Increased Plasma Levels of Triglyceride-Enriched Lipoproteins Associate with Systemic Inflammation, Lipopolysaccharides, and Gut Dysbiosis in Common Variable Immunodeficiency.

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.

Respiratory dysfunction three months after severe COVID-19 is associated with gut microbiota alterations.

BACKGROUND: Although coronavirus disease 2019 (COVID-19) is primarily a respiratory infection, mounting evidence suggests that the gastrointestinal tract is involved in the disease, with gut barrier dysfunction and gut microbiota alterations being related to disease severity. Whether these alterations persist and are related to long-term respiratory dysfunction remains unknown. METHODS: Plasma was collected during hospital admission and after 3 months from the NOR-Solidarity trial (n = 181) and analyzed for markers of gut barrier dysfunction and inflammation. At the 3-month follow-up, pulmonary function was assessed by measuring the diffusing capacity of the lungs for carbon monoxide (DLCO ). Rectal swabs for gut microbiota analyses were collected (n = 97) and analyzed by sequencing the 16S rRNA gene. RESULTS: Gut microbiota diversity was reduced in COVID-19 patients with respiratory dysfunction, defined as DLCO below the lower limit of normal 3 months after hospitalization. These patients also had an altered global gut microbiota composition, with reduced relative abundance of 20 bacterial taxa and increased abundance of five taxa, including Veillonella, potentially 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) <26.6 kPa. LBP levels remained elevated during and after hospitalization and were associated with low-grade inflammation and respiratory dysfunction after 3 months. CONCLUSION: Respiratory dysfunction after COVID-19 is associated with altered gut microbiota and persistently elevated LBP levels. Our results should be regarded as hypothesis generating, pointing to a potential gut-lung axis that should be further investigated in relation to long-term pulmonary dysfunction and long COVID.

Anti-PF4/polyanion antibodies in COVID-19 patients are associated with disease severity and pulmonary pathology.

Thromboembolic events are frequent and associated with poor outcome in severe COVID-19 disease. Anti-PF4/polyanion antibodies are related to heparin-induced thrombocytopenia (HIT) and thrombus formation, but data on these antibodies in unselected COVID-19 populations are scarce. We assessed the presence of anti-PF4/polyanion antibodies in prospectively collected serum from an unselected cohort of hospitalized COVID-19 patients and evaluated if elevated levels could give prognostic information on ICU admission and respiratory failure (RF), were associated with markers of inflammation, endothelial activation, platelet activation, coagulation and fibrosis and were associated with long-term pulmonary CT changes. Five out of 65 patients had anti-PF4/polyanion reactivity with OD ≥0.200. These patients had more severe disease as reflected by ICU admission without any evidence of HIT. They also had signs of enhanced inflammation and fibrinogenesis as reflected by elevated ferritin and osteopontin, respectively, during the first 10 days of hospitalization. Increased ferritin and osteopontin persisted in these patients at 3 months follow-up, concomitant with pulmonary CT pathology. Our finding shows that the presence of anti-PF4/polyanion antibodies in unselected hospitalized COVID-19 patients was not related to HIT, but was associated with disease severity, inflammation, and pulmonary pathology after 3 months.

Low Cellular NAD+ Compromises Lipopolysaccharide-Induced Inflammatory Responses via Inhibiting TLR4 Signal Transduction in Human Monocytes.

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-1ß 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, IKKß, 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.

NLRP3 Inflammasome: A Novel Player in Metabolically Induced Inflammation-Potential Influence on the Myocardium.

Metabolic and immune systems are among the most fundamental requirements for survival. Many metabolic and immune response pathways or nutrient- and pathogen-sensing systems are evolutionarily conserved throughout species. As a result, the immune response and metabolic regulation are highly integrated and the proper function of each is dependent on the other. This interaction between metabolic disturbances and the immune system has been most extensively studied in disorders related to obesity such as insulin resistance, type 2 diabetes, and nonalcoholic fatty liver disease. Metabolically induced inflammation seems also to play a role in the development and progression of atherosclerosis including its complications such as myocardial infarction (MI) and post-MI remodeling. There are several lines of evidence suggesting that NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is a sensor of metabolic stress linking metabolic disturbances to inflammation. Here, we will discuss the role of the NLRP3 inflammasome in the pathogenesis of obesity and diabetes, 2 important risk factors for atherosclerosis and MI. We will also discuss the role of NLRP3 inflammasome in the interaction between metabolic disturbances and myocardial inflammation during MI and during metabolically induced myocardial remodeling.

