Diapedesis-Induced Integrin Signaling via LFA-1 Facilitates Tissue Immunity by Inducing Intrinsic Complement C3 Expression in Immune Cells.

Intrinsic complement C3 activity is integral to human T helper type 1 (Th1) and cytotoxic T cell responses. Increased or decreased intracellular C3 results in autoimmunity and infections, respectively. The mechanisms regulating intracellular C3 expression remain undefined. We identified complement, including C3, as among the most significantly enriched biological pathway in tissue-occupying cells. We generated C3-reporter mice and confirmed that C3 expression was a defining feature of tissue-immune cells, including T cells and monocytes, occurred during transendothelial diapedesis, and depended on integrin lymphocyte-function-associated antigen 1 (LFA-1) signals. Immune cells from patients with leukocyte adhesion deficiency type 1 (LAD-1) had reduced C3 transcripts and diminished effector activities, which could be rescued proportionally by intracellular C3 provision. Conversely, increased C3 expression by T cells from arthritis patients correlated with disease severity. Our study defines integrins as key controllers of intracellular complement, demonstrates that perturbations in the LFA-1-C3-axis contribute to primary immunodeficiency, and identifies intracellular C3 as biomarker of severity in autoimmunity.

Regulatory B cells: origin, phenotype, and function.

Regulatory B (Breg) cells are immunosuppressive cells that support immunological tolerance. Through the production of interleukin-10 (IL-10), IL-35, and transforming growth factor ß (TGF-ß), Breg cells suppress immunopathology by prohibiting the expansion of pathogenic T cells and other pro-inflammatory lymphocytes. Recent work has shown that different inflammatory environments induce distinct Breg cell populations. Although these findings highlight the relevance of inflammatory signals in the differentiation of Breg cells, they also raise other questions about Breg cell biology and phenotype. For example, what are the functional properties and phenotype of Breg cells? Can a Breg cell arise at every stage in B cell development? Is inflammation the primary requisite for Breg cell differentiation? Here, we use these questions to discuss the advances in understanding Breg cell biology, with a particular emphasis on their ontogeny; we propose that multiple Breg cell subsets can be induced in response to inflammation at different stages in development.

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.

A clinical update on the significance of the gut microbiota in systemic autoimmunity.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease where a loss of tolerance to nuclear antigens leads to inflammation in multiple organ systems. The cause of SLE remains ill defined, although it is known that a complex interplay between genes and environment is necessary for disease development. In recent years, case studies have reported that the incidence of SLE in the USA, for example, has increased by approximately 3 fold. Although the reason for this is likely to be multifactorial, it has been hypothesized that the increasing incidence of autoimmune disease is due to considerable shifts in the bacterial communities resident the gut, collectively known as the gut microbiota, following a change in diet and the widespread introduction of antibiotics. Furthermore, a growing body of evidence suggests that the gut microbiota plays a role in the development of a range of autoimmune diseases including inflammatory bowel disease, multiple sclerosis, type one diabetes and rheumatoid arthritis. In this review, we summarize how advances in DNA-based sequencing technologies have been critical in providing baseline information concerning the gut microbiota in health and how variation amongst individuals in controlled by multiples factors including age, genetics, environment and the diet. We also discuss the importance of the gut microbiota in the development of a healthy immune system and how changes in particular bacterial phyla have been associated with immune abnormalities in animal models of autoimmune disease. Finally, in order to place the data in a clinical context, we highlight recent findings showing that abnormalities in the gut microbiota can be detected in patients with SLE, which provides the rationale for greater investigation into whether microbiota-targeted therapies could be used for the treatment/prevention of disease.

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.

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.

Active juvenile systemic lupus erythematosus is associated with distinct NK cell transcriptional and phenotypic alterations.

While adaptive immune responses have been studied extensively in SLE (systemic lupus erythematosus), there is limited and contradictory evidence regarding the contribution of natural killer (NK) cells to disease pathogenesis. There is even less evidence about the role of NK cells in the more severe phenotype with juvenile-onset (J)SLE. In this study, analysis of the phenotype and function of NK cells in a large cohort of JSLE patients demonstrated that total NK cells, as well as perforin and granzyme A expressing NK cell populations, were significantly diminished in JSLE patients compared to age- and sex-matched healthy controls. The reduction in NK cell frequency was associated with increased disease activity, and transcriptomic analysis of NK populations from active and low disease activity JSLE patients versus healthy controls confirmed that disease activity was the main driver of differential NK cell gene expression. Pathway analysis of differentially expressed genes revealed an upregulation of interferon-α responses and a downregulation of exocytosis in active disease compared to healthy controls. Further gene set enrichment analysis also demonstrated an overrepresentation of the apoptosis pathway in active disease. This points to increased propensity for apoptosis as a potential factor contributing to NK cell deficiency in JSLE.

