Phytosterols in human serum as measured using a liquid chromatography tandem mass spectrometry.

Phytosterols are lipophilic compounds found in plants with structural similarity to mammalian cholesterol. They cannot be endogenously produced by mammals and therefore always originate from diet. There has been increased interest in dietary phytosterols over the last few decades due to their association with a variety of beneficial health effects including low-density lipoprotein cholesterol lowering, anti-inflammatory and anti-cancerous effects. They are proposed as potential moderators for diseases associated with the central nervous system where cholesterol homeostasis is found to be imperative (multiple sclerosis, dementia, etc.) due to their ability to reach the brain. Here we utilised an enzyme-assisted derivatisation for sterol analysis (EADSA) in combination with a liquid chromatography tandem mass spectrometry (LC-MSn) to characterise phytosterol content in human serum. As little as 100 fg of plant sterol was injected on a reversed phase LC column. The method allows semi-quantitative measurements of phytosterols and their derivatives simultaneously with measurement of cholesterol metabolites. The identification of phytosterols in human serum was based on comparison of their LC retention times and MS2, MS3 spectra with a library of authentic standards. Free campesterol serum concentration was in the range from 0.30-4.10 µg/mL, ß-sitosterol 0.16-3.37 µg/mL and fucosterol was at lowest concentration range from 0.05-0.38 µg/mL in ten individuals. This analytical methodology could be applied to the analysis of other biological fluids and tissues.

Role of CD14+ monocyte-derived oxidised mitochondrial DNA in the inflammatory interferon type 1 signature in juvenile dermatomyositis.

OBJECTIVES: To define the host mechanisms contributing to the pathological interferon (IFN) type 1 signature in Juvenile dermatomyositis (JDM). METHODS: RNA-sequencing was performed on CD4+, CD8+, CD14+ and CD19+ cells sorted from pretreatment and on-treatment JDM (pretreatment n=10, on-treatment n=11) and age/sex-matched child healthy-control (CHC n=4) peripheral blood mononuclear cell (PBMC). Mitochondrial morphology and superoxide were assessed by fluorescence microscopy, cellular metabolism by 13C glucose uptake assays, and oxidised mitochondrial DNA (oxmtDNA) content by dot-blot. Healthy-control PBMC and JDM pretreatment PBMC were cultured with IFN-α, oxmtDNA, cGAS-inhibitor, TLR-9 antagonist and/or n-acetyl cysteine (NAC). IFN-stimulated gene (ISGs) expression was measured by qPCR. Total numbers of patient and controls for functional experiments, JDM n=82, total CHC n=35. RESULTS: Dysregulated mitochondrial-associated gene expression correlated with increased ISG expression in JDM CD14+ monocytes. Altered mitochondrial-associated gene expression was paralleled by altered mitochondrial biology, including 'megamitochondria', cellular metabolism and a decrease in gene expression of superoxide dismutase (SOD)1. This was associated with enhanced production of oxidised mitochondrial (oxmt)DNA. OxmtDNA induced ISG expression in healthy PBMC, which was blocked by targeting oxidative stress and intracellular nucleic acid sensing pathways. Complementary experiments showed that, under in vitro experimental conditions, targeting these pathways via the antioxidant drug NAC, TLR9 antagonist and to a lesser extent cGAS-inhibitor, suppressed ISG expression in pretreatment JDM PBMC. CONCLUSIONS: These results describe a novel pathway where altered mitochondrial biology in JDM CD14+ monocytes lead to oxmtDNA production and stimulates ISG expression. Targeting this pathway has therapeutical potential in JDM and other IFN type 1-driven autoimmune diseases.

Cytokine production by human B cells: role in health and autoimmune disease.

B cells are classically considered solely as antibody-producing cells driving humoral immune responses to foreign antigens in infections and vaccinations as well as self-antigens in pathological settings such as autoimmunity. However, it has now become clear that B cells can also secrete a vast array of cytokines, which influence both pro- and anti-inflammatory immune responses. Indeed, similarly to T cells, there is significant heterogeneity in cytokine-driven responses by B cells, ranging from the production of pro-inflammatory effector cytokines such as IL-6, through to the release of immunosuppressive cytokines such as IL-10. In this review, focusing on human B cells, we summarize the key findings that have revealed that cytokine-producing B cell subsets have critical functions in healthy immune responses and contribute to the pathophysiology of autoimmune diseases.

B cell contribution to immunometabolic dysfunction and impaired immune responses in obesity.

