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Diets high in cholesterol alter intestinal immunity. Here, we examined how the cholesterol metabolite 25-hydroxycholesterol (25-HC) impacts the intestinal B cell response. Mice lacking cholesterol 25-hydroxylase (CH25H), the enzyme generating 25-HC, had higher frequencies of IgA-secreting antigen-specific B cells upon immunization or infection. 25-HC did not affect class switch recombination, but rather restrained plasma cell (PC) differentiation. 25-HC was produced by follicular dendritic cells and increased in response to dietary cholesterol. Mechanistically, 25-HC restricted activation of the sterol sensing transcription factor SREBP2, thereby regulating B cell cholesterol biosynthesis. Ectopic expression of SREBP2 in germinal center B cells induced rapid PC differentiation, whereas SREBP2 deficiency reduced PC output in vitro and in vivo. High cholesterol diet impaired, whereas Ch25h deficiency enhanced, the IgA response against Salmonella and the resulting protection from systemic bacterial dissemination. Thus, a 25-HC-SREBP2 axis shapes the humoral response at the intestinal barrier, providing insight into the impact of high dietary cholesterol in intestinal immunity.

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.

Summary Regulatory B cells (Bregs) play a critical role in the control of autoimmunity and inflammation. IL-10 production is the hallmark for the identification of Bregs. However, the molecular determinants that regulate the transcription of IL-10 and control the Breg developmental program remain unknown. Here, we demonstrate that aryl hydrocarbon receptor (AhR) regulates the differentiation and function of IL-10-producing CD19+CD21hiCD24hiBregs and limits their differentiation into B cells that contribute to inflammation. Chromatin profiling and transcriptome analyses show that loss of AhR in B cells reduces expression of IL-10 by skewing the differentiation of CD19+CD21hiCD24hiB cells into a pro-inflammatory program, under Breg-inducing conditions. B cell AhR-deficient mice develop exacerbated arthritis, show significant reductions in IL-10-producing Bregs and regulatory T cells, and show an increase in T helper (Th) 1 and Th17 cells compared with B cell AhR-sufficient mice. Thus, we identify AhR as a relevant contributor to the transcriptional regulation of Breg differentiation., Graphical Abstract, Highlights • IL-10+ Bregs are identified by high expression of AhR • B cell AhR deficiency leads to exacerbated arthritis and impaired Breg function • AhR directly binds to and regulates the expression of IL-10 in Bregs • AhR maintains Breg phenotype by suppressing pro-inflammatory gene expression, The transcriptional control of interleukin-10 (IL-10) in regulatory B cells (Bregs) remains undefined. Piper et al. identify the aryl hydrocarbon receptor (AhR) as an important transcription factor involved in Breg differentiation and show a direct role of AhR in the regulation of IL-10 transcription.

Posté sur medRxiv le 07/04/2021Referred to byAntibodies against type I IFN: The bad guys self-restrain in systemic lupus erythematosusCell Reports Medicine, Volume 4, Issue 1, 17 January 2023, Pages 100903Simon Fillatreau; International audience; Systemic lupus erythematosus (SLE) is characterized by increased expression of type I interferon (IFN)-regulated genes in 50%-75% of patients. We report that out of 501 patients with SLE analyzed, 73 (14%) present autoantibodies against IFNα (anti-IFN-Abs). The presence of neutralizing-anti-IFN-Abs in 4.2% of patients inversely correlates with low circulating IFNα protein levels, inhibition of IFN-I downstream gene signatures, and inactive global disease score. Hallmarks of SLE pathogenesis, including increased immature, double-negative plasmablast B cell populations and reduction in regulatory B cell (Breg) frequencies, were normalized in patients with neutralizing anti-IFN-Abs compared with other patient groups. Immunoglobulin G (IgG) purified from sera of patients with SLE with neutralizing anti-IFN-Abs impedes CpGC-driven IFNα-dependent differentiation of B cells into immature B cells and plasmablasts, thus recapitulating the neutralizing effect of anti-IFN-Abs on B cell differentiation in vitro. Our findings highlight a role for neutralizing anti-IFN-Abs in controlling SLE pathogenesis and support the use of IFN-targeting therapies in patients with SLE lacking neutralizing-anti-IFN-Abs.

