attenuation of the cytokine response ex vivo.

Mediterranean spotted fever caused by Rickettsia conorii is a potentially lethal disease characterized by vascular inflammation affecting multiple organs. Studies of R. conorii so far have focused on activation of inflammatory cells and their release of inflammatory cytokines, but complement activation has not been investigated in R. conorii-infected patients. Here, we performed a comprehensive analysis of complement activation markers and the soluble cross-talking co-receptor CD14 (sCD14) in plasma from R. conorii-infected patients. The clinical data were supplemented with ex vivo experiments where the cytokine response was characterized in human whole blood stimulated with R. conorii. Complement activation markers at the level of C3 (C3bc, C3bBbP) and terminal pathway activation (sC5b-9), as well as sCD14, were markedly elevated (p <0.01 for all), and closely correlated (p <0.05 for all), in patients at admission compared with healthy matched controls. All tested markers were significantly reduced to baseline values at time of follow up. Rickettsia conorii incubated in human whole blood was shown to trigger complement activation accompanied by release of the inflammatory cytokines interleukin-1ß (IL-1ß), IL-6, IL-8 and tumour necrosis factor. Whereas inhibition of either C3 or CD14 had only a minor effect on released cytokines, combined inhibition of C3 and CD14 resulted in significant reduction, virtually to baseline levels, of the four cytokines (p <0.05 for all). Our data show that complement is markedly activated upon R. conorii infection and complement activation is, together with CD14, responsible for a major part of the cytokine response induced by R. conorii in human whole blood.

Altered gut microbiota profile in common variable immunodeficiency associates with levels of lipopolysaccharide and markers of systemic immune activation.

Common variable immunodeficiency (CVID) is the most common symptomatic primary immunodeficiency characterized by low immunoglobulin (Ig)G and IgA, and/or IgM. In addition to bacterial infections, a large subgroup has noninfectious inflammatory and autoimmune complications. We performed 16S ribosomal RNA-based profiling of stool samples in 44 CVID patients, 45 patients with inflammatory bowel disease (disease controls), and 263 healthy controls. We measured plasma lipopolysaccharide (LPS) and markers of immune cell activation (i.e., soluble (s) CD14 and sCD25) in an expanded cohort of 104 patients with CVID and in 30 healthy controls. We found a large shift in the microbiota of CVID patients characterized by a reduced within-individual bacterial diversity (alpha diversity, P<0.001) without obvious associations to antibiotics use. Plasma levels of both LPS (P=0.001) and sCD25 (P<0.0001) were elevated in CVID, correlating negatively with alpha diversity and positively with a dysbiosis index calculated from the taxonomic profile. Low alpha diversity and high dysbiosis index, LPS, and immune markers were most pronounced in the subgroup with inflammatory and autoimmune complications. Low level of IgA was associated with decreased alpha diversity, but not independently from sCD25 and LPS. Our findings suggest a link between immunodeficiency, systemic immune activation, LPS, and altered gut microbiota.

NLRP3 inflammasome activation during myocardial ischemia reperfusion is cardioprotective.

BACKGROUND: The innate immune receptor NLRP3 recognizes tissue damage and initiates inflammatory processes through formation multiprotein complexes with the adaptor protein ASC and caspase-1, i.e. NLRP3 inflammasomes, which through cleavage of pro-IL-1ß mediates release of bioactive IL-1ß. We hypothesized that NLRP3 mediates tissue damage during acute myocardial infarction (MI) and sought to investigate the mechanisms herein in an experimental MI model in mice. METHODS AND RESULTS: The left coronary artery (LCA) of WT, NLRP3(-/-) and ASC(-/-) mice of both genders was ligated for 30 min followed by 3 or 24 h reperfusion. For pre-conditioning studies, the TLR2 agonist Pam3CSK4 or PBS was injected intraperitoneally 60 min prior to LCA ligation. For mechanistic investigations, blood plasmas and left ventricle tissues were collected, and a hypothesis-driven selection of protein or mRNA targets was investigated. Surprisingly, hearts from NLRP3-deficient mice featured larger infarct size than WT mice (p = 0.0048). In general, there were only modest changes with no significant pattern in myocardial infiltration of neutrophils and macrophages and systemic and myocardial cytokine expression between the three genotypes. Preconditioning with the TLR2 agonist Pam3CSK4 induced Akt phosphorylation and reduced infarct size in WT but not NLRP3 -or ASC -deficient hearts. CONCLUSION: Absence of NLRP3 results in increased myocardial infarct size after in vivo ischemia reperfusion, seemingly due to dysfunction of the cardioprotective RISK pathway. Our data imply that NLRP3 contributes to cardio-protection during I/R and do not support a role for NLRP3 or ASC inhibition in the management of acute MI including revascularization therapy.

