Sex-specific association of visceral and subcutaneous adipose tissue volumes with systemic inflammation and innate immune cells in people living with obesity.

BACKGROUND AND AIMS: Obesity predisposes to metabolic and cardiovascular diseases. Adipose tissue inflammation and systemic inflammation contribute to these complications. There are strong sex differences in adipose tissue distribution and in systemic inflammation. Women have more subcutaneous adipose tissue (SAT) and less visceral adipose tissue (VAT) than men. We explored the sex differences in the association between the different adipose compartments and inflammatory markers that are important in cardiometabolic disease pathophysiology. METHODS: Single-center observational cohort study with 302 individuals with a BMI ≥ 27 kg/m2. We were unable to acquire MRI data from seven individuals and from another 18 the MRI data were not usable, resulting in 277 people (155 men, 122 women), aged 55-81 years. INTERVENTION: We performed the following measurements: abdominal magnetic resonance imaging to measure VAT, and SAT (deep and superficial) volumes; circulating leukocyte counts and cytokine production capacity of peripheral blood mononuclear cells (PBMCs), circulating cytokines, adipokines, and targeted proteomics; abdominal sSAT biopsies for histology and gene expression. RESULTS: Only in women, (s)SAT volume was associated with circulating leukocytes, monocytes, and neutrophils. Circulating IL-6 and IL-18BP were associated with SAT volume in women and VAT in men. Several circulating proteins, including monocyte-colony-stimulating factor 1 and hepatocyte growth factor, are associated with sSAT in women and VAT in men. Only in women, SAT volume is associated with SAT expression of inflammatory proteins, including leptin, CD68, TNFα and IL-1α. CONCLUSION: In women living with obesity, abdominal SAT volume, especially sSAT, is associated with circulating leukocytes and inflammatory proteins. In men, these parameters mainly show associations with VAT volume. This could be because only in women, sSAT volume is associated with sSAT expression of inflammatory proteins. These findings underscore that future research on adipose tissue in relation to cardiometabolic and cardiovascular disease should take sex differences into account.

A functional genomics approach reveals suggestive quantitative trait loci associated with combined TLR4 and BCP crystal-induced inflammation and osteoarthritis.

OBJECTIVE: Basic calcium phosphate (BCP) crystals can activate the NLRP3 inflammasome and are potentially involved in the pathogenesis of osteoarthritis (OA). In order to elucidate relevant inflammatory mechanisms in OA, we used a functional genomics approach to assess genetic variation influencing BCP crystal-induced cytokine production. METHOD: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers who were previously genotyped and stimulated with BCP crystals and/or lipopolysaccharide (LPS) after which cytokines release was assessed. Cytokine quantitative trait locus (cQTL) mapping was performed. For in vitro validation of the cQTL located in anoctamin 3 (ANO3), PBMCs were incubated with Tamoxifen and Benzbromarone prior to stimulation. Additionally, we performed co-localisation analysis of our top cQTLs with the most recent OA meta-analysis of genome-wide association studies (GWAS). RESULTS: We observed that BCP crystals and LPS synergistically induce IL-1ß in human PBMCs. cQTL analysis revealed several suggestive loci influencing cytokine release upon stimulation, among which are quantitative trait locus annotated to ANO3 and GLIS3. As functional validation, anoctamin inhibitors reduced IL-1ß release in PBMCs after stimulation. Co-localisation analysis showed that the GLIS3 locus was shared between LPS/BCP crystal-induced IL-1ß and genetic association with Knee OA. CONCLUSIONS: We identified and functionally validated a new locus, ANO3, associated with LPS/BCP crystal-induced inflammation in PBMCs. Moreover, the cQTL in the GLIS3 locus co-localises with the previously found locus associated with Knee OA, suggesting that this Knee OA locus might be explained through an inflammatory mechanism. These results form a basis for further exploration of inflammatory mechanisms in OA.

Superficial vs Deep Subcutaneous Adipose Tissue: Sex-Specific Associations With Hepatic Steatosis and Metabolic Traits.