Type I interferons are essential for host response to viral infections, while dysregulation of their response can result in autoinflammation or autoimmunity. Among IFNα (alpha) responses, 13 subtypes exist that signal through the same receptor, but have been reported to have different effector functions. However, the lack of available tools for discriminating these closely related subtypes, in particular at the protein level, has restricted the study of their differential roles in disease. We developed a digital ELISA with specificity and high sensitivity for the IFNα2 subtype. Application of this assay, in parallel with our previously described pan-IFNα assay, allowed us to study different IFNα protein responses following cellular stimulation and in diverse patient cohorts. We observed different ratios of IFNα protein responses between viral infection and autoimmune patients. This analysis also revealed a small percentage of autoimmune patients with high IFNα2 protein measurements but low pan-IFNα measurements. Correlation with an ISG score and functional activity showed that in this small sub group of patients, IFNα2 protein measurements did not reflect its biological activity. This unusual phenotype was partly explained by the presence of anti-IFNα auto-antibodies in a subset of autoimmune patients. This study reports ultrasensitive assays for the study of IFNα proteins in patient samples and highlights the insights that can be obtained from the use of multiple phenotypic readouts in translational and clinical studies.

Regulatory B cells (Bregs) have immunosuppressive capacity, primarily via the production of IL-10. IL-10 expression and immunosuppression have been described in a number of human B cell subsets, two of which include the CD19+CD24hiCD38hi and CD19+CD24hiCD27+ populations. In this chapter, we describe how to identify and isolate these subsets from peripheral blood B cells via flow cytometry. We also explain how to expand Bregs in culture and how to identify them based on intracellular expression of IL-10.

B cells have a central role in many autoimmune diseases, including in those with renal involvement, as well as in the immunological response to kidney transplantation. The majority of B cell studies have focused on their pathological role as antibody producers. However, these cells have broad functions in immune responses beyond immunoglobulin secretion, including antigen presentation to T cells and cytokine production. Importantly, not all B cell subsets enhance immune responses. Regulatory B (Breg) cells attenuate inflammation and contribute to the maintenance of immune tolerance. Breg cells are numerically deficient and/or dysfunctional in several autoimmune diseases that can affect the kidneys, including systemic lupus erythematosus and anti-neutrophil cytoplasmic antibody-associated vasculitis, as well as in some groups of renal transplant recipients with alloimmune graft damage. B cell-targeting biologics have been trialled with promising results in diverse immune-mediated renal conditions. These therapies can affect both pro-inflammatory B cells and Breg cells, potentially limiting their long-term efficacy. Future strategies might involve the modulation of pro-inflammatory B cells in combination with the stimulation of regulatory subsets. Additionally, the monitoring of individual B cell subsets in patients may lead to the discovery of novel biomarkers that could help to predict disease relapse or progression.

The rise of a new hypervirulent variant of Klebsiella pneumoniae (hvKp) was recently reported, mainly linked to the ST23 lineage. The hvKp variants can cause severe infections, including hepatic abscesses, bacteremia, and meningitis, with a particularly disconcerting propensity to cause community-acquired, life-threatening infection among young and otherwise healthy individuals. The present study aimed to report the clinical characteristics of a hypermucoviscous K. pneumoniae strain isolated in Italy and sustaining recurrent meningitis in a patient of Peruvian origin. A further objective was to retrospectively investigate, by means of whole-genome sequencing (WGS) analysis, the genomic features of such an isolate. The hypermucoviscosity phenotype of the strain (sk205y205t) was determined using the string test. Genomic information was obtained by WGS (Illumina) and bioinformatic analysis. Strain sk205y205t was susceptible to most antibiotics, despite the presence of some resistance genes, including blaSHV-11, blaSHV-67, fosA, and acrR. The isolate belonged to ST65 and serotype K2, and exhibited several virulence factors related to the hvKp variant. Among these, were the siderophore genes entB, irp2, iroN, iroB, and iucA; the capsule-regulating genes rmpA and rmpA2; and the type 1 and 3 fimbriae fimH27 and mrkD, respectively. A further operon, encoding the genotoxin colibactin (clbA-Q), was also identified. The virulence plasmids pK2044, pRJA166b, and pNDM. MAR were also detected. Phylogenetic investigation showed that this Italian strain is highly similar to a Chinese isolate, suggesting a hidden circulation of this hvKp ST65 K2 lineage.

Regulatory B cells (Breg) express high levels of CD1d that presents lipid antigens to invariant natural killer T (iNKT) cells. The function of CD1d in Breg biology and iNKT cell activity during inflammation remains unclear. Here we show, using chimeric mice, cell depletion and adoptive cell transfer, that CD1d–lipid presentation by Bregs induces iNKT cells to secrete interferon (IFN)-γ to contribute, partially, to the downregulation of T helper (Th)1 and Th17-adaptive immune responses and ameliorate experimental arthritis. Mice lacking CD1d-expressing B cells develop exacerbated disease compared to wild-type mice, and fail to respond to treatment with the prototypical iNKT cell agonist α-galactosylceramide. The absence of lipid presentation by B cells alters iNKT cell activation with disruption of metabolism regulation and cytokine responses. Thus, we identify a mechanism by which Bregs restrain excessive inflammation via lipid presentation.

