Highlights d Stool butyrate levels are reduced in patients with RA compared to healthy controls d Supplementation with butyrate suppresses arthritis severity in a mouse model d Suppression of arthritis by butyrate supplementation depends upon AhR + Bregs d Butyrate increases serotonin-derived 5-HIAA, which directly activates AhR + Bregs

SummaryRegulatory 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.

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.

1Abstract The Human Leukocyte Antigen HLA-B27(B27) is strongly associated with the spondyloarthritides. B27 can be expressed at the cell surface of antigen presenting cells (APC) as both classical β2m-associated B27 and as B27 free heavy chain forms (FHC) including disulphide-bonded heavy chain homodimers (termed B272). B27 FHC forms but not classical B27 bind to KIR3DL2. HLA-A3 which is not associated with spondyloarthritis (SpA) is also a ligand for KIR3DL2. Here we show that B272 and B27 FHC bind more strongly to KIR3DL2 than other HLA-class I, including HLA-A3. B272 tetramers bound KIR3DL2 transfected cells more strongly than HLA-A3. KIR3DL2Fc bound to HLA-B27-transfected cells more strongly than to cells transfected with other HLA-class I. KIR3DL2Fc pulled down multimeric, dimeric and monomeric free heavy chains from HLA-B27 expressing cell lines. Binding to B272 and B27 FHC stimulated greater KIR3DL2 phosphorylation than HLA-A3. B272 and B27 FHC stimulated KIR3DL2CD3ε–transduced T cell IL-2 production to a greater extent than control HLA-class I. KIR3DL2 binding to B27 inhibited NK IFNγ secretion and promoted greater survival of KIR3DL2+CD4 T and NK cells than binding to other HLA-class I. KIR3DL2+ T cells from B27+SpA patients proliferated more in response to antigen presented by syngeneic APC than the same T cell subset from healthy and disease controls. Our results suggest that expansion of KIR3DL2-expressing leukocytes observed in B27+ SpA may be explained by the stronger interaction of KIR3DL2 with B27 FHC.

ObjectiveGranulocyte–macrophage colony stimulating factor (GM-CSF) is a potent inflammatory mediator that is responsible for recruitment and activation of innate immune cells. Recent data from murine studies have identified Th17 cells as a key source of GM-CSF and suggest that T cell–derived GM-CSF is instrumental in the induction of autoimmune disease. The present study was undertaken to analyze the expression of T cell–derived GM-CSF in the joints of patients with juvenile idiopathic arthritis (JIA) and to investigate the differentiation of Th17 cells and how this relates to GM-CSF+ T helper cells.MethodsSynovial fluid (SF) and peripheral blood (PB) samples from 24 patients with JIA were analyzed, by flow cytometry and reverse transcription–polymerase chain reaction, for expression of GM-CSF and the Th17 marker CD161. A cytokine capture assay was used to purify Th17 cells and test the plasticity of cytokine production in response to interleukin-12 (IL-12) and IL-23.ResultsThe frequency of GM-CSF–producing T helper cells was significantly enriched in SF mononuclear cells compared to PB mononuclear cells from the patients with JIA (24.1% of CD4+ T cells versus 2.9%) and closely correlated with the erythrocyte sedimentation rate (r2 = 0.91, P < 0.001). Synovial GM-CSF+ T cells were predominantly CD161+ and coexpressed interferon-γ (IFNγ), but not IL-17. Culture of Th17 cells in the presence of IL-12 led to rapid up-regulation of GM-CSF and IFNγ, recapitulating the phenotype of GM-CSF–expressing cells within the joint.ConclusionOur results identify a novel outcome of Th17 plasticity in humans that may account for the enrichment of GM-CSF–expressing T cells in the joints of patients with JIA. The association of GM-CSF expression with systemic inflammation highlights the potential role of Th17-related cytokines in the pathology of JIA.

Possession of HLA-B27 (B27), strongly predisposes to the development of spondyloarthritis. B27 forms classical heterotrimeric complexes with beta-2-microglobulin (β2m) and peptide, and (β2m–free) free H chain (FHC) forms including B27 dimers (termed B272) at the cell surface. In this study we characterise the interaction of HLA-B27 with LILR, leukocyte Ig-like receptor (LILR)B1 and LILRB2 biophysically, biochemically and by FACS staining. LILRB1 bound to B27 heterotrimers with a KD of 5.3 ±1.5 μM but did not bind B27 FHC. LILRB2 bound to B272 and B27 FHC and B27 heterotrimers with KDs of 2.5, 2.6 and 22 ±6μM respectively. Domain exchange experiments showed that B272 bound to the two membrane distal Ig-like domains of LILRB2. In FACS staining experiments, B27 dimer protein and tetramers stained LILRB2 transfectants five times more strongly than B27 heterotrimers. Moreover, LILRB2Fc bound to dimeric and other B27 FHC forms on B27-expressing cell lines more strongly than other HLA-class I FHCs. B27 transfected cells expressing B27 dimers and FHC inhibited IL-2 production by LILRB2-expressing reporter cells to a greater extent than control HLA-class I transfectants. B27 heterotrimers complexed with the L6M variant of the GAG KK10 epitope bound with a similar affinity to complexes with the wild-type KK10 epitope (with KDs of 15.0±0.8 μM and 16.0±2.0 μM respectively). Disulfide-dependent B27 H chain dimers and multimers are stronger ligands for LILRB2 than HLA-class I heterotrimers and H chains. The stronger interaction of B27 dimers and FHC forms with LILRB2 compared with other HLA class I could play a role in spondyloarthritis pathogenesis.

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