NLRP3 inflammasome mediates oxidative stress-induced pancreatic islet dysfunction.

Inflammasomes are multiprotein inflammatory platforms that induce caspase-1 activation and subsequently interleukin (IL)-1ß and IL-18 processing. The NLRP3 inflammasome is activated by different forms of oxidative stress, and, based on the central role of IL-1ß in the destruction of pancreatic islets, it could be related to the development of diabetes. We therefore investigated responses in wild-type C57Bl/6 (WT) mice, NLRP3-/- mice, and mice deficient in apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) after exposing islets to short-term hypoxia or alloxan-induced islet damage. NLRP3-deficient islets compared with WT islets had preserved function ex vivo and were protected against hypoxia-induced cell death. Furthermore, NLRP3 and ASC-deficient mice were protected against oxidative stress-induced diabetes caused by repetitive low-dose alloxan administration, and this was associated with reduced ß-cell death and reduced macrophage infiltration. This suggests that the beneficial effect of NLRP3 inflammasome deficiency on oxidative stress-mediated ß-cell damage could involve reduced macrophage infiltration and activation. To support the role of macrophage activation in alloxan-induced diabetes, we injected WT mice with liposomal clodronate, which causes macrophage depletion before induction of a diabetic phenotype by alloxan treatment, resulting in improved glucose homeostasis in WT mice. We show here that the NLRP3 inflammasome acts as a mediator of hypoxia and oxidative stress in insulin-producing cells, suggesting that inhibition of the NLRP3 inflammasome could have beneficial effects on ß-cell preservation.

Leukocyte Overexpression of Intracellular NAMPT Attenuates Atherosclerosis by Regulating PPARγ-Dependent Monocyte Differentiation and Function.

OBJECTIVE: Extracellular nicotinamide phosphoribosyltransferase (eNAMPT) mediates inflammatory and potentially proatherogenic effects, whereas the role of intracellular NAMPT (iNAMPT), the rate limiting enzyme in the salvage pathway of nicotinamide adenine dinucleotide (NAD)+ generation, in atherogenesis is largely unknown. Here we investigated the effects of iNAMPT overexpression in leukocytes on inflammation and atherosclerosis. APPROACH AND RESULTS: Low-density lipoprotein receptor-deficient mice with hematopoietic overexpression of human iNAMPT (iNAMPThi), on a western type diet, showed attenuated plaque burden with features of lesion stabilization. This anti-atherogenic effect was caused by improved resistance of macrophages to apoptosis by attenuated chemokine (C-C motif) receptor 2-dependent monocyte chemotaxis and by skewing macrophage polarization toward an anti-inflammatory M2 phenotype. The iNAMPThi phenotype was almost fully reversed by treatment with the NAMPT inhibitor FK866, indicating that iNAMPT catalytic activity is instrumental in the atheroprotection. Importantly, iNAMPT overexpression did not induce any increase in eNAMPT, and eNAMPT had no effect on chemokine (C-C motif) receptor 2 expression and promoted an inflammatory M1 phenotype in macrophages. The iNAMPT-mediated effects at least partly involved sirtuin 1-dependent molecular crosstalk of NAMPT and peroxisome proliferator-activated receptor γ. Finally, iNAMPT and peroxisome proliferator-activated receptor γ showed a strong correlation in human atherosclerotic, but not healthy arteries, hinting to a relevance of iNAMPT/peroxisome proliferator-activated receptor γ pathway also in human carotid atherosclerosis. CONCLUSIONS: This study highlights the functional dichotomy of intracellular versus extracellular NAMPT, and unveils a critical role for the iNAMPT-peroxisome proliferator-activated receptor γ axis in atherosclerosis.

Palmitate promotes inflammatory responses and cellular senescence in cardiac fibroblasts.

