Differentially activated B cells develop regulatory phenotype and show varying immunosuppressive features: a comparative study

Regulatory B lymphocytes (Bregs) are B cells with well-pronounced immunosuppressive properties, allowing them to suppress the activity of effector cells. A broad repertoire of immunosuppressive mechanisms makes Bregs an attractive tool for adoptive cell therapy for diseases associated with excessive activation of immune reactions. Such therapy implies Breg extraction from the patient’s peripheral blood, ex vivo activation and expansion, and further infusion into the patient. At the same time, the utility of Bregs for therapeutic approaches is limited by their small numbers and extremely low survival rate, which is typical for all primary B cell cultures. Therefore, extracting CD19+ cells from the patient’s peripheral blood and specifically activating them ex vivo to make B cells acquire a suppressive phenotype seems to be far more productive. It will allow a much larger number of B cells to be obtained initially, which may significantly increase the likelihood of successful immunosuppression after adoptive Breg transfer. This comparative study focuses on finding ways to efficiently manipulate B cells in vitro to differentiate them into Bregs. We used CD40L, CpG, IL4, IL21, PMA, and ionomycin in various combinations to generate immunosuppressive phenotype in B cells and performed functional assays to test their regulatory capacity. This work shows that treatment of primary B cells using CD40L + CpG + IL21 mix was most effective in terms of induction of functionally active regulatory B lymphocytes with high immunosuppressive capacity ex vivo

Glucocorticoid Receptor Binding Inhibits an Intronic <i>IL33</i> Enhancer and is Disrupted by rs4742170 (T) Allele Associated with Specific Wheezing Phenotype in Early Childhood

Interleukin 33 (IL-33) is a cytokine constitutively expressed by various cells of barrier tissues that contribute to the development of inflammatory immune responses. According to its function as an alarmin secreted by lung and airway epithelium, IL-33 plays a significant role in pathogenesis of allergic disorders. IL-33 is strongly involved in the pathogenesis of asthma, anaphylaxis, allergy and dermatitis, and genetic variations in IL33 locus are associated with increased susceptibility to asthma. Genome-wide association studies have identified risk &#8220;T&#8222; allele of the single-nucleotide polymorphism rs4742170 located in putative IL33 enhancer area as susceptible variant for development of specific wheezing phenotype in early childhood. Here, we demonstrate that risk &#8220;T&#8222; rs4742170 allele disrupts binding of glucocorticoid receptor (GR) transcription factor to IL33 putative enhancer. The IL33 promoter/enhancer constructs containing either 4742170 (T) allele or point mutations in the GR-binding site, were significantly more active and did not respond to cortisol in a pulmonary epithelial cell line. At the same time, the constructs containing rs4742170 (C) allele with a functional GR-binding site were less active and further inhibitable by cortisol. The latter effect was GR-dependent as it was completely abolished by GR-specific siRNA. This mechanism may explain the negative effect of the rs4742170 (T) risk allele on the development of wheezing phenotype that strongly correlates with allergic sensitization in childhood

Association between Alcohol Consumption and Body Composition in Russian Adults and Patients Treated for Alcohol-Related Disorders: The Know Your Heart Cross-Sectional Study

There is conflicting evidence about the association between alcohol consumption and body composition (BC). We aimed to investigate this association in Russian adults. The study population included 2357 residents of Arkhangelsk aged 35–69 years, and 272 in-patients treated for alcohol problems (narcological patients) who participated in the Know Your Heart (KYH) cross-sectional study in 2015–2017. The participants were divided into five subgroups based on their alcohol use characteristics: non-drinkers, non-problem drinkers, hazardous drinkers, harmful drinkers, and narcological patients. Considering men, hazardous drinkers had a larger waist circumference (WC), waist-to-hip ratio (WHR), and percentage of body fat mass (%FM) compared to non-problem drinkers. In harmful drinking men, these differences were the opposite: a lower body mass index (BMI), hip circumference (HC), and %FM. Men among narcological patients had the lowest mean BMI, WC, HC, WHR, and %FM compared to other subgroups of men. As for women, non-drinkers had a lower BMI, WC, HC, and %FM compared to non-problem drinkers. Women among narcological patients had the lowest mean BMI and HC but an increased WHR compared to other subgroups of women. In conclusion, alcohol consumption levels had an inverted J-shaped association with adiposity-related BC parameters: they were elevated in hazardous drinkers but were reduced in harmful drinkers, and were even lower in patients with alcohol-related diagnoses