Circulating annexin A5 predicts mortality in patients with heart failure.

BACKGROUND: Natriuretic peptides are currently used to predict mortality in patients with heart failure (HF). However, novel independent biomarkers are needed to improve risk stratification in these patients. We hypothesized that annexin A5 (anxA5) would be highly expressed by organs which are generally affected by HF and that circulating anxA5 levels would predict mortality in HF patients. METHODS: We prospectively determined the diagnostic value of anxA5, N-terminal pro-B-type natriuretic peptide (NT-proBNP), C-reactive protein (CRP) and estimated glomerular filtration rate (eGFR) to predict mortality in 180 HF patients during a median follow-up of 3.6 years. Studies were conducted with anxA5(-/-) mice to investigate the underlying mechanisms. RESULTS: AnxA5 levels were significantly elevated in HF patients compared to healthy control subjects. Cox regression analysis demonstrated that anxA5, NT-proBNP and eGFR all predict mortality independently. AnxA5 significantly improved the diagnostic efficiency of NT-proBNP alone (improvement of c-statistic from 0.662 to 0.705, P < 0.001) and also combined with eGFR and CRP (improvement of c-statistic from 0.675 to 0.738, P < 0.001) to predict mortality in the Cox regression model. Receiver operating characteristic curve analysis showed that anxA5 predicted 3-year survival (area under curve 0.708) with an optimal cut-off value of 2.24 ng mL(-1) . Using anxA5(-/-) mice, we demonstrated that anxA5 is highly expressed in organs that are often affected by HF including lung, kidney, liver and spleen. Lysis of these organs in vitro resulted in a marked and significant increase in anxA5 concentrations. CONCLUSION: AnxA5 improves the diagnostic efficiency of conventional biomarkers to predict mortality in HF patients. Whereas natriuretic peptides originate from the myocardium, high circulating anxA5 levels in patients with HF are likely to reflect peripheral organ damage secondary to HF.

Association between cytokine levels, verbal memory and hippocampus volume in psychotic disorders and healthy controls.

OBJECTIVE: We investigated whether elevated plasma levels of immune markers were associated with verbal memory and hippocampal subfield volumes in patients with severe mental illnesses and in healthy controls. METHOD: In total, 230 patients with a broad DSM-IV schizophrenia spectrum illness or bipolar disorder and 236 healthy controls were recruited. Memory was assessed using the Wechsler Memory Scale-Third Edition (WMS-III) Logical Memory immediate and delayed recall, and the California Verbal Learning Test summed recall over learning list (CVLT learning) and delayed free recall. We measured plasma levels of soluble tumor necrosis factor receptor 1 (sTNF-R1), interleukin-1 receptor antagonist, interleukin-6, von Willebrand factor, osteoprotegerin, high-sensitivity C-reactive protein and sCD40 ligand. Hippocampal subfield estimates were obtained using FreeSurfer. RESULTS: We found a moderate negative association between sTNF-R1 and performance on verbal memory learning and recall tests as measured by the WMS-III Logical Memory after controlling for age, sex and diagnosis. We observed no interaction effect of diagnosis and sTNF-R1 on memory scores. We also found a nominally significant positive association between CVLT learning and hippocampal volumes. CONCLUSION: The findings suggest a role for immune involvement in memory independent of severe mental disorders and may support the 'bigger is better' hypothesis of hippocampal subfield volumes.