Palmitate triggers inflammatory responses in several cell types, but its effects on cardiac fibroblasts are at present unknown. The aims of the study were to (1) assess the potential of palmitate to promote inflammatory signaling in cardiac fibroblasts through TLR4 and the NLRP3 inflammasome and (2) characterize the cellular phenotype of cardiac fibroblasts exposed to palmitate. We examined whether palmitate induces inflammatory responses in cardiac fibroblasts from WT, NLRP3-/- and ASC-/-mice (C57BL/6 background). Exposure to palmitate caused production of TNF, IL-6 and CXCL2 via TLR4 activation. NLRP3 inflammasomes are activated in a two-step manner. Whereas palmitate did not prime the NLRP3 inflammasome, it induced activation in LPS-primed cardiac fibroblasts as indicated by IL-1ß, IL-18 production and NLRP3-ASC co-localization. Palmitate-induced NLRP3 inflammasome activation in LPS-primed cardiac fibroblasts was associated with reduced AMPK activity, mitochondrial reactive oxygen species production and mitochondrial dysfunction. The cardiac fibroblast phenotype caused by palmitate, in an LPS and NLRP3 independent manner, was characterized by decreased cellular proliferation, contractility, collagen and MMP-2 expression, as well as increased senescence-associated ß-galactosidase activity, and consistent with a state of cellular senescence. This study establishes that in vitro palmitate exposure of cardiac fibroblasts provides inflammatory responses via TLR4 and NLRP3 inflammasome activation. Palmitate also modulates cardiac fibroblast functionality, in a NLRP3 independent manner, resulting in a phenotype related to cellular senescence. These effects of palmitate could be of importance for myocardial dysfunction in obese and diabetic patients.

Toll-Like Receptor 9 Promotes Survival in SERCA2a KO Heart Failure Mice.

Aim. Inflammation is important in heart failure (HF). The role of the immune receptor toll-like receptor 9 (TLR9) in HF is not understood and not investigated in diastolic HF. We investigated the role of TLR9 in a murine diastolic HF model caused by cardiomyocyte SERCA2a excision. Methods and Results. We crossed SERCA2a KO and TLR9 KO mice to generate four mouse lines. Tamoxifen-induced cardiomyocyte SERCA2a gene excision was carried out in mice, causing diastolic HF. After 7.6 weeks, cardiac functions and dimensions were analyzed by echocardiography and heart tissues were processed. HF mice depleted of TLR9 demonstrated reduced survival compared to SERC2a KO mice, with a median life expectancy of 58 days compared to 63 days. Both HF groups displayed increased left atrium size, lung weight, fetal gene expressions, monocyte/macrophage infiltration, and fibrosis. However, there were no significant differences between the groups. Conclusion. In mice with SERCA2a KO-induced diastolic HF, the absence of TLR9 reduced median life expectancy. The cause remains elusive, as all investigated HF parameters were unaltered. Still, these findings support a salutary role of TLR9 in some subsets of HF conditions and underline the importance for future studies on the mechanisms of TLR9 in diastolic HF.

Absence of the inflammasome adaptor ASC reduces hypoxia-induced pulmonary hypertension in mice.

Pulmonary hypertension is a serious condition that can lead to premature death. The mechanisms involved are incompletely understood although a role for the immune system has been suggested. Inflammasomes are part of the innate immune system and consist of the effector caspase-1 and a receptor, where nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) is the best characterized and interacts with the adaptor protein apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC). To investigate whether ASC and NLRP3 inflammasome components are involved in hypoxia-induced pulmonary hypertension, we utilized mice deficient in ASC and NLRP3. Active caspase-1, IL-18, and IL-1ß, which are regulated by inflammasomes, were measured in lung homogenates in wild-type (WT), ASC(-/-), and NLRP3(-/-) mice, and phenotypical changes related to pulmonary hypertension and right ventricular remodeling were characterized after hypoxic exposure. Right ventricular systolic pressure (RVSP) of ASC(-/-) mice was significantly lower than in WT exposed to hypoxia (40.8 ± 1.5 mmHg vs. 55.8 ± 2.4 mmHg, P < 0.001), indicating a substantially reduced pulmonary hypertension in mice lacking ASC. Magnetic resonance imaging further supported these findings by demonstrating reduced right ventricular remodeling. RVSP of NLRP3(-/-) mice exposed to hypoxia was not significantly altered compared with WT hypoxia. Whereas hypoxia increased protein levels of caspase-1, IL-18, and IL-1ß in WT and NLRP3(-/-) mice, this response was absent in ASC(-/-) mice. Moreover, ASC(-/-) mice displayed reduced muscularization and collagen deposition around arteries. In conclusion, hypoxia-induced elevated right ventricular pressure and remodeling were attenuated in mice lacking the inflammasome adaptor protein ASC, suggesting that inflammasomes play an important role in the pathogenesis of pulmonary hypertension.

