Phosphorylation and linear ubiquitin direct A20 inhibition of inflammation.

Inactivation of the TNFAIP3 gene, encoding the A20 protein, is associated with critical inflammatory diseases including multiple sclerosis, rheumatoid arthritis and Crohn's disease. However, the role of A20 in attenuating inflammatory signalling is unclear owing to paradoxical in vitro and in vivo findings. Here we utilize genetically engineered mice bearing mutations in the A20 ovarian tumour (OTU)-type deubiquitinase domain or in the zinc finger-4 (ZnF4) ubiquitin-binding motif to investigate these discrepancies. We find that phosphorylation of A20 promotes cleavage of Lys63-linked polyubiquitin chains by the OTU domain and enhances ZnF4-mediated substrate ubiquitination. Additionally, levels of linear ubiquitination dictate whether A20-deficient cells die in response to tumour necrosis factor. Mechanistically, linear ubiquitin chains preserve the architecture of the TNFR1 signalling complex by blocking A20-mediated disassembly of Lys63-linked polyubiquitin scaffolds. Collectively, our studies reveal molecular mechanisms whereby A20 deubiquitinase activity and ubiquitin binding, linear ubiquitination, and cellular kinases cooperate to regulate inflammation and cell death.

PILRα negatively regulates mouse inflammatory arthritis.

Paired Ig-like type 2 receptor (PILR)α inhibitory receptor and its counterpart PILRß activating receptor are coexpressed on myeloid cells. In this article, we report that PILRα, but not PILRß, is elevated in human rheumatoid arthritis synovial tissue and correlates with inflammatory cell infiltration. Pilrα(-/-) mice produce more pathogenic cytokines during inflammation and are prone to enhanced autoimmune arthritis. Correspondingly, engaging PILRα with anti-PILRα mAb ameliorates inflammation in mouse arthritis models and suppresses the production of proinflammatory cytokines. Our studies suggest that PILRα mediates an important inhibitory pathway that can dampen inflammatory responses.

Marginal-zone B cells.

Recent advances in genomics and proteomics, combined with the facilitated generation and analysis of transgenic and gene-knockout animals, have revealed new complexities in classical biological systems, including the B-cell compartment. Studies on an 'old', but poorly characterized, B-cell subset--the naive, marginal-zone (MZ) B-cell subset--over the past two years have spawned an avalanche of data that encompass the generation and function of these cells. Now that the initial 'infatuation' is over, it is time to reconsider these data and generate some conclusions that can be incorporated into a working model of the B-cell system.

Chondroitin sulfate synthase 1 (Chsy1) is required for bone development and digit patterning.

Joint and skeletal development is highly regulated by extracellular matrix (ECM) proteoglycans, of which chondroitin sulfate proteoglycans (CSPGs) are a major class. Despite the requirement of joint CSPGs for skeletal flexibility and structure, relatively little is understood regarding their role in establishing joint positioning or in modulating signaling and cell behavior during joint formation. Chondroitin sulfate synthase 1 (Chsy1) is one of a family of enzymes that catalyze the extension of chondroitin and dermatan sulfate glycosaminoglycans. Recently, human syndromic brachydactylies have been described to have loss-of-function mutations at the CHSY1 locus. In concordance with these observations, we demonstrate that mice lacking Chsy1, though viable, display chondrodysplasia and decreased bone density. Notably, Chsy1(-/-) mice show a profound limb patterning defect in which orthogonally shifted ectopic joints form in the distal digits. Associated with the digit-patterning defect is a shift in cell orientation and an imbalance in chondroitin sulfation. Our results place Chsy1 as an essential regulator of joint patterning and provide a mouse model of human brachydactylies caused by mutations in CHSY1.

Comparative transcriptomic analyses of atopic dermatitis and psoriasis reveal shared neutrophilic inflammation.

