Hema Kothari

The adaptive immune response plays an important role in atherosclerosis. In response to a high-fat/high-cholesterol (HF/HC) diet, marginal zone B (MZB) cells activate an atheroprotective programme by regulating the differentiation and accumulation of ‘poorly differentiated’ T follicular helper (Tfh) cells. On the other hand, Tfh cells activate the germinal centre response, which promotes atherosclerosis through the production of class-switched high-affinity antibodies. We therefore investigated the direct role of Tfh cells and the role of IL18 in Tfh differentiation in atherosclerosis. We generated atherosclerotic mouse models with selective genetic deletion of Tfh cells, MZB cells, or IL18 signalling in Tfh cells. Surprisingly, mice lacking Tfh cells had increased atherosclerosis. Lack of Tfh not only reduced class-switched IgG antibodies against oxidation-specific epitopes (OSEs) but also reduced atheroprotective natural IgM-type anti-phosphorylcholine (PC) antibodies, despite no alteration of natural B1 cells. Moreover, the absence of Tfh cells was associated with an accumulation of MZB cells with substantially reduced ability to secrete antibodies. In the same manner, MZB cell deficiency in Ldlr^−/− mice was associated with a significant decrease in atheroprotective IgM antibodies, including natural anti-PC IgM antibodies. In humans, we found a positive correlation between circulating MZB-like cells and anti-OSE IgM antibodies. Finally, we identified an important role for IL18 signalling in HF/HC diet–induced Tfh. Our findings reveal a previously unsuspected role of MZB cells in regulating atheroprotective ‘natural’ IgM antibody production in a Tfh-dependent manner, which could have important pathophysiological and therapeutic implications.

IL-1β is a key mediator of the cytokine storm linked to high morbidity and mortality from COVID-19, and IL-1β blockade with anakinra and canakinumab during COVID-19 infection has entered clinical trials. Using mass cytometry of human peripheral blood mononuclear cells, we identified effector memory CD4⁺ T cells and CD4⁻CD8^low/−CD161⁺ T cells, specifically those positive for the chemokine receptor CCR6, as the circulating immune subtypes with the greatest response to IL-1β. This response manifested as increased phosphorylation and, thus, activation of the proinflammatory transcription factor NF-κB and was also seen in other subsets, including CD11c⁺ myeloid dendritic cells, classical monocytes, two subsets of natural killer cells (CD16⁻CD56^brightCD161⁻ and CD16⁻CD56^dimCD161⁺), and lineage⁻ (Lin⁻) cells expressing CD161 and CD25. IL-1β also induced a rapid but less robust increase in the phosphorylation of the kinase p38 as compared to that of NF-κB in most of these immune cell subsets. Prolonged IL-1β stimulation increased the phosphorylation of the transcription factor STAT3 and to a lesser extent that of STAT1 and STAT5 across various immune cell types. IL-1β–induced production of IL-6 likely led to the activation of STAT1 and STAT3 at later time points. Interindividual heterogeneity and inhibition of STAT activation by anakinra raise the possibility that assays measuring NF-κB phosphorylation in response to IL-1β in CCR6⁺ T cell subtypes could identify those patients at higher risk of cytokine storm and most likely to benefit from IL-1β–neutralizing therapies.

IL-1β has emerged as a key mediator of the cytokine storm linked to high morbidity and mortality from COVID-19 and blockade of the IL-1 receptor (IL-1R) with Anakinra has entered clinical trials in COVID-19 subjects. Yet, knowledge of the specific immune cell subsets targeted by IL-1β and IL-1β-induced signaling pathways in humans is limited. Utilizing mass cytometry (CyTOF) of human peripheral blood mononuclear cells, we identified effector memory CD4 T cells and CD4 ⁻ CD8 ^low/- CD161 ⁺ T cells as the circulating immune subtypes with the greatest expression of p-NF-κB in response to IL-1β stimulation. Notably, CCR6 distinctly identified T cells most responsive to IL-1β. Other subsets including CD11c myeloid dendritic cells (mDCs), classical monocytes (CM), two subsets of natural killer cells (CD16 ⁻ CD56 ^bright CD161 ⁻ and CD16 ⁻ CD56 ^dim CD161 ⁺ ) and a population of lineage ⁻ (Lin-) cells expressing CD161 and CD25 also showed IL-1β-induced expression of p-NF-kB. The IL-1R antagonist, Anakinra significantly inhibited IL-1β-induced p-NF-kB in the CCR6 ⁺ T cells and CD11c mDCs with a trending inhibition in CD14 monocytes and Lin ⁻ CD161 ⁺ CD25 ⁺ cells. IL-1β also induced a rapid but much less robust increase in p-p38 expression as compared to p-NF-kB in the majority of these same immune cell subsets. Prolonged IL-1β stimulation greatly increased p-STAT3 and to a much lesser extent p-STAT1 and p-STAT5 in T cell subsets, monocytes, DCs and the Lin ⁻ CD161 ⁺ CD25 ⁺ cells suggesting IL-1β-induced production of downstream STAT-activating cytokines, consistent with its role in cytokine storm. Interindividual heterogeneity and inhibition of this activation by Anakinra raises the intriguing possibility that assays to measure IL-1β-induced p-NF-kB in CCR6 ⁺ T cell subtypes could identify those at higher risk of cytokine storm and those most likely to benefit from Anakinra therapy.