Increased expression of the homeostatic chemokines CCL19 and CCL21 in clinical and experimental Rickettsia conorii infection.

BACKGROUND: Based on their essential role in concerting immunological and inflammatory responses we hypothesized that the homeostatic chemokines CCL19 and CCL21 may play a pathogenic role in rickettsiae infection. METHODS: Serum levels of CCL19 and CCL21 in patients with R. africae and R. conorii infection were analyzed by enzyme immunoassays. Lungs from R. conorii infected mice were examined for CCL19, CCL21 and CCR7 expression by immunohistochemistry. RESULTS: We found that patients with R. africae infection (n = 15) and in particular those with R. conorii infection (n = 16) had elevated serum levels of CCL19 on admission, with a decline during follow-up. While a similar pattern was seen for CCL21 in R. africae infection, patients with R. conorii infection showed persistently increased CCL21 levels during follow-up. In experimental R. conorii infection, we found strong immunostaining of CCL19 and CCL21 in the lungs, particularly in individuals that had received lethal doses. Immunofluorescence showed co-localization of CCR7 to endothelial cells, macrophages and fibroblasts within the lung tissue of R. conorii infected mice. CONCLUSIONS: Our findings suggest that the CCL19/CCL21/CCR7 axis is up-regulated during R. africae and in particular during R. conorii infection, which may potentially contribute to the pathogenesis of these disorders.

Extracellular matrix remodelling pathway in peripheral blood mononuclear cells from severe COVID-19 patients: an explorative study.

There is a reciprocal relationship between extracellular matrix (ECM) remodelling and inflammation that could be operating in the progression of severe COVID-19. To explore the immune-driven ECM remodelling in COVID-19, we in this explorative study analysed these interactions in hospitalised COVID-19 patients. RNA sequencing and flow analysis were performed on peripheral blood mononuclear cells. Inflammatory mediators in plasma were measured by ELISA and MSD, and clinical information from hospitalised COVID-19 patients (N=15) at admission was included in the analysis. Further, we reanalysed two publicly available datasets: (1) lung tissue RNA-sequencing dataset (N=5) and (2) proteomics dataset from PBCM. ECM remodelling pathways were enriched in PBMC from COVID-19 patients compared to healthy controls. Patients treated at the intensive care unit (ICU) expressed distinct ECM remodelling gene profiles compared to patients in the hospital ward. Several markers were strongly correlated to immune cell subsets, and the dysregulation in the ICU patients was positively associated with plasma levels of inflammatory cytokines and negatively associated with B-cell activating factors. Finally, our analysis of publicly accessible datasets revealed (i) an augmented ECM remodelling signature in inflamed lung tissue compared to non-inflamed tissue and (ii) proteomics analysis of PBMC from severe COVID-19 patients demonstrated an up-regulation in an ECM remodelling pathway. Our results may suggest the presence of an interaction between ECM remodelling, inflammation, and immune cells, potentially initiating or perpetuating pulmonary pathology in severe COVID-19.

Breakthrough infections with the omicron and delta variants of SARS-CoV-2 result in similar re-activation of vaccine-induced immunity.