BACKGROUND: Atopic dermatitis (AD) and psoriasis are common inflammatory diseases canonically described as involving distinct T(H) polarization and granulocytic infiltration. Acute AD lesions are associated with T(H)2 and eosinophilic inflammation, whereas psoriatic lesions are associated with T(H)1/T(H)17 and neutrophilic inflammation. Despite intensive investigation, these pathways remain incompletely understood in vivo in human subjects. OBJECTIVE: Using AD and psoriatic lesional skin as exemplar T(H)2 and T(H)1/T(H)17 diseased tissue, we sought to clarify common and unique molecular and pathophysiologic features in inflamed skin with different types of inflammatory polarization. METHODS: We conducted gene expression microarray analyses to identify distinct and commonly dysregulated expression in AD (based on Hanifin and Rajka criteria) and psoriatic lesions. We defined gene sets (GSs) as comprising genes encoding cytokines, chemokines, and growth factors that were uniquely or jointly dysregulated in patients with AD and those with psoriasis and calculated aggregate GS expression scores for lesional skin of patients with these dermatoses and healthy control skin. RESULTS: The atopic dermatitis gene set (AD-GS) score correlated with systemic and local measures of allergic inflammation, including serum IgE levels, blood eosinophil counts, and tissue eosinophil counts. Unexpectedly, genes encoding neutrophil chemoattractants among the common GS were highly expressed in AD lesional skin. Hematoxylin and eosin and immunohistochemical analyses showed the numbers of neutrophils in AD lesional skin were comparable with those in psoriatic lesional skin, and both were correlated with the extent of expression of neutrophil chemoattractant genes. CONCLUSION: These data are evidence that neutrophilic inflammation is a feature of lesional AD pathology comorbid with allergic inflammation.

An agonistic anti-signal regulatory protein α antibody for chronic inflammatory diseases.

Signal regulatory protein (SIRPα) is an immune inhibitory receptor expressed by myeloid cells to inhibit immune cell phagocytosis, migration, and activation. Despite the progress of SIRPα and CD47 antagonist antibodies to promote anti-cancer immunity, it is not yet known whether SIRPα receptor agonism could restrain excessive autoimmune tissue inflammation. Here, we report that neutrophil- and monocyte-associated genes including SIRPA are increased in inflamed tissue biopsies from patients with rheumatoid arthritis and inflammatory bowel diseases, and elevated SIRPA is associated with treatment-refractory ulcerative colitis. We next identify an agonistic anti-SIRPα antibody that exhibits potent anti-inflammatory effects in reducing neutrophil and monocyte chemotaxis and tissue infiltration. In preclinical models of arthritis and colitis, anti-SIRPα agonistic antibody ameliorates autoimmune joint inflammation and inflammatory colitis by reducing neutrophils and monocytes in tissues. Our work provides a proof of concept for SIRPα receptor agonism for suppressing excessive innate immune activation and chronic inflammatory disease treatment.

The adaptor protein Sh2d3c is critical for marginal zone B cell development and function.

Sh2d3c is an adaptor protein that has been implicated in T cell activation and shown to associate with different components of the integrin signaling pathway ex vivo. However, the in vivo significance of Sh2d3c expression in the regulation of the immune response and/or hematopoietic cell lineage development is not known. In this study, we show that expression of Sh2d3c is more critical for development and function of marginal zone B (MZB) cells than for T cell maturation. Mice deficient in Sh2d3c expression (Sh2d3c(-/-)) had a reduced number of MZB cells, and the residual MZB cells failed to properly capture polysaccharide Ags. Activation-induced proliferation, cytokine production, and migration of Sh2d3c(-)(/)(-) splenic B cells were also significantly reduced in vitro compared with wild-type (Sh2d3c(+/+)) cells. In contrast, T cell development and function were largely normal in Sh2d3c(-/-) mice. The thymi of Sh2d3c(-/-) mice showed no maturational abnormalities, the number of splenic T cells was only modestly reduced, and the T cells responded normally to in vitro polyclonal activation. The observed B cell deficiency in the Sh2d3c(-/-) mice led to diminished humoral immune response against thymus-independent type 2, but not thymus-dependent Ags, which highlights the primary in vivo role of Sh2d3c in regulating B cell development and function.

B-cell activation influences T-cell polarization and outcome of anti-CD20 B-cell depletion in central nervous system autoimmunity.