In light of recent data regarding inflammatory signalling pathways in cardiovascular disease and the recently demonstrated impact of pharmacologic inhibition of interleukin-1β (IL-1β) in heart failure, the primary aim was to assess the physiologic effects of cardiac resynchronization therapy (CRT) on the expression of systemic inflammatory, immune-modulatory, metabolic, and apoptotic genes in peripheral blood mononuclear cells (PBMCs) of patients with heart failure. We used RNA sequencing (RNA-Seq) and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to identify gene expression changes in PBMCs in response to CRT. In total, 27 patients were analysed: 12 with heart failure undergoing CRT, 6 with heart failure undergoing standard implanted cardioverter defibrillators, and 9 with coronary artery disease but not heart failure. In CRT patients (median age 65.5 years, interquartile range 63.0–66.8 years, 33% female), RNA-Seq analysis identified 40 genes, including multiple genes associated with the IL-1β pathway, with significant correlations (false discovery rate < 0.05) with four key CRT response measures. CRT was associated with suppression of PBMC expression of IL-1β (1.80-fold decrease, P = 0.047), FOS proto-oncogene (FOS) (3.25-fold decrease, P = 0.01), dual specificity phosphatase 1 (DUSP1) (2.05-fold decrease, P = 0.001), and early growth response 1 (EGR1) (7.38-fold decrease, P = 0.03), and suppression was greater in responders vs. non-responders (P = 0.03 for IL-1β, P = 0.02 for FOS, P = 0.02 for DUSP1, and P = 0.11 for EGR1). Baseline FOS and DUSP-1 levels were greater in responders vs. non-responders (6.15-fold higher, FOS, P = 0.002; 2.60-fold higher, DUSP1, P = 0.0001). CRT responders but not non-responders showed higher baseline gene expression of FOS (P = 0.04) and DUSP1 (P = 0.06) compared with control patients without heart failure. Baseline serum high-sensitivity C-reactive protein levels were 3.47-fold higher in CRT responders vs. non-responders (P = 0.008). Treatment of heart failure with CRT resulted in decreased PBMC expression of genes linked to inflammation. Moreover, CRT responders had higher expression of these inflammatory genes prior to CRT and greater suppression of these genes after CRT compared with non-responders.

D-dimer has emerged as a biomarker of cardiovascular event risk, yet pathophysiological factors associated with plasma D-dimer levels in stable coronary artery disease (CAD) subjects are poorly understood. In 106 stable CAD subjects undergoing intravascular ultrasound with virtual histology (IVUS-VH), we measured D-dimer, lipoprotein(a) (Lp(a)), plasminogen, biomarkers reflecting oxidation-specific epitopes (OSE) such as oxidized phospholipids on apolipoprotein B-100 (OxPL-apoB), OxPL on plasminogen (OxPL-PLG), and autoantibodies to phosphorylcholine-BSA [PC-BSA] and a malondialdehyde [MDA] mimotope. In univariate analysis, D-dimer was positively associated with Lp(a), OxPL-apoB, OxPL-PLG, and coronary artery calcium, and inversely associated with autoantibodies to OSE and plaque fibrosis. D-dimer levels > 500 ng/ml also showed positive association with plaque necrosis. After multivariate analysis, D-dimer remained significantly associated with Lp(a) and plaque calcium. While further studies are needed, results provide evidence that plasma D-dimer levels are associated with levels of OxPLs and IVUS-VH indices linked to plaque erosion and rupture.