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The subset of pro-inflammatory B cells, called late memory, tissue-like or double negative (DN), accumulates in the blood of elderly individuals. Here we show that DN B cells do not proliferate and do not make antibodies to influenza antigens, but they secrete antibodies with autoimmune reactivity, in agreement with their membrane phenotype (CD95+CD21-CD11c+) and their spontaneous expression of the transcription factor T-bet. These cells also increase in the blood of individuals with obesity and autoimmune diseases, but causative mechanisms and signaling pathways involved are known only in part. In the present paper we compare frequencies and metabolic requirements of these cells in the blood of healthy individuals of different ages and in the blood and the subcutaneous adipose tissue (SAT) of individuals with obesity. Results show that DN B cells from young individuals have minimal metabolic requirements, DN B cells from elderly and obese individuals utilize higher amounts of glucose to perform autoimmune antibody production and enroll in aerobic glycolysis to support their function. DN B cells from the SAT have the highest metabolic requirements as they activate oxidative phosphorylation, aerobic glycolysis and fatty acid oxidation. DN B cells from the SAT also show the highest levels of ROS and the highest levels of phosphorylated AMPK (5'-AMP activated kinase) and Sestrin 1, both able to mitigate stress and cell death. This metabolic advantage drives DN B cell survival and function (secretion of autoimmune antibodies).

SARS-CoV-2 (Severe Acute Respiratory Syndrome Corona Virus-2), cause of COVID-19 (Coronavirus Disease of 2019), represents a significant risk to people living with pre-existing conditions associated with exacerbated inflammatory responses and consequent dysfunctional immunity. In this paper, we have evaluated the influence of obesity, a condition associated with chronic systemic inflammation, on the secretion of SARS-CoV-2-specific IgG antibodies in the blood of COVID-19 patients. Our hypothesis is that obesity is associated with reduced amounts of specific IgG antibodies. Results have confirmed our hypothesis and have shown that SARS-CoV-2 IgG antibodies are negatively associated with Body Mass Index (BMI) in COVID-19 obese patients, as expected based on the known influence of obesity on humoral immunity. Antibodies in COVID-19 obese patients are also negatively associated with serum levels of pro-inflammatory and metabolic markers of inflammaging and pulmonary inflammation, such as SAA (serum amyloid A protein), CRP (C-reactive protein), and ferritin, but positively associated with NEFA (nonesterified fatty acids). These results altogether could help to identify an inflammatory signature with strong predictive value for immune dysfunction. Inflammatory markers identified may subsequently be targeted to improve humoral immunity in individuals with obesity and in individuals with other chronic inflammatory conditions.

The adipose tissue (AT) contributes to systemic and B cell intrinsic inflammation, reduced B cell responses and secretion of autoimmune antibodies. In this study we show that adipocytes in the human obese subcutaneous AT (SAT) secrete several pro-inflammatory cytokines and chemokines, which contribute to the establishment and maintenance of local and systemic inflammation, and consequent suboptimal immune responses in obese individuals, as we have previously shown. We also show that pro-inflammatory chemokines recruit immune cells expressing the corresponding receptors to the SAT, where they also contribute to local and systemic inflammation, secreting additional pro-inflammatory mediators. Moreover, we show that the SAT generates autoimmune antibodies. During the development of obesity, reduced oxygen and consequent hypoxia and cell death lead to further release of pro-inflammatory cytokines, \"self\" protein antigens, cell-free DNA and lipids. All these stimulate class switch and the production of autoimmune IgG antibodies which have been described to be pathogenic. In addition to hypoxia, we have measured cell cytotoxicity and DNA damage mechanisms, which may also contribute to the release of \"self\" antigens in the SAT. All these processes are significantly elevated in the SAT as compared to the blood. We definitively found that fat-specific IgG antibodies are secreted by B cells in the SAT and that B cells express mRNA for the transcription factor T-bet and the membrane marker CD11c, both involved in the production of autoimmune IgG antibodies. Finally, the SAT also expresses RNA for cytokines known to promote Germinal Center formation, isotype class switch, and plasma cell differentiation. Our results show novel mechanisms for the generation of autoimmune antibody responses in the human SAT and allow the identification of new pathways to possibly manipulate in order to reduce systemic inflammation and autoantibody production in obese individuals.