IL-12p35 subunit contributes to autoimmunity by limiting IL-27-driven regulatory responses.

Contrasting results have emerged from studies performed using IL-12p35(-/-) mice. Animals lacking the IL-12p35 subunit can either be protected from or develop exacerbated autoimmune diseases, intracellular infections, and delayed-type hypersensitivity responses. In this study, we report that mice lacking the IL-12p35 subunit develop a significantly milder Ag-induced arthritis compared with wild-type (WT) mice. Lack of severe inflammation is accompanied by an increase in the mRNA levels of the Ebi-3 and p28 subunits and increased secretion of IL-27 and IL-10. This anti-inflammatory environment contributed to increased differentiation of regulatory T and B cells with intact suppressive function. Furthermore, IL-12p35(-/-) mice display reduced numbers of Th17 cells compared with WT arthritic mice. Neutralization of IL-27, but not the systemic administration of IL-12, restored inflammation and Th17 to levels seen in WT mice. The restoration of disease phenotype after anti-IL-27 administration indicates that the IL-12p35 subunit acts as negative regulator of the developing IL-27 response in this model of arthritis.

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.

Mini-Review: Gut-Microbiota and the Sex-Bias in Autoimmunity - Lessons Learnt From Animal Models.

It is well appreciated that there is a female preponderance in the development of most autoimmune diseases. Thought to be due to a complex interplay between sex chromosome complement and sex-hormones, however, the exact mechanisms underlying this sex-bias remain unknown. In recent years, there has been a focus on understanding the central pathogenic role of the bacteria that live in the gut, or the gut-microbiota, in the development of autoimmunity. In this review, we discuss evidence from animal models demonstrating that the gut-microbiota is sexually dimorphic, that there is a bidirectional relationship between the production of sex-hormones and the gut-microbiota, and that this sexual dimorphism within the gut-microbiota may influence the sex-bias observed in autoimmune disease development. Collectively, these data underline the importance of considering sex as a variable when investigating biological pathways that contribute to autoimmune disease risk.

Gender-Diverse Inclusion in Immunological Research: Benefits to Science and Health.

The differences between male and female immune systems are an under-researched field, ripe for discovery. This is evidenced by the stark sex biases seen in autoimmunity and infectious disease. Both the sex hormones (oestrogen and testosterone), as well as the sex chromosomes have been demonstrated to impact immune responses, in multiple ways. Historical shortcomings in reporting basic and clinical scientific findings in a sex-disaggregated manner have led not only to limited discovery of disease aetiology, but to potential inaccuracies in the estimation of the effects of diseases or interventions on females and gender-diverse groups. Here we propose not only that research subjects should include both cis-gender men and cis-gender women, but also transgender and gender-diverse people alongside them. The known interaction between the hormonal milieu and the sex chromosomes is inseparable in cis-gender human research, without the confounders of puberty and age. By inclusion of those pursuing hormonal affirmation of their gender identity- the individual and interactive investigation of hormones and chromosomes is permitted. Not only does this allow for a fine-tuned dissection of these individual effects, but it allows for discovery that is both pertinent and relevant to a far wider portion of the population. There is an unmet need for detailed treatment follow-up of the transgender community- little is known of the potential benefits and risks of hormonal supplementation on the immune system, nor indeed on many other health and disease outcomes. Our research team has pioneered the inclusion of gender-diverse persons in our basic research in adolescent autoimmune rheumatic diseases. We review here the many avenues that remain unexplored, and suggest ways in which other groups and teams can broaden their horizons and invest in a future for medicine that is both fruitful and inclusive.

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.

Regulatory B Cells in Experimental Mouse Models of Arthritis.