Background: Results showing that sera from double vaccinated individuals have minimal neutralizing activity against Omicron have been interpreted as indicating the need for a third vaccine dose for protection. However, there is little information about early immune responses to Omicron infection in double vaccinated individuals. Methods: We measured inflammatory mediators, antibodies to the SARS-CoV-2 spike and nucleocapsid proteins, and spike peptide-induced release of interferon gamma in whole blood in 51 double-vaccinated individuals infected with Omicron, in 14 infected with Delta, and in 18 healthy controls. The median time points for the first and second samples were 7 and 14 days after symptom onset, respectively. Findings: Infection with Omicron or Delta led to a rapid and similar increase in antibodies to the receptor-binding domain (RBD) of Omicron protein and spike peptide-induced interferon gamma in whole blood. Both the Omicron- and the Delta-infected patients had a mild and transient increase in inflammatory parameters. Interpretation: The results suggest that two vaccine doses are sufficient to mount a rapid and potent immune response upon infection in healthy individuals of with the Omicron variant. Funding: The study was funded by the Oslo University Hospital, and by grants from The Coalition for Epidemic Preparedness Innovations, Research Council of Norway (no 312780, 324272), South-Eastern Norway Regional Health Authority (no 2019067, 2021071, 10357, 2021047, 33612, 2021087, 2017092), EU Horizon 2020 grant no 848099, a philantropic donation from Vivaldi Invest A/S, and The European Virus Archive Global.

Nicotinamide phosphoribosyltransferase and lipid accumulation in macrophages.

BACKGROUND: Accumulation of modified low-density lipoprotein (LDL) in macrophages is a key event in all stages of atherogenesis. We have previously shown increased level of nicotinamide phosphoribosyltransferase (nampt) in symptomatic, compared to asymptomatic, atherosclerotic carotid lesions, primarily located to macrophages. Here we sought to investigate the regulation and effect of intracellular nampt in macrophages. MATERIALS AND METHODS: The expression of nampt was assessed by real-time PCR in THP-1 cells that was exposed to different pro-atherogenic stimuli. The effect of nampt on lipid accumulation was examined in phorbol myristate acetate-differentiated THP-1 and in primary monocyte-derived macrophages. RESULTS: Our main findings were: (1) Nampt is increased during macrophage differentiation. (2) Tumor necrosis factor (TNF)α, oxidized (ox)LDL, and hypoxia increased nampt expression in macrophages, with further enhancing effect when these stimuli were combined. The effect of hypoxia seems to involve hypoxia inducing factor 1α. (3) Silencing of nampt increased lipid accumulation in macrophages as shown by increased protein levels of the lipid droplet marker adipose differentiation-related protein (ADRP). (4) A specific inhibitor of nampt enzymatic activity, increased ADRP and cholesterol levels in oxLDL stimulated macrophages, and enhanced the binding of acetylated LDL in these cells. (5). Nampt inhibition decreased the relative amount of cholesterol efflux in oxLDL-exposed macrophages. CONCLUSIONS: Our data suggest that the regulation of nampt in macrophages is linked to pro-atherogenic stimuli, potentially mediating counteracting effects on lipid accumulation and foam cell formation. These findings suggest a rather complex role of nampt in atherogenesis, potentially mediating both adaptive and maladaptive responses.

NLRP3 inflammasome deficiency attenuates metabolic disturbances involving alterations in the gut microbial profile in mice exposed to high fat diet.

Obesity-related diseases (e.g. type 2 diabetes mellitus and cardiovascular disorders) represent an increasing health problem worldwide. NLRP3 inflammasome activation may underlie obesity-induced inflammation and insulin resistance, and NLRP3 deficient mice exposed to high fat diet (HFD) appear to be protected from left ventricle (LV) concentric remodeling. Herein, we investigated if these beneficial effects were associated with alterations in plasma metabolites, using metabolomic and lipidomic analysis, and gut microbiota composition, using 16S rRNA sequencing of cecum content, comparing NLRP3 deficient and wild type (WT) mice on HFD and control diet. Obese NLRP3 deficient mice had lower systemic ceramide levels, potentially resulting attenuating inflammation, altered hepatic expression of fatty acids (FA) with lower mono-saturated FA and higher polyunsaturated FA levels, potentially counteracting development of liver steatosis, downregulated myocardial energy metabolism as assessed by proteomic analyses of LV heart tissue, and different levels of bile acids as compared with WT mice. These changes were accompanied by an altered composition of gut microbiota associated with decreased systemic levels of tri-methylamine-N-oxide and lipopolysaccharide, potentially inducing attenuating systemic inflammation and beneficial effects on lipid metabolism. Our findings support a role of NLRP3 inflammasome in the interface between metabolic and inflammatory stress, involving an altered gut microbiota composition.