Obesity increases the risk of type 2 diabetes mellitus, cardiovascular disease, fatty liver disease, and cancer. It is also linked with more severe complications from infections, including COVID-19, and poor vaccine responses. Chronic, low-grade inflammation and associated immune perturbations play an important role in determining morbidity in people living with obesity. The contribution of B cells to immune dysregulation and meta-inflammation associated with obesity has been documented by studies over the past decade. With a focus on human studies, here we consolidate the observations demonstrating that there is altered B cell subset composition, differentiation, and function both systemically and in the adipose tissue of individuals living with obesity. Finally, we discuss the potential factors that drive B cell dysfunction in obesity and propose a model by which altered B cell subset composition in obesity underlies dysfunctional B cell responses to novel pathogens.

Favorable antibody responses to human coronaviruses in children and adolescents with autoimmune rheumatic diseases.

BACKGROUND: Differences in humoral immunity to coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), between children and adults remain unexplained, and the effect of underlying immune dysfunction or suppression is unknown. Here, we sought to examine the antibody immune competence of children and adolescents with prevalent inflammatory rheumatic diseases, juvenile idiopathic arthritis (JIA), juvenile dermatomyositis (JDM), and juvenile systemic lupus erythematosus (JSLE) against the seasonal human coronavirus (HCoV)-OC43 that frequently infects this age group. METHODS: Sera were collected from JIA (n = 118), JDM (n = 49), and JSLE (n = 30) patients and from healthy control (n = 54) children and adolescents prior to the coronavirus disease 19 (COVID-19) pandemic. We used sensitive flow-cytometry-based assays to determine titers of antibodies that reacted with the spike and nucleoprotein of HCoV-OC43 and cross-reacted with the spike and nucleoprotein of SARS-CoV-2, and we compared them with respective titers in sera from patients with multisystem inflammatory syndrome in children and adolescents (MIS-C). FINDINGS: Despite immune dysfunction and immunosuppressive treatment, JIA, JDM, and JSLE patients maintained comparable or stronger humoral responses than healthier peers, which was dominated by immunoglobulin G (IgG) antibodies to HCoV-OC43 spike, and harbored IgG antibodies that cross-reacted with SARS-CoV-2 spike. In contrast, responses to HCoV-OC43 and SARS-CoV-2 nucleoproteins exhibited delayed age-dependent class-switching and were not elevated in JIA, JDM, and JSLE patients, which argues against increased exposure. CONCLUSIONS: Consequently, autoimmune rheumatic diseases and their treatment were associated with a favorable ratio of spike to nucleoprotein antibodies. FUNDING: This work was supported by a Centre of Excellence Centre for Adolescent Rheumatology Versus Arthritis grant, 21593, UKRI funding reference MR/R013926/1, the Great Ormond Street Children's Charity, Cure JM Foundation, Myositis UK, Lupus UK, and the NIHR Biomedical Research Centres at GOSH and UCLH. This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK, the UK Medical Research Council, and the Wellcome Trust.

The emerging field of regulatory B cell immunometabolism.

B cells are well known as critical mediators of humoral immune responses via the production of antibodies. However, numerous studies have also identified populations of B cells that are characterized by their anti-inflammatory properties. These "regulatory B cells" restrain excessive inflammatory responses in a wide range of health conditions. A significant knowledge gap remains concerning the nature of the signals that determine whether a B cell exerts a pro-inflammatory or anti-inflammatory function. In this perspective, we explore the concept that in addition to the cytokine microenvironment, intracellular and extracellular metabolic signals play a pivotal role in controlling the balance between regulatory and antibody-producing B cell subsets. Determining the metabolites and tissue-specific signals that influence B cell fate could establish novel therapeutic targets for the treatment of diseases where abnormal B cell responses contribute to pathogenesis.

Identification and Isolation of Regulatory B Cells in Mouse and Human.

Regulatory B cells (Bregs) suppress immune response via the provision of IL-10. Due to the phenotypic heterogeneity of described Bregs, it is important to have standardized protocols for their isolation and identification. Previous work by our laboratory has shown that the immature B-cell populations in the murine spleen and human peripheral blood produce the highest levels of IL-10 on engagement of CD40, and can suppress pro-inflammatory T-cell differentiation. In this chapter, we describe the methods necessary for the isolation of this subset of Bregs and their activation via CD40 in vitro.

B regulatory cells are increased in hypercholesterolaemic mice and protect from lesion development via IL-10.