The frequency of definitive childlessness in women with multiple sclerosis (MS) may be higher than in the general population. MS may also affect decisions on the delivery procedure and on breast-feeding issues. Aim of the study was to assess the frequency of childlessness and its possible causes, the proportion of cesarean deliveries (CD), and the frequency of breast-feeding in patients and controls who have reached the end of their reproductive period. Female MS patients (>43 years) and controls (>45 years) filled out a questionnaire. We enrolled 303 patients and 500 controls. MS was associated with a higher frequency of childlessness (22 vs 13%) and less patients were in a stable relationship (83 vs 89%). There was no difference in the reported rates of infertility and miscarriages, while elective abortions were more frequent in patients (20 vs 12%). MS did not significantly affect the frequency of CD or of breast-feeding. MS-related reasons for childlessness, reported by 16% of childless patients, included disability/fear of future disability, fear of genetically transmitting MS, fear of not starting/discontinuing treatments, and discouragement by physician. Definitive childlessness is more frequent in women with MS compared to controls. A portion of voluntary childlessness may be avoided through correct/tailored information to patients.

Regulatory B cells (Bregs) modulate immune responses predominantly, although not exclusively, via the release of IL-10. The importance of human Bregs in the maintenance of immune homeostasis comes from a variety of immune-related pathologies, such as autoimmune diseases, cancers, and chronic infections that are often associated with abnormalities in Breg numbers or function. A continuous effort toward understanding Breg biology in healthy individuals will provide new opportunities to develop Breg immunotherapy that could prove beneficial in treating various immune-mediated pathologies. In this Review, we discuss findings regarding human Bregs, including their mechanisms of suppression and role in different disease settings. We also propose several therapeutic strategies targeting Bregs for better management of immune disorders.

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.

Summary 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)., Graphical abstract, Highlights Serum gut-permeability markers LPB, LPS, and I-FABP are increased in RA Mice with arthritis have increased gut permeability and intestinal inflammation Both bacteria and leukocytes are needed to disrupt gut-barrier integrity Prevention of gut-barrier dysfunction in arthritis ameliorates joint inflammation, Context and significance Rheumatoid arthritis is an autoimmune disorder characterized by chronic joint inflammation. Accumulating evidence suggests that changes in the composition of the bacteria residing in the gut could be responsible for joint inflammation. Currently, it is unclear how bacteria or their products instruct cells of the immune system to become harmful and induce arthritis. Researchers at University College London have shown that, in arthritis, there is profound damage to the gut lining, which fails to work properly as a barrier, as well as an accumulation in the gut of white blood cells that cause inflammation. The authors show that, in arthritis, bacteria cross the prohibited border of the intestinal lining and that repairing gut permeability defects with specific drugs inhibits joint inflammation., Changes to the gut bacteria have been associated with the development of arthritis; however, the mechanistic connection with disease remains unknown. Matei et al. identify pathological changes to the gut tissue in arthritis, including loss of gut-barrier integrity and inflammatory-cell infiltration, and show that restoration of gut homeostasis ameliorates disease.

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.

Background The immune surveillance reactivator lefitolimod (MGN1703), a DNA-based TLR9 agonist, might foster innate and adaptive immune response and thus improve immune-mediated control of residual cancer disease. The IMPULSE phase II study evaluated the efficacy and safety of lefitolimod as maintenance treatment in extensive-stage small-cell lung cancer (ES-SCLC) after objective response to first-line chemotherapy, an indication with a high unmet medical need and stagnant treatment improvement in the last decades. Patients and methods 103 patients with ES-SCLC and objective tumor response (as per RECIST 1.1) following four cycles of platinum-based first-line induction therapy were randomized to receive either lefitolimod maintenance therapy or local standard of care at a ratio of 3 : 2 until progression or unacceptable toxicity. Results From 103 patients enrolled, 62 were randomized to lefitolimod, 41 to the control arm. Patient demographics and response patterns to first-line therapy were balanced. Lefitolimod exhibited a favorable safety profile and pharmacodynamic assessment confirmed the mode-of-action showing a clear activation of monocytes and production of interferon-gamma-induced protein 10 (IP-10). While in the intent-to-treat (ITT) population no relevant effect of lefitolimod on progression-free and overall survival (OS) could be observed, two predefined patient subgroups indicated promising results, favoring lefitolimod with respect to OS: in patients with a low frequency of activated CD86+ B cells (hazard ratio, HR 0.53, 95% CI: 0.26–1.08; n = 38 of 88 analyzed) and in patients with reported chronic obstructive pulmonary disease (COPD) (HR 0.48, 95% CI: 0.20–1.17, n = 25 of 103). Conclusions The IMPULSE study showed no relevant effect of lefitolimod on the main efficacy end point OS in the ITT, but (1) the expected pharmacodynamic response to lefitolimod, (2) positive OS efficacy signals in two predefined subgroups and (3) a favorable safety profile. These data support further exploration of lefitolimod in SCLC.