Regulatory B cells (Breg) have been shown to have a role in the suppression of a wide variety of immune responses, yet they are deficient or defective in autoimmune diseases such as rheumatoid arthritis. For the study of autoimmune inflammation, experimental models of arthritis have acted as a valuable tool in understanding the development of Bregs and their role in maintaining immune homeostasis. In this chapter, we will focus on the study of transitional-2 marginal zone precursor (T2-MZP) Bregs in the context of two experimental arthritis models: antigen-induced arthritis (AIA) and collagen-induced arthritis (CIA). We will specifically focus on how to induce arthritis, as well as on methods for the isolation and functional study of Bregs both in vitro and in vivo.

A systematic review exploring the bidirectional relationship between puberty and autoimmune rheumatic diseases.

BACKGROUND: Autoimmune rheumatic diseases (ARDs) are associated with a significant sex-bias, which becomes more evident post-puberty. This systematic review aims to elucidate the bidirectional relationship between puberty and ARD-related outcomes. METHODS: Studies published in English until October 2019 were identified using a systematic search of endocrinology and rheumatology literature. Information was extracted on study design, sample size, demographics, puberty outcome measures, disease outcome measures, and main findings. The methodological quality of the studies included was analysed using the Newcastle-Ottawa Scale (NOS). RESULTS: Sixteen non-randomised studies reporting on the impact of puberty on ARD outcomes (n = 7), ARD impact on puberty-related outcomes (n = 8), or both (n = 1) have been identified. The impact of puberty on ARD outcomes were investigated in patients with juvenile idiopathic arthritis (JIA)-associated uveitis (n = 1), juvenile systemic lupus erythematosus (JSLE) (n = 5) or in healthy controls who developed adult-onset SLE (n = 1) or had non-specific symptoms (n = 1). The impact of ARD on puberty outcomes was explored in JIA (n = 4) and JSLE (n = 3). Quality assessment of studies showed a small to moderate risk of bias overall (NOS 4-9/9). Due to large heterogeneity of the studies it was not possible to perform a meta-analysis. Multiple studies reported on delayed puberty in patients with JIA/JSLE, menstrual and hormonal abnormalities, and lower height and weight than controls. Earlier (pre-pubertal) onset of JSLE was correlated with more severe disease and more need for systemic treatment. CONCLUSION: A bidirectional relationship exists between puberty and ARDs; however, more and better research is required to elucidate the complexity of this relationship. We propose puberty-related clinical assessments in patients with ARDs, which can improve patient outcomes and facilitate future research.

Intestinal barrier dysfunction plays an integral role in arthritis pathology and can be targeted to ameliorate disease.

BACKGROUND: Evidence suggests an important role for gut-microbiota dysbiosis in the development of rheumatoid arthritis (RA). The link between changes in gut bacteria and the development of joint inflammation is missing. Here, we address whether there are changes to the gut environment and how they contribute to arthritis pathogenesis. METHODS: We analyzed changes in markers of gut permeability, damage, and inflammation in peripheral blood and serum of RA patients. Serum, intestines, and lymphoid organs isolated from K/BxN mice with spontaneous arthritis or from wild-type, genetically modified interleukin (IL)-10R-/-or claudin-8-/-mice with induced arthritis were analyzed by immunofluorescence/histology, ELISA, and flow cytometry. FINDINGS: RA patients display increased levels of serum markers of gut permeability and damage and cellular gut-homing markers, both parameters positively correlating with disease severity. Arthritic mice display increased gut permeability from early stages of disease, as well as bacterial translocation, inflammatory gut damage, increases in interferon γ (IFNγ)+and decreases in IL-10+intestinal-infiltrating leukocyte frequency, and reduced intestinal epithelial IL-10R expression. Mechanistically, both arthritogenic bacteria and leukocytes are required to disrupt gut-barrier integrity. We show that exposing intestinal organoids to IFNγ reduces IL-10R expression by epithelial cells and that mice lacking epithelial IL-10R display increased intestinal permeability and exacerbated arthritis. Claudin-8-/-mice with constitutively increased gut permeability also develop worse joint disease. Treatment of mice with AT-1001, a molecule that prevents development of gut permeability, ameliorates arthritis. CONCLUSIONS: We suggest that breakdown of gut-barrier integrity contributes to arthritis development and propose restoration of gut-barrier homeostasis as a new therapeutic approach for RA. FUNDING: Funded by Versus Arthritis (21140 and 21257) and UKRI/MRC (MR/T000910/1).

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.