CONTEXT: Subcutaneous adipose tissue (SAT) is not homogeneous, as the fascia scarpa separates the deep SAT (dSAT) from the superficial SAT (sSAT). OBJECTIVE: The aim of this study is to evaluate the sex-specific associations of sSAT and dSAT with hepatic steatosis and metabolic syndrome in overweight individuals. METHODS: We recruited 285 individuals with a body mass index (BMI) greater than or equal to 27 and aged 55 to 81 years. Abdominal magnetic resonance imaging was performed around level L4 to L5 to measure visceral adipose tissue (VAT), dSAT, and sSAT volumes. The amount of hepatic fat was quantified by MR spectroscopy. RESULTS: Men had significantly higher volumes of VAT (122.6 cm3 vs 98.7 cm3, P < .001) and had only half the volume of sSAT compared to women adjusted for BMI (50.3 cm3 in men vs 97.0 cm3 in women, P < .001). dSAT correlated significantly with hepatic fat content in univariate analysis (standardized ß = .190, P < .05), while VAT correlated significantly with hepatic steatosis in a multivariate model, adjusted for age, alcohol use, and other abdominal fat compartments (standardized ß = .184, P = .037). Moreover, dSAT in men correlated negatively with HDL cholesterol (standardized ß = -0.165, P = .038) in multivariate analyses. In women with a BMI between 30 and 40, in a multivariate model adjusted for age, alcohol use, and other abdominal fat compartments, VAT correlated positively (standardized ß = -.404, P = .003), and sSAT negatively (standardized ß = -.300, P = .04) with hepatic fat content. CONCLUSION: In men, dSAT is associated with hepatic steatosis and adverse metabolic traits, such as lower HDL cholesterol levels, whereas in women with obesity sSAT shows a beneficial relation with respect to hepatic fat content.

The anti-inflammatory cytokine interleukin-37 is an inhibitor of trained immunity.

Trained immunity (TI) is a de facto innate immune memory program induced in monocytes/macrophages by exposure to pathogens or vaccines, which evolved as protection against infections. TI is characterized by immunometabolic changes and histone post-translational modifications, which enhance production of pro-inflammatory cytokines. As aberrant activation of TI is implicated in inflammatory diseases, tight regulation is critical; however, the mechanisms responsible for this modulation remain elusive. Interleukin-37 (IL-37) is an anti-inflammatory cytokine that curbs inflammation and modulates metabolic pathways. In this study, we show that administration of recombinant IL-37 abrogates the protective effects of TI in vivo, as revealed by reduced host pro-inflammatory responses and survival to disseminated candidiasis. Mechanistically, IL-37 reverses the immunometabolic changes and histone post-translational modifications characteristic of TI in monocytes, thus suppressing cytokine production in response to infection. IL-37 thereby emerges as an inhibitor of TI and as a potential therapeutic target in immune-mediated pathologies.

An integrative model of cardiometabolic traits identifies two types of metabolic syndrome.

Human diseases arise in a complex ecosystem composed of disease mechanisms and the whole-body state. However, the precise nature of the whole-body state and its relations with disease remain obscure. Here we map similarities among clinical parameters in normal physiological settings, including a large collection of metabolic, hemodynamic, and immune parameters, and then use the mapping to dissect phenotypic states. We find that the whole-body state is faithfully represented by a quantitative two-dimensional model. One component of the whole-body state represents 'metabolic syndrome' (MetS) - a conventional way to determine the cardiometabolic state. The second component is decoupled from the classical MetS, suggesting a novel 'non-classical MetS' that is characterized by dozens of parameters, including dysregulated lipoprotein parameters (e.g. low free cholesterol in small high-density lipoproteins) and attenuated cytokine responses of immune cells to ex vivo stimulations. Both components are associated with disease, but differ in their particular associations, thus opening new avenues for improved personalized diagnosis and treatment. These results provide a practical paradigm to describe whole-body states and to dissect complex disease within the ecosystem of the human body.

Reduced concentrations of the B cell cytokine interleukin 38 are associated with cardiovascular disease risk in overweight subjects.