B cells are considered central to the pathogenesis of patients with rheumatic diseases (RD) including Systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). In addition to producing autoantibodies, B cells suppressing inflammatory responses, known as regulatory B cells (Bregs) are numerically and functionally defective in rheumatic diseases. The production of interleukin 10 (IL-10) classically defines a Breg, however the stability and/or plasticity of this population is not well understood. In addition to producing autoantibodies, B cells suppressing inflammatory responses, known as regulatory B cells (Bregs) are numerically and functionally defective in RD patients. The production of interleukin 10 (IL-10) classically defines a Breg, however the stability and/or plasticity of this population is not well understood. Additionally, IL-10 +B cells have been shown to co-express pro-inflammatory cytokines such as TNFa and IL-6, further complicating Breg classification. Characterising the signals inducing Breg differentiation and the subsequent stability and/or plasticity of this population may aid in understanding the factors contributing to Breg dysfunction in RD patients. Novel findings unravelling the signals required and the stability of B regs versus B effector cells in RDs will be discussed. Disclosure of Interest None declared

Juvenile dermatomyositis (JDM) is a rare form of childhood autoimmune myositis that presents with proximal muscle weakness and skin rash. B cells are strongly implicated in the pathogenesis of the disease, but the underlying mechanisms are unknown. Therefore, the main objective of our study was to investigate mechanisms driving B cell lymphocytosis and define pathological features of B cells in JDM patients. Patients were recruited through the UK JDM Cohort and Biomarker study. Peripheral blood B cell subpopulations were immunophenotyped by flow cytometry. The results identified that immature transitional B cells were significantly expanded in active JDM, actively dividing, and correlated positively with disease activity. Protein and RNAseq analysis revealed high interferon alpha (IFNα) and TLR7-pathway signatures pre-treatment. Stimulation of B cells through TLR7/8 promoted both IL-10 and IL-6 production in controls but failed to induce IL-10 in JDM patient cells. Interrogation of the CD40–CD40L pathway (known to induce B cell IL-10 and IL-6) revealed similar expression of IL-10 and IL-6 in B cells cultured with CD40L from both JDM patients and controls. In conclusion, JDM patients with active disease have a significantly expanded immature transitional B cell population which correlated with the type I IFN signature. Activation through TLR7 and IFNα may drive the expansion of immature transitional B cells in JDM and skew the cells toward a pro-inflammatory phenotype.

Multiple sclerosis (MS) is an autoimmune disease characterized by CNS inflammation leading to demyelination and axonal damage. IFN-β is an established treatment for MS; however, up to 30% of IFN-β-treated MS patients develop neutralizing antidrug antibodies (nADA), leading to reduced drug bioactivity and efficacy. Mechanisms driving antidrug immunogenicity remain uncertain, and reliable biomarkers to predict immunogenicity development are lacking. Using high-throughput flow cytometry, NOTCH2 expression on CD14+ monocytes and increased frequency of proinflammatory monocyte subsets were identified as baseline predictors of nADA development in MS patients treated with IFN-β. The association of this monocyte profile with nADA development was validated in 2 independent cross-sectional MS patient cohorts and a prospective cohort followed before and after IFN-β administration. Reduced monocyte NOTCH2 expression in nADA+ MS patients was associated with NOTCH2 activation measured by increased expression of Notch-responsive genes, polarization of monocytes toward a nonclassical phenotype, and increased proinflammatory IL-6 production. NOTCH2 activation was T cell dependent and was only triggered in the presence of serum from nADA+ patients. Thus, nADA development was driven by a proinflammatory environment that triggered activation of the NOTCH2 signaling pathway prior to first IFN-β administration.