YKL-40 (Chitinase-3-Like Protein 1) Serum Levels in Aortic Stenosis.

BACKGROUND: Identification of novel biomarkers could provide prognostic information and improve risk stratification in patients with aortic stenosis (AS). YKL-40 (chitinase-3-like protein 1), a protein involved in atherogenesis, is upregulated in human calcific aortic valves. We hypothesized that circulating YKL-40 would be elevated and associated with the degree of AS severity and outcome in patients with symptomatic AS. METHODS: Plasma YKL-40 was analyzed in 2 AS populations, one severe AS (n=572) with outcome measures and one with mixed severity (n=67). YKL-40 expression in calcified valves and in an experimental pressure overload model was assessed. RESULTS: We found (1) patients with AS had upregulated circulating YKL-40 compared with healthy controls (median 109 versus 34 ng/mL, P<0.001), but levels were not related to the degree of AS severity. (2) High YKL-40 levels (quartile 4) were associated with long-term (median follow-up 4.7 years) all-cause mortality (adjusted hazard ratio, 1.93 [95% CI, 1.37-2.73], P<0.001). (3) YKL-40 protein expression in human calcific valves co-localized with its putative receptor IL-13rα2 in close proximity to valve interstitial cells. (4) Myocardial YKL-40 increased in experimental pressure overload (6-fold in decompensated versus sham mice). CONCLUSIONS: YKL-40 levels were elevated in AS and associated with mortality but not with other metrics of disease severity including the degree of AS severity. Despite scientific rationale for its role in AS, the clinical utility of circulating YKL-40 as a biomarker is limited. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT01794832.

Effects of intronic mutations in the LDLR gene on pre-mRNA splicing: Comparison of wet-lab and bioinformatics analyses.

Screening for mutations in the low density lipoprotein receptor (LDLR) gene has identified more than 1000 mutations as the cause of familial hypercholesterolemia (FH). In addition, numerous intronic mutations with uncertain effects on pre-mRNA splicing have also been identified. In this study, we have selected 18 intronic mutations in the LDLR gene for comprehensive studies of their effects on pre-mRNA splicing. Epstein-Barr virus (EBV) transformed lymphocytes from subjects heterozygous for these mutations were established and mRNA was studied by Northern blot analyses and reverse transcription polymerase chain reactions. Furthermore, functional studies of the LDLRs were performed by flow cytometry. The results of the wet-lab analyses were compared to the predictions obtained from bioinformatics analyses using the programs MaxEntScan, NetGene2 and NNSplice 0.9, which are commonly used software packages for prediction of abnormal splice sites. Thirteen of the 18 intronic mutations were found to affect pre-mRNA splicing in a biologically relevant way as determined by wet-lab analyses. Skipping of one or two exons was observed for eight of the mutations, intron inclusion was observed for four of the mutations and activation of a cryptic splice site was observed for two of the mutations. Transcripts from eight of the mutant alleles were subjected to degradation. The computational analyses of the normal and mutant splice sites, predicted abnormal splicing with a sensitivity of 100% and a specificity of 60%. Thus, bioinformatics analyses are valuable tools as a first screening of the effects of intronic mutations in the LDLR gene on pre-mRNA splicing.

Nonsense-mediated decay of human LDL receptor mRNA.