We have previously shown that obesity is associated with increased secretion of IgG antibodies with anti-self-reactivity. In this paper, we confirm and extend our previous findings. We show that the plasma of individuals with obesity is enriched in autoimmune antibodies whose levels are positively associated with blood frequencies of the subset of Double Negative (DN) B cells, which is the most pro-inflammatory B cell subset. We also show that DN B cells, significantly increased in the blood of obese versus lean individuals, are characterized by higher expression of immune activation markers and of the transcription factor T-bet, both associated with autoimmunity. The removal of DN B cells from the peripheral B cell pool significantly decreases in vitro secretion of anti-self IgG antibodies. These results altogether confirm the crucial role of DN B cells in the secretion of anti-self IgG antibodies in individuals with obesity.

Background We have previously shown that obesity accelerates age-associated defects in B cell function and antibody production leading to decreased secretion of protective antibodies and increased autoimmunity. We wanted to evaluate if obese adults enrolled in a voluntary weight reduction program had higher protective and lower autoimmune antibody responses similar to those observed in lean adults. Methods Experiments were performed using blood isolated from an established cohort of female lean adult and elderly individuals, as well as from the blood of female adults with obesity, before and after a voluntary weight reduction program in which their Body Mass Index (BMI) was reduced 10–34% in 12 months. All participants were vaccinated with the Trivalent Inactivated Influenza vaccine. Serum samples were evaluated for the presence of pro-inflammatory cytokines and adipokines, vaccine-specific antibodies and autoimmune antibodies. We evaluated the composition of the B cell pool by flow cytometry, the expression of RNA for class switch transcription factors and pro-inflammatory markers by qPCR, the in vitro secretion of pro- and anti-inflammatory cytokines and their capacity to induce pro-inflammatory T cells. Results Obesity, similar to aging, induced increased serum levels of pro-inflammatory cytokines and autoimmune antibodies, while vaccine-specific antibodies were reduced. In agreement with the serum results, the B cell pool of obese adults and elderly individuals was enriched in pro-inflammatory B cell subsets and was characterized by higher expression of markers associated with cell senescence, higher levels of T-bet, the transcription factor for autoimmune antibodies and lower levels of E47, the transcription factor associated with protective responses to the influenza vaccine. B cells from obese adults and elderly individuals were also able to secrete inflammatory cytokines and support the generation of inflammatory T cells. All these pro-inflammatory characteristics of B cells from obese individuals were significantly attenuated, but not completely reversed, by weight loss. Conclusions Although the results from our small observational study show that obesity-induced dysfunctional B cell responses, similar to those occurring during aging, are ameliorated in some but not all obese individuals after weight loss, the effects of body weight loss on mechanistic pathways are largely missing and deserve further investigation.

In this study, we have compared frequencies, phenotype, function and metabolic requirements of B cells isolated from the breast and abdominal subcutaneous adipose tissue (AT) of women with obesity who underwent weight reduction surgeries. Results show that B cells from the abdominal AT are more inflammatory than those from the breast, characterized by higher frequencies of inflammatory B cell subsets and higher expression of RNA for inflammatory markers associated with senescence. Secretion of autoimmune antibodies is also higher in the abdominal AT as compared to the breast, and is associated with higher frequencies of autoimmune B cells with the membrane phenotype CD21lowCD95+ B cells expressing the transcription factor T-bet. Moreover, glucose uptake is higher in B cells from the abdominal AT as compared to the breast, thereby suggesting a better capacity to perform glycolysis, needed to support intrinsic B cell inflammation and autoimmune antibody secretion.

Standardization of immunophenotyping requires careful attention to reagents, sample handling, instrument setup, and data analysis, and is essential for successful cross-study and cross-center comparison of data. Experts developed five standardized, eight-color panels for identification of major immune cell subsets in peripheral blood. These were produced as pre-configured, lyophilized, reagents in 96-well plates. We present the results of a coordinated analysis of samples across nine laboratories using these panels with standardized operating procedures (SOPs). Manual gating was performed by each site and by a central site. Automated gating algorithms were developed and tested by the FlowCAP consortium. Centralized manual gating can reduce cross-center variability, and we sought to determine whether automated methods could streamline and standardize the analysis. Within-site variability was low in all experiments, but cross-site variability was lower when central analysis was performed in comparison with site-specific analysis. It was also lower for clearly defined cell subsets than those based on dim markers and for rare populations. Automated gating was able to match the performance of central manual analysis for all tested panels, exhibiting little to no bias and comparable variability. Standardized staining, data collection, and automated gating can increase power, reduce variability, and streamline analysis for immunophenotyping.