Summary The differentiation of IL-10-producing regulatory B cells (Bregs) in response to gut-microbiota-derived signals supports the maintenance of tolerance. However, whether microbiota-derived metabolites can modulate Breg suppressive function remains unknown. Here, we demonstrate that rheumatoid arthritis (RA) patients and arthritic mice have a reduction in microbial-derived short-chain fatty acids (SCFAs) compared to healthy controls and that in mice, supplementation with the SCFA butyrate reduces arthritis severity. Butyrate supplementation suppresses arthritis in a Breg-dependent manner by increasing the level of the serotonin-derived metabolite 5-Hydroxyindole-3-acetic acid (5-HIAA), which activates the aryl-hydrocarbon receptor (AhR), a newly discovered transcriptional marker for Breg function. Thus, butyrate supplementation via AhR activation controls a molecular program that supports Breg function while inhibiting germinal center (GC) B cell and plasmablast differentiation. Our study demonstrates that butyrate supplementation may serve as a viable therapy for the amelioration of systemic autoimmune disorders., Graphical Abstract, Highlights • Stool butyrate levels are reduced in patients with RA compared to healthy controls • Supplementation with butyrate suppresses arthritis severity in a mouse model • Suppression of arthritis by butyrate supplementation depends upon AhR+Bregs • Butyrate increases serotonin-derived 5-HIAA, which directly activates AhR+Bregs, The environmental signals that influence Breg function are not yet fully defined. Here, Rosser et al. demonstrate that the short-chain fatty acid butyrate supports Breg function by increasing the level of the serotonin-derived metabolite 5-Hydroxyindole-3-acetic acid (5-HIAA). 5-HIAA, in turn, activates the aryl-hydrocarbon receptor, a newly discovered transcriptional regulator for Bregs.

Objectives: B cells are central to the pathology of ANCA-associated vasculitis (AAV), a disease characterized by autoantibodies and effectively treated by rituximab. In addition to promoting inflammation, a subset of B cells act to suppress harmful autoimmune responses (Breg). The balance of effector and regulatory B cell subsets in AAV is not known. This study was conducted to assess the relative frequency of these subsets during different states of disease activity. / Methods: B memory (Bmem), naive (Bnaive) and regulatory (Breg) subsets were defined by their relative expression of CD24 and CD38. Function was assessed by cytokine production and suppressive action on CD4(+) Th1 activation evaluated in a co-culture system. / Results: Compared with healthy controls, the frequency of Breg (CD24(hi)CD38(hi)) was significantly reduced during disease remission in both proteinase 3 (PR3)- and MPO-ANCA patients and during acute disease in PR3-ANCA patients, while the frequency of memory cells (CD24(hi)CD38(lo)) was reduced during active disease and restored during remission. Breg cell frequency showed a positive correlation, while Bmem had an inverse correlation with IL-10 production in vitro. B and T cell co-cultures revealed that memory and naive B cell subsets augmented Th1 activation in vitro, which was prevented by Breg, and this pattern did not differ between remission AAV patients and controls. / Conclusion: In remission there is a numerical, but not functional, deficiency in Breg and preservation of Bmem associated with reduced IL-10 production and increased Th1 activation in vitro. This imbalance may contribute to the high rate of relapse observed in AAV, especially in PR3-ANCA patients.

Systemic lupus erythematosus (SLE) is a severe autoimmune disease that is associated with increased circulating apoptotic cell autoantigens (AC-Ags) as well as increased type I IFN signaling. Here, we describe a pathogenic mechanism in which follicular translocation of marginal zone (MZ) B cells in the spleens of BXD2 lupus mice disrupts marginal zone macrophages (MZMs), which normally clear AC debris and prevent follicular entry of AC-Ags. Phagocytosis of ACs by splenic MZMs required the megakaryoblastic leukemia 1 (MKL1) transcriptional coactivator-mediated mechanosensing pathway, which was maintained by MZ B cells through expression of membrane lymphotoxin-α1β2 (mLT). Specifically, type I IFN-induced follicular shuttling of mLT-expressing MZ B cells disengaged interactions between these MZ B cells and LTβ receptor-expressing MZMs, thereby downregulating MKL1 in MZMs. Loss of MKL1 expression in MZMs led to defective F-actin polymerization, inability to clear ACs, and, eventually, MZM dissipation. Aggregation of plasmacytoid DCs in the splenic perifollicular region, follicular translocation of MZ B cells, and loss of MKL1 and MZMs were also observed in an additional murine lupus model and in the spleens of patients with SLE. Collectively, the results suggest that lupus might be interrupted by strategies that maintain or enhance mechanosensing signaling in the MZM barrier to prevent follicular entry of AC-Ags.