The IL-1 family member IL-38 (IL1F10) suppresses inflammatory and autoimmune conditions. Here, we report that plasma concentrations of IL-38 in 288 healthy Europeans correlate positively with circulating memory B cells and plasmablasts. IL-38 correlated negatively with age (p = 0.02) and was stable in 48 subjects for 1 year. In comparison with primary keratinocytes, IL1F10 expression in CD19+ B cells from PBMC was lower, whereas cell-associated IL-38 expression was comparable. In vitro, IL-38 is released from CD19+ B cells after stimulation with rituximab. Intravenous LPS in humans failed to induce circulating IL-38, compared to 100-fold induction of IL-6 and IL-1 receptor antagonist. In a cohort of 296 subjects with body mass index > 27 at high risk for cardiovascular disease, IL-38 plasma concentrations were significantly lower than in healthy subjects (p < 0.0001), and lowest in those with metabolic syndrome (p < 0.05). IL-38 also correlated inversely with high sensitivity C-reactive protein (p < 0.01), IL-6, IL-1Ra, and leptin (p < 0.05). We conclude that a relative deficiency of the B cell product IL-38 is associated with increased systemic inflammation in aging, cardiovascular and metabolic disease, and is consistent with IL-38 as an anti-inflammatory cytokine.

Dapansutrile, an oral selective NLRP3 inflammasome inhibitor, for treatment of gout flares: an open-label, dose-adaptive, proof-of-concept, phase 2a trial.

BACKGROUND: Gout flares are driven by interleukin (IL)-1ß. Dapansutrile inhibits the NLRP3 inflammasome and subsequent activation of IL-1ß. In this study we aimed to investigate the safety and efficacy of orally administered dapansutrile in patients with a gout flare. METHODS: In this open-label, proof-of-concept, phase 2a trial, adult patients (aged 18-80 years) with a monoarticular monosodium urate crystal-proven gout flare were enrolled at an outpatient clinic in the Netherlands and sequentially assigned using a dose-adaptive design to receive 100 mg/day, 300 mg/day, 1000 mg/day, or 2000 mg/day oral dapansutrile for 8 days. The coprimary outcomes were change in patient-reported target joint pain from baseline to day 3 and from baseline to day 7, assessed in the per-protocol population (all patients who received at least 80% of the study drug and had no major protocol deviations). Safety was assessed in the intention-to-treat population. This trial is registered with the EU Clinical Trials Register, EudraCT 2016-000943-14, and is completed. FINDINGS: Between May 18, 2017, and Jan 21, 2019, 144 patients were assessed for eligibility, of whom 34 were enrolled and 29 were included in the per-protocol population (three patients were excluded due to receiving <80% of study drug and two had major protocol deviations): eight patients received 100 mg/day, seven received 300 mg/day, six received 1000 mg/day, and eight received 2000 mg/day. Between baseline and day 3, there was a mean reduction in patient-reported target joint pain of 52·4% (SD 32·94; p=0∙016) for the 100 mg/day group, 68·4% (34·29; p=0∙016) for the 300 mg/day group, 55·8% (44·90; p=0∙063) for the 1000 mg/day group, and 57·6% (38·72; p=0∙016) for the 2000 mg/day group. At day 7, there was a mean reduction of 82·1% (22·68; p=0∙031) for the 100 mg/day group, 84·2% (16·33; p=0∙016) for the 300 mg/day group, 68·9% (34·89; p=0∙031) for the 1000 mg/day group, and 83·9% (15·44; p=0∙008) for the 2000 mg/day group, compared to baseline. 25 (73·5%) of 34 patients reported a total of 45 treatment-emergent adverse events, most of which were metabolism and nutrition disorders (17 [37·8%]) and gastrointestinal disorders (ten [22·2%]). Two serious adverse events occurred during the study, admission to hospital because of worsening of gout flare at day 3, and admission to hospital because of coronary stenosis 18 days after the patient received their last dose; these were considered moderate in severity and unrelated to the study drug. INTERPRETATION: Dapansutrile is a specific NLRP3 inflammasome inhibitor with a satisfactory safety profile and efficacy in the reduction of target joint pain in this study. Future studies are needed to confirm the clinical potential of dapansutrile.

Genetic and Microbial Associations to Plasma and Fecal Bile Acids in Obesity Relate to Plasma Lipids and Liver Fat Content.