OBJECTIVE: Clinical studies indicate that anti-CD20 B-cell depletion may be an effective multiple sclerosis (MS) therapy. We investigated mechanisms of anti-CD20-mediated immune modulation using 2 paradigms of experimental autoimmune encephalomyelitis (EAE). METHODS: Murine EAE was induced by recombinant myelin oligodendrocyte glycoprotein (rMOG), a model in which B cells are considered to contribute pathogenically, or MOG peptide (p)35-55, which does not require B cells. RESULTS: In EAE induced by rMOG, B cells became activated and, when serving as antigen-presenting cells (APCs), promoted differentiation of proinflammatory MOG-specific Th1 and Th17 cells. B-cell depletion prevented or reversed established rMOG-induced EAE, which was associated with less central nervous system (CNS) inflammation, elimination of meningeal B cells, and reduction of MOG-specific Th1 and Th17 cells. In contrast, in MOG p35-55-induced EAE, B cells did not become activated or efficiently polarize proinflammatory MOG-specific T cells, similar to naive B cells. In this setting, anti-CD20 treatment exacerbated EAE, and did not impede development of Th1 or Th17 cells. Irrespective of the EAE model used, B-cell depletion reduced the frequency of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg), and increased the proinflammatory polarizing capacity of remaining myeloid APCs. INTERPRETATION: Our study highlights distinct roles for B cells in CNS autoimmunity. Clinical benefit from anti-CD20 treatment may relate to inhibition of proinflammatory B cell APC function. In certain clinical settings, however, elimination of unactivated B cells, which participate in regulation of T cells and other APC, may be undesirable. Differences in immune responses to MOG protein and peptide may be important considerations when choosing an EAE model for testing novel B cell-targeting agents for MS.

In vivo blockade of OX40 ligand inhibits thymic stromal lymphopoietin driven atopic inflammation.

Thymic stromal lymphopoietin (TSLP) potently induces deregulation of Th2 responses, a hallmark feature of allergic inflammatory diseases such as asthma, atopic dermatitis, and allergic rhinitis. However, direct downstream in vivo mediators in the TSLP-induced atopic immune cascade have not been identified. In our current study, we have shown that OX40 ligand (OX40L) is a critical in vivo mediator of TSLP-mediated Th2 responses. Treating mice with OX40L-blocking antibodies substantially inhibited immune responses induced by TSLP in the lung and skin, including Th2 inflammatory cell infiltration, cytokine secretion, and IgE production. OX40L-blocking antibodies also inhibited antigen-driven Th2 inflammation in mouse and nonhuman primate models of asthma. This treatment resulted in both blockade of the OX40-OX40L receptor-ligand interaction and depletion of OX40L-positive cells. The use of a blocking, OX40L-specific mAb thus presents a promising strategy for the treatment of allergic diseases associated with pathologic Th2 immune responses.

Function of CSF1 and IL34 in Macrophage Homeostasis, Inflammation, and Cancer.

Colony-stimulating factor 1 (CSF1) and interleukin 34 (IL34) signal via the CSF1 receptor to regulate macrophage differentiation. Studies in IL34- or CSF1-deficient mice have revealed that IL34 function is limited to the central nervous system and skin during development. However, the roles of IL34 and CSF1 at homeostasis or in the context of inflammatory diseases or cancer in wild-type mice have not been clarified in vivo. By neutralizing CSF1 and/or IL34 in adult mice, we identified that they play important roles in macrophage differentiation, specifically in steady-state microglia, Langerhans cells, and kidney macrophages. In several inflammatory models, neutralization of both CSF1 and IL34 contributed to maximal disease protection. However, in a myeloid cell-rich tumor model, CSF1 but not IL34 was required for tumor-associated macrophage accumulation and immune homeostasis. Analysis of human inflammatory conditions reveals IL34 upregulation that may account for the protection requirement of IL34 blockade. Furthermore, evaluation of IL34 and CSF1 blockade treatment during Listeria infection reveals no substantial safety concerns. Thus, IL34 and CSF1 play non-redundant roles in macrophage differentiation, and therapeutic intervention targeting IL34 and/or CSF1 may provide an effective treatment in macrophage-driven immune-pathologies.