Objective. To define the mechanisms of action of 2 novel drugs, cyclosporine and anti-tumor necrosis factor alpha (TNF alpha), in collagen-induced arthritis and to determine the effect of combination therapy.Methods. Type II collagen-immunized DBA/1 mice with established arthritis,were treated with cyclosporine alone, anti-TNF alpha alone, cyclosporine plus anti-TNF alpha, or saline.Results. Cyclosporine was found to ameliorate arthritis, suppress interferon-gamma (IFN gamma) production by CD4+ T cells, and reduce TNF alpha expression in arthritic joints. However, cyclosporine did not directly inhibit TNF alpha production by macrophages, indicating that the decrease in TNF alpha expression observed in vivo was probably an indirect consequence of the reduction in type 1 T helper cell activity. Anti-TNF alpha also reduced IFN gamma production by T cells, indicating that TNF alpha is involved in the cellular immune response to collagen. Combined treatment with cyclosporine plus anti-TNF alpha! had an additive therapeutic effect.Conclusion. Although cyclosporine and anti-TNF alpha target different points in the inflammatory pathway, there is an overlap in the consequences of their actions in vivo.

Summary Signals controlling the generation of regulatory B (Breg) cells remain ill-defined. Here we report an “auto”-regulatory feedback mechanism between plasmacytoid dendritic cells (pDCs) and Breg cells. In healthy individuals, pDCs drive the differentiation of CD19+CD24hiCD38hi (immature) B cells into IL-10-producing CD24+CD38hi Breg cells and plasmablasts, via the release of IFN-α and CD40 engagement. CD24+CD38hi Breg cells conversely restrained IFN-α production by pDCs via IL-10 release. In systemic lupus erythematosus (SLE), this cross-talk was compromised; pDCs promoted plasmablast differentiation but failed to induce Breg cells. This defect was recapitulated in healthy B cells upon exposure to a high concentration of IFN-α. Defective pDC-mediated expansion of CD24+CD38hi Breg cell numbers in SLE was associated with altered STAT1 and STAT3 activation. Both altered pDC-CD24+CD38hi Breg cell interactions and STAT1-STAT3 activation were normalized in SLE patients responding to rituximab. We propose that alteration in pDC-CD24+CD38hi Breg cell interaction contributes to the pathogenesis of SLE., Graphical Abstract, Highlights • pDCs induce the differentiation of Breg cells in an IFN-α-dependent manner • Breg cells limit pDC-derived IFN-α in an IL-10-dependent mechanism • pDCs are hyperactivated in SLE and fail to induce Breg cells • Patients responding to rituximab display a normalized pDC-Breg cell interaction, The signals required for Breg cell differentiation in humans are currently unknown. Mauri and colleagues show that plasmacytoid dendritic cells, via the provision of IFN-α, govern the differentiation of immature B cells into regulatory B cells that restrain inflammation.

Objective The importance of interleukin-17 (IL-17) is underscored both by its resistance to control by Treg cells and the propensity of Treg cells to produce this highly inflammatory cytokine. This study sought to address whether Th17 cells are inhibited by Treg cells in rheumatoid arthritis (RA) patients responding to anti–tumor necrosis factor (anti-TNF) therapy, and if so defining the underlying mechanisms of suppression. Methods Inhibition of Th17 cell responses was determined by Treg cell suppression assays. The Treg cell phenotype was analyzed using flow cytometry and enzyme-linked immunosorbent assay. Mechanisms of suppression were tested by cytokine addition or antibody blockade. Results Th17 responses were inhibited by Treg cells from RA patients responding to the anti-TNF antibody adalimumab (Tregada), but not by Treg cells from healthy individuals or patients with active RA. Furthermore, Tregada cells secreted less IL-17, even when exposed to proinflammatory monocytes from patients with active RA. Tregada cells suppressed Th17 cells through the inhibition of monocyte-derived IL-6, but this effect was independent of IL-10 and transforming growth factor β, which mediated the suppression of Th1 responses. Adalimumab therapy led to a reduction in retinoic acid receptor–related orphan nuclear receptor C–positive Th17 cells and an increase in FoxP3+ Treg cells lacking expression of the transcription factor Helios. However, this acquisition of IL-17–suppressor function was not observed in RA patients responding to treatment with etanercept, a modified TNF receptor–Fc fusion protein. Indeed, there was no alteration in Treg cell number, function, or phenotype in etanercept-treated patients, and Th17 responses remained unchecked. Conclusion Th1 and Th17 responses are controlled through distinct mechanisms by Treg cells from patients responding to anti-TNF antibody therapy. Adalimumab therapy, but not etanercept therapy, induces a potent and stable Treg cell population with the potential to restrain the progression of IL-17–associated inflammation in RA via regulation of monocyte-derived IL-6.