Bile acids (BAs) are implicated in the etiology of obesity-related conditions such as non-alcoholic fatty liver disease. Differently structured BA species display variable signaling activities via farnesoid X receptor (FXR) and Takeda G protein-coupled BA receptor 1 (TGR5). This study profiles plasma and fecal BAs and plasma 7α-hydroxy-4-cholesten-3-one (C4) in 297 persons with obesity, identifies underlying genetic and microbial determinants, and establishes BA correlations with liver fat and plasma lipid parameters. We identify 27 genetic associations (p < 5 × 10-8) and 439 microbial correlations (FDR < 0.05) for 50 BA entities. Additionally, we report 111 correlations between BA and 88 lipid parameters (FDR < 0.05), mainly for C4 reflecting hepatic BA synthesis. Inter-individual variability in the plasma BA profile does not reflect hepatic BA synthetic pathways, but rather transport and metabolism within the enterohepatic circulation. Our study reveals genetic and microbial determinants of BAs in obesity and their relationship to disease-relevant lipid parameters that are important for the design of personalized therapies targeting BA-signaling pathways.

Vasculometabolic and Inflammatory Effects of Aldosterone in Obesity.

CONTEXT: Not all obese individuals develop cardiovascular disease (CVD). Hyperaldosteronism is suggested to cause inflammation and metabolic dysregulation, and might contribute to CVD development in obese individuals. OBJECTIVE: We aimed to investigate the association of aldosterone concentrations with inflammation, metabolic disturbances, and atherosclerosis in overweight and obese individuals. Additionally, we measured renin concentrations to investigate whether the observed effects reflected general activation of the renin-angiotensin-aldosterone system (RAAS). DESIGN: A cross-sectional cohort study (300-OB study) was conducted. Various inflammatory parameters, traits of the metabolic syndrome, lipidome and metabolome parameters, fat distribution, and carotid atherosclerosis were associated with plasma aldosterone and renin levels. SETTING: The setting of this study was the Radboudumc (i.o. Radboudumc), the Netherlands. PATIENTS: A total of 302 individuals with a body mass index greater than or equal to 27 kg/m2 participated. MAIN OUTCOME MEASURES AND RESULTS: Aldosterone was associated with various markers of inflammation and metabolic dysregulation, which partly differed from the associations observed for renin. Although both were associated with inflammatory cell numbers, only renin was associated with classical markers of systemic inflammation. Both were associated with the metabolic syndrome and hepatic steatosis. Of the traits that constitute metabolic syndrome, aldosterone, but not renin, was associated with triglyceride concentrations. Accordingly, aldosterone was associated with large very low-density lipoprotein particles; metabolomics studies further associated aldosterone with urate concentrations and derivatives of the linoleic acid metabolism pathway. Neither aldosterone nor renin was associated with atherosclerotic plaque thickness. CONCLUSIONS: Aldosterone is not an important driver of systemic inflammation in the obese, whereas aldosterone concentrations and metabolic dysregulation are strongly intertwined in these individuals. Although prospective studies are necessary to validate these results, the independent effects of aldosterone on carotid atherosclerosis appear modest.

Sex-Specific Regulation of Inflammation and Metabolic Syndrome in Obesity.

OBJECTIVE: Metabolic dysregulation and inflammation are important consequences of obesity and impact susceptibility to cardiovascular disease. Anti-inflammatory therapy in cardiovascular disease is being developed under the assumption that inflammatory pathways are identical in women and men, but it is not known if this is indeed the case. In this study, we assessed the sex-specific relation between inflammation and metabolic dysregulation in obesity. Approach and Results: Three hundred two individuals were included, half with a BMI 27 to 30 kg/m2 and half with a BMI>30 kg/m2, 45% were women. The presence of metabolic syndrome was assessed according to the National Cholesterol Education Program-ATPIII criteria, and inflammation was studied using circulating markers of inflammation, cell counts, and ex vivo cytokine production capacity of isolated immune cells. Additionally, lipidomic and metabolomic data were gathered, and subcutaneous fat biopsies were histologically assessed. Metabolic syndrome is associated with an increased inflammatory profile that profoundly differs between women and men: women with metabolic syndrome show a lower concentration of the anti-inflammatory adiponectin, whereas men show increased levels of several pro-inflammatory markers such as IL (interleukin)-6 and leptin. Adipose tissue inflammation showed similar sex-specific associations with these markers. Peripheral blood mononuclear cells isolated from men, but not women, with metabolic syndrome display enhanced cytokine production capacity. CONCLUSIONS: We identified sex-specific pathways that influence inflammation in obesity. Excessive production of proinflammatory cytokines was observed in men with metabolic syndrome. In contrast, women typically showed reduced levels of the anti-inflammatory adipokine adiponectin. These different mechanisms of inflammatory dysregulation between women and men with obesity argue for sex-specific therapeutic strategies.