In this study, we have compared frequencies, phenotype, function and metabolic requirements of B cells isolated from the breast and abdominal subcutaneous adipose tissue (AT) of women with obesity who underwent weight reduction surgeries. Results show that B cells from the abdominal AT are more inflammatory than those from the breast, characterized by higher frequencies of inflammatory B cell subsets and higher expression of RNA for inflammatory markers associated with senescence. Secretion of autoimmune antibodies is also higher in the abdominal AT as compared to the breast, and is associated with higher frequencies of autoimmune B cells with the membrane phenotype CD21 low CD95+ B cells expressing the transcription factor T-bet. Moreover, glucose uptake is higher in B cells from the abdominal AT as compared to the breast, thereby suggesting a better capacity to perform glycolysis, needed to support intrinsic B cell inflammation and autoimmune antibody secretion.

Background Aging is associated with increased intrinsic B cell inflammation, decreased protective antibody responses and increased autoimmune antibody responses. The effects of aging on the metabolic phenotype of B cells and on the metabolic programs that lead to the secretion of protective versus autoimmune antibodies are not known. Methods Splenic B cells and the major splenic B cell subsets, Follicular (FO) and Age-associated B cells (ABCs), were isolated from the spleens of young and old mice and left unstimulated. The RNA was collected to measure the expression of markers associated with intrinsic inflammation and autoimmune antibody production by qPCR. B cells and B cell subsets were also stimulated with CpG and supernatants collected after 7 days to measure autoimmune IgG secretion by ELISA. Metabolic measures (oxygen consumption rate, extracellular acidification rate and glucose uptake) were performed using a Seahorse XFp extracellular flux analyzer. Results Results have identified the subset of ABCs, whose frequencies and numbers increase with age and represent the most pro-inflammatory B cell subset, as the cell type mainly if not exclusively responsible for the expression of inflammatory markers and for the secretion of autoimmune antibodies in the spleen of old mice. Hyper-inflammatory ABCs from old mice are also hyper-metabolic, as compared to those from young mice and to the subset of FO B cells, a feature needed not only to support their higher expression of RNA for inflammatory markers but also their higher autoimmune antibody secretion. Conclusions These results identify a relationship between intrinsic inflammation, metabolism and autoimmune B cells and suggest possible ways to understand cellular mechanisms that lead to the generation of pathogenic B cells, that are hyper-inflammatory and hyper-metabolic, and secrete IgG antibodies with autoimmune specificities.

Non-metastatic breast cancer patients often experience psychological distress which may influence disease progression and survival. Cognitive-behavioral stress management (CBSM) improves psychological adaptation and lowers distress during breast cancer treatment and long-term follow-ups. We examined whether breast cancer patients randomized to CBSM had improved survival and recurrence 8–15 years post-enrollment. From 1998 to 2005, women ( N  = 240) 2–10 weeks post-surgery for non-metastatic Stage 0–IIIb breast cancer were randomized to a 10-week, group-based CBSM intervention ( n  = 120) or a 1-day psychoeducational seminar control ( n  = 120). In 2013, 8–15 years post-study enrollment (11-year median), recurrence and survival data were collected. Cox Proportional Hazards Models and Weibull Accelerated Failure Time tests were used to assess group differences in all-cause mortality, breast cancer-specific mortality, and disease-free interval, controlling for biomedical confounders. Relative to the control, the CBSM group was found to have a reduced risk of all-cause mortality (HR = 0.21; 95 % CI [0.05, 0.93]; p  = .040). Restricting analyses to women with invasive disease revealed significant effects of CBSM on breast cancer-related mortality ( p = .006) and disease-free interval ( p  = .011). CBSM intervention delivered post-surgery may provide long-term clinical benefit for non-metastatic breast cancer patients in addition to previously established psychological benefits. Results should be interpreted with caution; however, the findings contribute to the limited evidence regarding physical benefits of psychosocial intervention post-surgery for non-metastatic breast cancer. Additional research is necessary to confirm these results and investigate potential explanatory mechanisms, including physiological pathways, health behaviors, and treatment adherence changes.