Summary B cells perform many immunological functions, including presenting lipid antigen to CD1d-restricted invariant natural killer T (iNKT) cells, known to contribute to maintaining tolerance in autoimmunity. Patients with systemic lupus erythematous (SLE) display dysregulated B cell responses and reduced peripheral iNKT cell frequencies. The significance of these defects and how they relate to SLE pathogenesis remain elusive. We report that B cells are essential for iNKT cell expansion and activation in healthy donors but fail to exert a similar effect in SLE patients. Defective B cell-mediated stimulation of iNKT cells in SLE patients was associated with altered CD1d recycling, a defect recapitulated in B cells from healthy donors after stimulation with interferon-α (IFN-α) and anti-immunoglobulin (Ig). iNKT cell number and function were restored in SLE patients responding to anti-CD20 treatment upon normalization of CD1d expression exclusively in repopulated immature B cells. We propose that healthy B cells are pivotal for iNKT cell homeostasis., Highlights ► B cells sustain iNKT cell homeostasis and activation in healthy individuals ► SLE B cells fail to sustain iNKT cell homeostasis and activation ► SLE B cells were characterized by a profound decrease in surface CD1d expression ► Correct trafficking of CD1d is important for the maintenance of iNKT cell homeostasis

Regulatory B cells have largely been reported as B cells at a developmental stage before plasma cell differentiation. Matsumoto et al. (2014) report that IL-10(+) plasmablasts restrain autoimmune inflammation and suggest an ontological connection between immature B cells and regulatory plasmablasts.

CTLA-4 is a critical gatekeeper of T-cell activation and immunological tolerance and has been implicated in patients with a variety of autoimmune diseases through genetic association. Since T cells from patients with the autoimmune disease systemic lupus erythematosus (SLE) display a characteristic hyperactive phenotype, we investigated the function of CTLA-4 in SLE. Our results reveal increased CTLA-4 expression in FOXP3(-) responder T cells from patients with SLE compared with other autoimmune rheumatic diseases and healthy controls. However, CTLA-4 was unable to regulate T-cell proliferation, lipid microdomain formation and phosphorylation of TCR-zeta following CD3/CD28 co-stimulation, in contrast to healthy T cells. Although lupus T cells responded in vitro to CD3/CD28 co-stimulation, there was no parallel increase in CTLA-4 expression, which would normally provide a break on T-cell proliferation. These defects were associated with exclusion of CTLA-4 from lipid microdomains providing an anatomical basis for its loss of function. Collectively our data identify CTLA-4 dysfunction as a potential cause for abnormal T-cell activation in patients with SLE, which could be targeted for therapy.

We have previously reported that IL-10+ regulatory B cells, known to play an important role in controlling autoimmunity and inflammatory disorders, are contained within the transitional 2 immature (T2) B cell pool (T2 Bregs). Therapeutic strategies facilitating their enrichment or enhancing their suppressive activity are highly attractive. In this study, we report that agonistic anti-CD40 specifically targets T2 B cells and enriches Bregs upon short-term in vitro culture. Although transfer of unmanipulated T2 B cells, isolated from mice with established lupus, failed to confer protection to diseased mice, transfer of in vitro anti-CD40-generated T2 B cells (T2-like-Bregs) significantly improved renal disease and survival by an IL-10-dependent mechanism. T2-like-Bregs readily accumulated in the spleen after transfer, suppressed Th1 responses, induced the differentiation of IL-10+CD4+T cells, and conveyed a regulatory effect to CD4+T cells. In addition, in vivo administration of agonistic anti-CD40, currently on trial for the treatment of cancer, halted and reversed established lupus. Taken together, our results suggest a novel cellular approach for the amelioration of experimental lupus.

The expanding family of regulatory B cells, Over the last decade it has become evident that in addition to producing antibody, B cells activate the immune system by producing cytokines and via antigen presentation. In addition, B cells also exhibit immunosuppressive functions via diverse regulatory mechanisms. This subset of B cells, known as regulatory B cells (Bregs), contributes to the maintenance of tolerance, primarily via the production of IL-10. Studies in experimental animal models, as well as in patients with autoimmune diseases, have identified multiple Breg subsets exhibiting diverse mechanisms of immune suppression. In this review, we describe the different Breg subsets identified in mice and humans, and their diverse mechanisms of suppression in different disease settings.