The Risk G Allele of the Single-Nucleotide Polymorphism rs928413 Creates a CREB1-Binding Site That Activates IL33 Promoter in Lung Epithelial Cells

Cytokine interleukin 33 (IL-33) is constitutively expressed by epithelial barrier cells, and promotes the development of humoral immune responses. Along with other proinflammatory mediators released by the epithelium of airways and lungs, it plays an important role in a number of respiratory pathologies. In particular, IL-33 significantly contributes to pathogenesis of allergy and asthma; genetic variations in the IL33 locus are associated with increased susceptibility to asthma. Large-scale genome-wide association studies have identified minor &ldquo;G&rdquo; allele of the single-nucleotide polymorphism rs928413, located in the IL33 promoter area, as a susceptible variant for early childhood and atopic asthma development. Here, we demonstrate that the rs928413(G) allele creates a binding site for the cAMP response element-binding protein 1 (CREB1) transcription factor. In a pulmonary epithelial cell line, activation of CREB1, presumably via the p38 mitogen-activated protein kinases (MAPK) cascade, activates the IL33 promoter containing the rs928413(G) allele specifically and in a CREB1-dependent manner. This mechanism may explain the negative effect of the rs928413 minor &ldquo;G&rdquo; allele on asthma development

Western blot analysis of CLU isoform.

<p>(A) Electrophoresis of stromal proteins from spleen and mesenteric lymph nodes (MLN) was performed in reducing and non-reducing conditions. Immunopositive bands mobility corresponds to secreted CLU isoform (sCLU). (B) Quantitative comparison of splenic sCLU expression in wild type (WT) and LTβR-deficient (LTβR-KO) mice. Data is normalized to the average WT expression and represented as mean±SD. * – Difference from the wild type is significant at <i>p</i><0.05.</p

Changes in sCLU protein level in spleen and MLN of WT mice at day 8 after immunization with SRBC.

<p>Western blot of total tissue homogenates shows an increase in sCLU amount in spleen but not MLN. Data is representative of 2 independent experiments.</p

Relative mRNA levels of known and potential LTβR target genes in various mouse tissues.

<p>Real-time PCR data on mRNA levels in various tissues from wild type, TNFR1-KO, and LTβR-KO mice of selected genes down-regulated in LTβR-KO splenic stroma. Data was normalized to GAPDH, which expression level was taken as 100%. Note high expression of clusterin in wild type spleen and its dramatic reduction in spleen upon LTβR knockout. Data is represented as mean±SD. * – Difference from the wild type is significant at <i>p</i><0.05.</p

Interfollicular channel region of mouse mesenteric lymph node.

<p>MLN was stained with anti-clusterin and ER-TR7 antibodies after immunization with SRBC. Note clear immunopositivity of conduits (arrows) for clusterin. Lower row represents the close up of the indicated square region. Data is representative of at least 2 experiments. Scale bar: 100 µm.</p

Cellular and tissue distribution of clusterin.

<p>Immunohistochemical staining of wild type spleen (A) and MLN (B) after immunization with SRBC. (C) Shows an image of clusterin-positive spleen stromal cell under high power (original magnification 630×), showing that clusterin is located near cell membrane. (D) Double staining for MRC marker ER-TR7 (green) and clusterin (red). Ubiquitous presence of clusterin-positive cells can be seen in all stromal compartments except for splenic marginal zone (D), with the brightest staining seen in germinal centers. Scale bar: 100 µm. GC – germinal center, CA – central arteriole, RP – red pulp, F – follicle, arrows – high endothelial venules, MZ – marginal zone. Data is representative of at least 2 experiments.</p