Genetic variation in Interleukin-32 influence the immune response against New World Leishmania species and susceptibility to American Tegumentary Leishmaniasis.

Interleukin-32 is a novel inflammatory mediator that has been described to be important in the immunopathogenesis and control of infections caused by Leishmania parasites. By performing experiments with primary human cells in vitro, we demonstrate that the expression of IL-32 isoforms is dependent on the time exposed to L. amazonensis and L. braziliensis antigens. Moreover, for the first time we show the functional consequences of three different genetic variations in the IL32 (rs4786370, rs4349147, rs1555001) modulating IL-32γ expression, influencing innate and adaptive cytokine production after Leishmania exposure. Using a Brazilian cohort of 107 American Tegumentary Leishmaniasis patients and a control cohort of 245 healthy individuals, the IL32 rs4786370 genetic variant was associated with protection against ATL, whereas the IL32 rs4349147 was associated with susceptibility to the development of localized cutaneous and mucosal leishmaniasis. These novel insights may help improve therapeutic strategies and lead to benefits for patients suffering from Leishmania infections.

Increased proteinase 3 and neutrophil elastase plasma concentrations are associated with non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes.

INTRODUCTION: Non-alcoholic fatty liver disease (NAFLD) is becoming a major health problem worldwide. Inflammation plays an important role in disease pathogenesis and recent studies have shown a potential role for the neutrophil serine proteases (NSPs) proteinase-3 (PR3) and neutrophil elastase (NE) in NAFLD as well as an imbalance between NSPs and their natural inhibitor alpha-1 antitrypsin (AAT). The aim of this study was to investigate whether PR3 and NE plasma concentrations are associated with NAFLD and/or type 2 diabetes. METHODS: To explore this hypothesis we used several cohorts: a cohort of 271 obese individuals with liver steatosis, a cohort of 41 patients with biopsy-proven NAFLD, a cohort of 401 obese type 2 diabetes patients and a cohort of 205 lean healthy controls; and measured PR3 and NE plasma concentrations. In addition, we measured AAT plasma concentrations in order to investigate if the ratios between NSPs and their natural inhibitor were altered in NAFLD and type 2 diabetes when compared to healthy controls. RESULTS: Our data shows an increase in PR3 and NE concentrations and a decrease in AAT concentrations in obese patients when compared to controls. Moreover, PR3 plasma concentrations are increased in patients with liver steatosis. Furthermore, PR3 and NE concentrations in the liver are associated with the advanced stages of NAFLD characterized by NASH and/ or liver fibrosis. Additionally, PR3 and NE concentrations were up-regulated in patients with type 2 diabetes when compared to lean and obese controls. CONCLUSION: We conclude that circulating levels of NSPs associate with obesity-related metabolic disorders. Further research is needed to clearly establish the role of these proteases and investigate whether they could be used as non-invasive markers for NAFLD and/or type 2 diabetes.

Gut Microbial Associations to Plasma Metabolites Linked to Cardiovascular Phenotypes and Risk.

RATIONALE: Altered gut microbial composition has been linked to cardiovascular diseases (CVDs), but its functional links to host metabolism and immunity in relation to CVD development remain unclear. OBJECTIVES: To systematically assess functional links between the microbiome and the plasma metabolome, cardiometabolic phenotypes, and CVD risk and to identify diet-microbe-metabolism-immune interactions in well-documented cohorts. METHODS AND RESULTS: We assessed metagenomics-based microbial associations between 231 plasma metabolites and microbial species and pathways in the population-based LLD (Lifelines DEEP) cohort (n=978) and a clinical obesity cohort (n=297). After correcting for age, sex, and body mass index, the gut microbiome could explain ≤11.1% and 16.4% of the variation in plasma metabolites in the population-based and obesity cohorts, respectively. Obese-specific microbial associations were found for lipid compositions in the VLDL, IDL, and LDL lipoprotein subclasses. Bacterial L-methionine biosynthesis and a Ruminococcus species were associated to cardiovascular phenotypes in obese individuals, namely atherosclerosis and liver fat content, respectively. Integration of microbiome-diet-inflammation analysis in relation to metabolic risk score of CVD in the population cohort revealed 48 microbial pathways associated to CVD risk that were largely independent of diet and inflammation. Our data also showed that plasma levels rather than fecal levels of short-chain fatty acids were relevant to inflammation and CVD risk. CONCLUSIONS: This study presents the largest metagenome-based association study on plasma metabolism and microbiome relevance to diet, inflammation, CVD risk, and cardiometabolic phenotypes in both population-based and clinical obesity cohorts. Our findings identified novel bacterial species and pathways that associated to specific lipoprotein subclasses and revealed functional links between the gut microbiome and host health that provide a basis for developing microbiome-targeted therapy for disease prevention and treatment.