Systemic lupus erythematosus is a multisystem autoimmune disease characterized by a wide range of immunological abnormalities that underlie the loss of tolerance. In this study we show that administration of atorvastatin to lupus-prone NZB/W F1 mice resulted in a significant reduction in serum IgG anti-dsDNA Abs and decreased proteinuria. Histologically, the treatment was associated with reduced glomerular Ig deposition and less glomerular injury. Disease improvement was paralleled by decreased expression of MHC class II on monocytes and B lymphocytes and reduced expression of CD80 and CD86 on B lymphocytes. Consequent upon this inhibition of Ag presentation, T cell proliferation was strongly impaired by atorvastatin in vitro and in vivo. A significant decrease in MHC class II expression was also observed in the target organ of lupus disease (i.e., the glomerulus). Serum cholesterol in atorvastatin-treated lupus mice fell to the level found in young NZB/W mice before disease onset. This is the first demonstration that atorvastatin can delay the progression of a spontaneous autoimmune disease and may specifically benefit patients with systemic lupus erythematosus.

Regulatory T cells have been clearly implicated in the control of disease in murine models of autoimmunity. The paucity of data regarding the role of these lymphocytes in human autoimmune disease has prompted us to examine their function in patients with rheumatoid arthritis (RA). Regulatory (CD4+CD25+) T cells isolated from patients with active RA displayed an anergic phenotype upon stimulation with anti-CD3 and anti-CD28 antibodies, and suppressed the proliferation of effector T cells in vitro. However, they were unable to suppress proinflammatory cytokine secretion from activated T cells and monocytes, or to convey a suppressive phenotype to effector CD4+CD25− T cells. Treatment with antitumor necrosis factor α (TNFα; Infliximab) restored the capacity of regulatory T cells to inhibit cytokine production and to convey a suppressive phenotype to “conventional” T cells. Furthermore, anti-TNFα treatment led to a significant rise in the number of peripheral blood regulatory T cells in RA patients responding to this treatment, which correlated with a reduction in C reactive protein. These data are the first to demonstrate that regulatory T cells are functionally compromised in RA, and indicate that modulation of regulatory T cells by anti-TNFα therapy may be a further mechanism by which this disease is ameliorated.

Nuclear factor kappa B (NF-kappa B) activation has been observed in human atherosclerotic plaques and is enhanced in unstable coronary plaques, but whether such activation has a protective or pathophysiological role remains to be determined. We addressed this question by developing a short-term culture system of cells isolated from human atherosclerotic tissue, allowing efficient gene transfer to directly investigate signaling pathways in human atherosclerosis. We found that NF-kappa B is activated in these cells and that this activity involves p65, p50, and c-Rel but not p52 or RelB. This NF-kappa B activation can be blocked by overexpression of I kappa B alpha or dominant-negative I kappa B kinase (IKK)-2 but not dominant-negative IKK-1 or NF-kappa B-inducing kinase, resulting in selective inhibition of inflammatory cytokines (tumor necrosis factor alpha, IL-6, and IL-8), tissue factor, and matrix metalloproteinases without affecting the antiinflammatory cytokine IL-10 or tissue inhibitor of matrix metalloproteinases. Our results demonstrate that the canonical pathway of NF-kappa B activation that involves p65, p50, c-Rel, and IKK-2 is activated in human atherosclerosis and results in selective up-regulation of major proinflammatory and prothrombotic mediators of the disease.

In this study we have shown that activation of arthritogenic splenocytes with antigen and agonistic anti-CD40 gives raise to a B cell population that produce high levels of interleukin (IL)-10 and low levels of interferon (IFN)-γ. Transfer of these B cells into DBA/1-TcR-β-Tg mice, immunized with bovine collagen (CII) emulsified in complete Freund's adjuvant inhibited T helper type 1 differentiation, prevented arthritis development, and was also effective in ameliorating established disease. IL-10 is essential for the regulatory function of this subset of B cells, as the B cells population isolated from IL-10 knockout mice failed to mediate this protective function. Furthermore, B cells isolated from arthritogenic splenocytes treated in vitro with anti–IL-10/anti–IL-10R were unable to protect recipient mice from developing arthritis. Our results suggest a new role of a subset of B cells in controlling T cell differentiation and autoimmune disorders.

Juvenile dermatomyositis (JDM) is a childhood autoimmune disease characterised by proximal muscle weakness and cutaneous manifestations. Previous studies have identified an increase in circulating B cells in JDM patients, but their provenance and functional characteristics have not been examined. In this study we investigated whether an immature B cell subset (CD24hiCD38hi), known to be enriched for interleukin (IL)-10 producing regulatory B cells (Breg)1, accounted for the expansion of circulating B cells seen in JDM. The aryl hydrocarbon receptor (AhR) is a ligand based transcription factor, which induces IL-10 expression in T cells2. We investigated the effects of modulating the AhR pathway on IL-10 expression in B cells.