The Effect of Everolimus Initiation and Calcineurin Inhibitor Elimination on Cardiac Allograft Vasculopathy in De Novo Recipients: One-Year Results of a Scandinavian Randomized Trial.

Early initiation of everolimus with calcineurin inhibitor therapy has been shown to reduce the progression of cardiac allograft vasculopathy (CAV) in de novo heart transplant recipients. The effect of de novo everolimus therapy and early total elimination of calcineurin inhibitor therapy has, however, not been investigated and is relevant given the morbidity and lack of efficacy of current protocols in preventing CAV. This 12-month multicenter Scandinavian trial randomized 115 de novo heart transplant recipients to everolimus with complete calcineurin inhibitor elimination 7-11 weeks after HTx or standard cyclosporine immunosuppression. Ninety-five (83%) patients had matched intravascular ultrasound examinations at baseline and 12 months. Mean (± SD) recipient age was 49.9 ± 13.1 years. The everolimus group (n = 47) demonstrated significantly reduced CAV progression as compared to the calcineurin inhibitor group (n = 48) (ΔMaximal Intimal Thickness 0.03 ± 0.06 and 0.08 ± 0.12 mm, ΔPercent Atheroma Volume 1.3 ± 2.3 and 4.2 ± 5.0%, ΔTotal Atheroma Volume 1.1 ± 19.2 mm(3) and 13.8 ± 28.0 mm(3) [all p-values ≤ 0.01]). Everolimus patients also had a significantly greater decline in levels of soluble tumor necrosis factor receptor-1 as compared to the calcineurin inhibitor group (p = 0.02). These preliminary results suggest that an everolimus-based CNI-free can potentially be considered in suitable de novo HTx recipients.

Circulating levels of inflammatory cytokines and cytokine receptors in patients with ankylosing spondylitis: a cross-sectional comparative study.

OBJECTIVES: Insight into the most important inflammatory pathways in ankylosing spondylitis (AS) could be of importance in risk stratification and the development of treatment strategies. Therefore, we aimed to compare circulating levels of inflammatory biomarkers between AS patients and controls, and explore associations between these biomarkers and clinical measures of disease activity. METHOD: In a cross-sectional study, 143 AS patients were compared with 124 population controls. Blood samples were analysed by immunoassays for interleukin (IL)-6, IL-17a, IL-23, soluble tumour necrosis factor receptor 1 (sTNF-R1) and 2 (sTNF-R2), and osteoprotegerin (OPG). Disease activity was measured by the AS Disease Activity Score (ASDAS) and the Bath AS Disease Activity Index (BASDAI). RESULTS: Analysis of covariance (ANCOVA) demonstrated elevated plasma levels of sTNF-R1 [geometrical mean 0.94 (95% CI 0.88-1.00) vs. 0.83 (95% CI 0.78-0.89) ng/mL, p < 0.01] and OPG (2.3, 95% CI 2.1-2.4 vs. 2.0, 95% CI 1.9-2.2 ng/mL, p = 0.02) and, although not significant, of IL-23 (122, 95% CI 108-139 vs. 106, 95% CI 93-120 pg/mL, p = 0.07) in AS patients vs. CONTROLS: More AS patients had a high level of sTNF-R2 than controls (22 vs. 1, p < 0.01). No differences between the groups were seen for IL-6 and IL-17a. In patients, no significant associations were seen between inflammatory markers and disease activity measures after adjusting for personal characteristics. CONCLUSION: Significantly higher plasma levels of sTNF-R1, sTNF-R2, and OPG and numerically but non-significantly higher levels of IL-23 were found in AS patients compared to controls, indicating that these cytokines and cytokine receptors are important inflammatory pathways. Clinical measures of disease activity were not significantly correlated with circulating inflammatory markers.