Predictive value of ex-vivo drug-inhibited cytokine production for clinical response to biologic DMARD therapy in rheumatoid arthritis.

OBJECTIVES: To investigate ex-vivo drug-inhibited cytokine production before the start of a biological DMARD (bDMARD) as predictor of treatment response in rheumatoid arthritis (RA). METHODS: In a prospective RA cohort study [BIO-TOP], blood samples were obtained from patients before the start of a bDMARD (abatacept, adalimumab, etanercept, rituximab or tocilizumab). Peripheral blood mononuclear cells were pre-incubated for 1 hour with the therapeutic in-vivo concentration of the bDMARD and stimulated for 24 hours with heat-killed Candida albicans or Pam3Cys. Concentrations of IL-1ß, IL-6, TNFα, IL-17 and IFNγ were determined by ELISA. EULAR response (good vs. moderate/no) was assessed at month 6. Area under the receiver operating characteristic curves (AUCs) were generated to evaluate the predictive value of baseline characteristics and ex-vivo cytokine production (including stimulated cytokine concentrations and absolute changes after inhibition by a bDMARD). Logistic prediction models were created to assess the added value of potential cytokine predictors. RESULTS: 277 RA patients were included with 330 blood samples. Good response was reached in 39% of the cases. DAS28-CRP was predictive for response to adalimumab (AUC 0.70, 95%CI 0.57-0.83), etanercept (AUC 0.68, 95%CI 0.58-0.78) and rituximab (AUC 0.76, 95%CI 0.65-0.86). ACPA was modestly predictive for response to abatacept (AUC 0.63, 95%CI 0.52-0.75). In the ex-vivo analysis, 4 of 64 (6%) tests showed some predictive value but these had no added value to clinical factors routinely measured in RA, such as DAS28-CRP. CONCLUSIONS: Ex-vivo inhibition of cytokine production by bDMARDs is unable to help prediction of treatment response to bDMARDs in RA.

Effects of oral butyrate supplementation on inflammatory potential of circulating peripheral blood mononuclear cells in healthy and obese males.

Sodium butyrate is well-known for its immune-modulatory properties. Studies until now only focused on the in vitro effects of butyrate or assessed local effects in the gut upon butyrate administration. In this trial, we studied the systemic anti-inflammatory effects induced by sodium butyrate supplementation in humans. Nine healthy (Lean) and ten obese (metabolic syndrome group, MetSyn) males were given 4 grams sodium butyrate daily for 4 weeks. PBMCs were isolated before and after supplementation for direct stimulation experiments and induction of trained immunity by oxidized low-density lipoprotein (oxLDL), ß-glucan, or Bacillus Calmette-Guérin vaccine (BCG). Butyrate supplementation moderately affected some of the cytokine responses in the MetSyn group. In the direct stimulation setup, effects of butyrate supplementation were limited. Interestingly, butyrate supplementation decreased oxLDL-induced trained immunity in the MetSyn group for LPS-induced IL-6 responses and Pam3CSK4-induced TNF-α responses. Induction of trained immunity by ß-glucan was decreased by butyrate in the MetSyn group for Pam3CSK4-induced IL-10 production. In this study, while having only limited effects on the direct stimulation of cytokine production, butyrate supplementation significantly affected trained immunity in monocytes of obese individuals with metabolic complications. Therefore, oral butyrate supplementation may be beneficial in reducing the overall inflammatory status of circulating monocytes in patients with metabolic syndrome.

Genetic variant in IL-32 is associated with the ex vivo cytokine production of anti-TNF treated PBMCs from rheumatoid arthritis patients.