OBJECTIVE: The objective of this project was to determine whether nonsense mutation in the low density lipoprotein receptor (LDLR) induces nonsense-mediated mRNA decay (NMD). MATERIAL AND METHODS: Four known nonsense mutations (W23X, S78X, E207X and W541X) in the LDLR gene, which are found in Norwegian familial hypercholesterolaemia (FH) patients, were investigated. Epstein-Barr virus (EBV) transformed lymphocytes from patients heterozygous for these mutations in the LDLR gene were analysed. Flow cytometric analysis was used to determine the amount and function of the cell surface LDLRs. The expression of LDLR mRNA in lymphocytes was quantified by real-time polymerase chain reaction (PCR). The presence of NMD was tested using the inhibitors gentamicin, emetine or cycloheximide. RESULTS: Cells from heterozygous FH patients with nonsense mutations in the LDLR gene contained significantly less LDLR protein (p<0.05). In addition, flow cytometric analysis revealed that these patients had a reduced LDL-uptake compared to controls (p<0.005). Cells from heterozygous FH patients with nonsense mutations W23X, S78X or W541X in the LDLR gene showed significantly decreased levels of LDLR mRNA (p<0.005). LDLR mRNA was reduced in the mutant lymphocyte S78X prior to treatment with pharmacological inhibitors, and after treatment the level of LDLR mRNA increased to the same level as that of normal cells. CONCLUSION: In the present study, NMD was confirmed in the LDLR gene. Translation inhibitors showed reduced NMD caused by nonsense mutated LDLR transcripts. Knowledge of NMD might have an important impact in clinical medicine as genetic intervention develops.

The effect of bafilomycin A1 and protease inhibitors on the degradation and recycling of a Class 5-mutant LDLR.

The low-density lipoprotein receptor (LDLR) mediates cholesterol homeostasis through endocytosis of lipoprotein particles, particularly low-density lipoproteins (LDLs). Normally, the lipoprotein particles are released in the endosomes and the receptors recycle to the cell surface. Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in the gene encoding the LDLR. These mutations are divided into five functional classes where Class 5 mutations encode receptors that suffer from ligand-induced degradation and recycling deficiency. The aim of this study was to investigate whether it is possible to prevent the fast ligand-induced degradation of Class 5-mutant LDLR and to restore its ability to recycle to the cell surface. E387K is a naturally occurring Class 5 mutation found in FH patients, and in the present study, we used Chinese hamster ovary cells transfected with an E387K-mutant LDLR. Abrogation of endosomal acidification by adding bafilomycin A1 or addition of the irreversible serine protease inhibitors, 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) and 3,4-dichloroisocoumarin (DCI), prevented the degradation of the E387K-mutant LDLR. However, the undegraded receptor did not recycle to the cell surface in the presence of LDL. Unexpectedly, AEBSF caused aggregation of early endosome antigen-1- positive endosomes and the intracellular trapped LDLR co-localized with these aggregated early endosomes.

Neuromodulatory Effect of NLRP3 and ASC in Neonatal Hypoxic Ischemic Encephalopathy.

BACKGROUND: Following birth asphyxia there is a robust inflammatory response. NLRP3 is a receptor of the innate immune system. Upon activation, NLRP3 forms an inflammasome together with ASC and procaspase-1 to mediate release of IL-1ß and IL-18. NLRP3 has previously been shown to be upregulated following neonatal hypoxic-ischemic (HI) brain injury in mice, but with no early effect on brain injury. OBJECTIVE: We aimed to evaluate if deficiency of NLRP3 or ASC protects against neonatal HI brain damage 7 days after hypoxia-ischemia. METHODS: C57BL/6J, NLRP3-/-, and ASC-/- mice were subjected to unilateral common carotid artery ligation followed by hypoxia at P9. Brain infarction, apoptosis, and microglial response were evaluated, as well as total RNA sequencing and examination of plasma levels of systemic proinflammatory cytokines. RESULTS: NLRP3-/- mice showed significantly increased brain infarction volumes compared to wild-type (Wt) mice, while ASC-/- mice showed reduced brain infarction volumes after neonatal hypoxia-ischemia. The amount of activated microglia was increased in NLRP3-/- mice, while decreased in ASC-/- mice compared to Wt mice. Total RNA sequencing showed an impaired inflammatory transcriptional response in the hippocampus of NLRP3-/- mice. Plasma levels of IL-1ß and IL-18 were not affected, but TNF was lower in NLRP3-/- and ASC-/- mice compared to Wt mice. CONCLUSION: ASC deficiency is neuroprotective in neonatal HI brain damage in mice, while NLRP3 deficiency increases brain damage.