Whilst innate B1-B cells are atheroprotective, adaptive B2-B cells are considered pro-atherogenic. Different subsets of B regulatory cells (B(reg)) have been described. In experimental arthritis and lupus-like disease, B(reg) are contained within the CD21(hi)CD23(hi)CD24(hi) B cell pool. The existence and role of B(reg) in vascular disease is not known. We sought to investigate the existence, identity and location of B(reg) in vascular disease. The representation of B2-B cell subsets in the spleens and lymph nodes (LNs) of Apolipoprotein E(-/-) (ApoE(-/-)) mice compared to controls was characterised by flow cytometry. Additionally, we utilised a model of neointima formation based on the placement of a perivascular collar around the carotid artery in ApoE(-/-) mice to ascertain whether B cells and B cell subsets confer protection against lesion development. Adoptive transfer of B cells was performed from wild type or genetically modified mice. We showed that CD21(hi)CD23(hi)CD24(hi) B cells are unexpectedly increased in the draining LNs of ApoE(-/-) mice. Adoptive transfer of LN-derived B2-B cells or purified CD21(hi)CD23(hi)CD24(hi) B cells to syngeneic mice reduced lesion size and inflammation without changing serum cholesterol levels. Follicular B2-B cells did not confer protection. IL-10 blockade or transfer of IL10-deficient B cells prevented LN-derived B cell-mediated protection. This is the first identification of a specific LN-derived B2-B(reg) subset that confers IL-10 mediated protection from neointima formation. This may open the way for immune modulatory approaches in cardiovascular disease.

Regulatory B cells are induced by gut microbiota-driven interleukin-1ß and interleukin-6 production.

Regulatory B cells (Breg cells) differentiate in response to inflammation and subsequently restrain excessive immune responses via the release of interleukin-10 (IL-10). However, the precise inflammatory signals governing their differentiation remain to be elucidated. Here we show that the gut microbiota promotes the differentiation of Breg cells in the spleen as well as in the mesenteric lymph nodes. Perturbation of the gut microbiome imposed either by antibiotic treatment or by changes in the sterility of housing conditions reduces the number and function of Breg cells. Following the induction of arthritis, IL-1ß and IL-6 are produced only in conventionally housed mice and both cytokines directly promote Breg cell differentiation and IL-10 production. Mice lacking IL-6 receptor (IL-6R) or IL-1 receptor 1 (IL-1R1) specifically on B cells have a reduced number of IL-10-producing B cells and develop exacerbated arthritis compared to control animals. Thus, in response to inflammatory signals induced by both the gut flora and arthritis, Breg cells increase in number and restrain excessive inflammation.

B regulatory cells and the tumor-promoting actions of TNF-α during squamous carcinogenesis.

The inflammatory cytokine TNF-α has been recognized as a critical tumor promoter, but the effector cells that mediate its action have not been fully characterized. Because B cells regulate squamous and prostate carcinogenesis, and Tnf(-/-) mice harbor B-cell defects, we investigated the hypothesis that B cells are important effector cells for TNF-α-mediated promotion of cancer development. Using an adoptive transfer strategy and the 7,12-dimethylbenz[α]anthracene/terephthalic acid (DMBA/TPA) two-stage model of skin carcinogenesis, we found that both B cells and TNF-α are critical for the development of DMBA/TPA-induced papilloma. Transfer of B cells from DMBA/TPA-treated wild-type mice to Tnf(-/-) mice rescued papilloma development to a wild-type level, a result not observed when B cells from Tnf(-/-) mice were transferred to Rag2(-/-) mice or when TNF-α was eliminated selectively in B cells. Resistance to papilloma development in Tnf(-/-) mice was associated with increased IFN-γ and CD8(+) T cells in skin and a significant reduction in IL-10-producing B regulatory cells alongside an increase in IFN-γ-producing CD8(+) T cells in the spleen. These data indicate that during DMBA/TPA-induced squamous carcinogenesis TNF-α mediates tumor-promoting activity via regulatory B cells that repress antitumor immunity.

Mice lacking endogenous IL-10-producing regulatory B cells develop exacerbated disease and present with an increased frequency of Th1/Th17 but a decrease in regulatory T cells.

IL-10-producing B cells, also known as regulatory B cells (Bregs), play a key role in controlling autoimmunity. In this study, we report that chimeric mice specifically lacking IL-10-producing B cells (IL-10(-/-)B cell) developed an exacerbated arthritis compared with chimeric wild-type (WT) B cell mice. A significant decrease in the absolute numbers of Foxp3 regulatory T cells (Tregs), in their expression level of Foxp3, and a marked increase in inflammatory Th1 and Th17 cells were detected in IL-10(-/-) B cell mice compared with WT B cell mice. Reconstitution of arthritic B cell deficient (µMT) mice with different B cell subsets revealed that the ability to modulate Treg frequencies in vivo is exclusively restricted to transitional 2 marginal zone precursor Bregs. Moreover, transfer of WT transitional 2 marginal zone precursor Bregs to arthritic IL-10(-/-) mice increased Foxp3(+) Tregs and reduced Th1 and Th17 cell frequencies to levels measured in arthritic WT mice and inhibited inflammation. In vitro, IL-10(+/+) B cells established longer contact times with arthritogenic CD4(+)CD25(-) T cells compared with IL-10(-/-) B cells in response to Ag stimulation, and using the same culture conditions, we observed upregulation of Foxp3 on CD4(+) T cells. Thus, IL-10-producing B cells restrain inflammation by promoting differentiation of immunoregulatory over proinflammatory T cells.