About 60% of RA patients don't achieve good response with biological disease-modifying anti-rheumatic drugs bDMARD treatment (including TNF inhibitors, TNFi's). Previously, a link between TNFα and interleukin (IL)-32 was reported in RA. However, the exact mechanism linking IL-32 to response to treatment as not been studied yet. Therefore, we explored the influence of a promoter single nucleotide polymorphism (SNP) rs4786370 in IL-32 on clinical responsiveness to TNFi's in RA patients, potentially serving as new biomarker in RA. Expression of pro-inflammatory cytokines by peripheral mononuclear cells (PBMCs) from RA patients and healthy individuals were studied. Moreover, "ex vivo response" and clinical response to anti-TNFα therapy (etanercept, adalimumab) were measured and stratified for the IL-32 SNP. Higher IL-32 protein production was observed in RA patients. Additionally, patients bearing the CC genotype showed higher IL-32 protein and cytokine expression. DAS28 was independent of the promoter SNP, however, the "ex vivo" cytokine response was not. IL-32 mRNA and protein production was higher in RA patients, with a trend towards higher concentrations in patients bearing the CC genotype. Furthermore, genotype dependent IL-1 beta production might predict clinical response to etanercept/adalimumab. This indicates that IL-32 could play a role in predicting response to treatment in RA.

Ex vivo inhibited cytokine profiling may explain inferior treatment response to golimumab after adalimumab failure in rheumatoid arthritis.

OBJECTIVES: Clinical data suggest that the response of rheumatoid arthritis patients to treatment with golimumab is much lower among those who switched from adalimumab than among those who switched from etanercept. To elucidate the mechanism behind this difference in response to sequential biologic treatment, we examined the effect of TNF inhibitors on ex vivo cytokine production profiling. METHODS: In a prospective cohort study, blood samples were obtained from patients before the start of a biologic. Peripheral blood mononuclear cells were pre-incubated for 1 hour with the therapeutic in vivo concentration of adalimumab, etanercept or golimumab and stimulated for 24 hours with heat killed Candida albicans or Pam3Cys. Cytokine concentrations of IL-1ß, IL-6 and TNFα were determined by ELISA. RESULTS: Ex vivo cytokine profiling was performed in 71 patients. Golimumab, adalimumab and etanercept significantly (p<0.01) decreased Candida albicans-induced IL-1ß and IL-6 production and Pam3Cys-induced IL-6 production. In contrast to etanercept, golimumab and adalimumab decreased the concentration of TNFα below the detection limit. Absolute changes in cytokine levels after inhibition by golimumab or adalimumab were all significantly correlated (Spearman rank rs: 0.52-0.99, p<0.001). These correlations were much lower or non-significant between etanercept and either golimumab or adalimumab. CONCLUSIONS: High similarity between ex vivo inhibited cytokine profiling by golimumab and adalimumab, compared to etanercept, may explain the previously found inferior treatment response to golimumab after adalimumab failure. This suggests that patients who are non-responsive to adalimumab should preferably not switch to golimumab and vice versa.

Genome-wide identification of genes essential for the survival of Streptococcus pneumoniae in human saliva.

Since Streptococcus pneumoniae transmits through droplet spread, this respiratory tract pathogen may be able to survive in saliva. Here, we show that saliva supports survival of clinically relevant S. pneumoniae strains for more than 24 h in a capsule-independent manner. Moreover, saliva induced growth of S. pneumoniae in growth-permissive conditions, suggesting that S. pneumoniae is well adapted for uptake of nutrients from this bodily fluid. By using Tn-seq, a method for genome-wide negative selection screening, we identified 147 genes potentially required for growth and survival of S. pneumoniae in saliva, among which genes predicted to be involved in cell envelope biosynthesis, cell transport, amino acid metabolism, and stress response predominated. The Tn-seq findings were validated by testing a panel of directed gene deletion mutants for their ability to survive in saliva under two testing conditions: at room temperature without CO2, representing transmission, and at 37 °C with CO2, representing in-host carriage. These validation experiments confirmed that the plsX gene and the amiACDEF and aroDEBC operons, involved in respectively fatty acid metabolism, oligopeptide transport, and biosynthesis of aromatic amino acids play an important role in the growth and survival of S. pneumoniae in saliva at 37 °C. In conclusion, this study shows that S. pneumoniae is well-adapted for growth and survival in human saliva and provides a genome-wide list of genes potentially involved in adaptation. This notion supports earlier evidence that S. pneumoniae can use human saliva as a vector for transmission.