APRIL-deficient mice have normal immune system development.

APRIL (a proliferation-inducing ligand) is a member of the tumor necrosis factor (TNF) superfamily. APRIL mRNA shows high levels of expression in tumors of different origin and a low level of expression in normal cells. APRIL shares two TNF receptor family members, TACI and BCMA, with another TNF homolog, BLyS/BAFF. BLyS is involved in regulation of B-cell activation and survival and also binds to a third receptor, BR3/BAFF-R, which is not shared with APRIL. Recombinant APRIL and BLyS induce accumulation of B cells in mice, while BLyS deficiency results in severe B-cell dysfunction. To investigate the physiological role of APRIL, we generated mice that are deficient in its encoding gene. APRIL(-/-) mice were viable and fertile and lacked any gross abnormality. Detailed histological analysis did not reveal any defects in major tissues and organs, including the primary and secondary immune organs. T- and B-cell development and in vitro function were normal as well, as were T-cell-dependent and -independent in vivo humoral responses to antigenic challenge. These data indicate that APRIL is dispensable in the mouse for proper development. Thus, BLyS may be capable of fulfilling APRIL's main functions.

Dual B cell immunotherapy is superior to individual anti-CD20 depletion or BAFF blockade in murine models of spontaneous or accelerated lupus.

OBJECTIVE: To determine whether a combination of B cell depletion and BAFF blockade is more effective than monotherapy in treating models of spontaneous or accelerated systemic lupus erythematosus (SLE) in (NZB × NZW)F1 mice. METHODS: Clinical parameters such as disease progression-free survival, proteinuria, and renal injury were assessed in models of spontaneous, interferon-α (IFNα)-accelerated, or pristane-accelerated lupus in (NZB × NZW)F1 mice. Treatment arms included anti-CD20 (B cell depletion), B lymphocyte stimulator receptor 3 fusion protein (BR-3-Fc) (BAFF blockade), the combination of anti-CD20 and BR-3-Fc, isotype control, or cyclophosphamide. In models of spontaneous, IFNα-accelerated, or pristane-accelerated lupus, mice were treated for 24 weeks, 8 weeks, or 12 weeks, respectively. Peripheral and resident B cell subsets and various autoantibodies were examined. RESULTS: Compared to B cell depletion or BAFF blockade alone, combined therapy significantly improved disease manifestations in all 3 lupus models. In addition, marginal zone B cells, plasmablasts, and circulating and tissue plasma cells were decreased more effectively. Dual B cell immunotherapy also reduced multiple classes of pathogenic autoantibodies, consistent with its observed effectiveness in reducing immune complex-mediated renal injury. CONCLUSION: Dual immunotherapy via B cell depletion and BAFF blockade is more efficacious than single agent immunotherapy in murine SLE models, and this combination treatment is predicted to be an effective strategy for immunotherapy in human SLE.

Synovial phenotypes in rheumatoid arthritis correlate with response to biologic therapeutics.

INTRODUCTION: Rheumatoid arthritis (RA) is a complex and clinically heterogeneous autoimmune disease. Currently, the relationship between pathogenic molecular drivers of disease in RA and therapeutic response is poorly understood. METHODS: We analyzed synovial tissue samples from two RA cohorts of 49 and 20 patients using a combination of global gene expression, histologic and cellular analyses, and analysis of gene expression data from two further publicly available RA cohorts. To identify candidate serum biomarkers that correspond to differential synovial biology and clinical response to targeted therapies, we performed pre-treatment biomarker analysis compared with therapeutic outcome at week 24 in serum samples from 198 patients from the ADACTA (ADalimumab ACTemrA) phase 4 trial of tocilizumab (anti-IL-6R) monotherapy versus adalimumab (anti-TNFα) monotherapy. RESULTS: We documented evidence for four major phenotypes of RA synovium - lymphoid, myeloid, low inflammatory, and fibroid - each with distinct underlying gene expression signatures. We observed that baseline synovial myeloid, but not lymphoid, gene signature expression was higher in patients with good compared with poor European league against rheumatism (EULAR) clinical response to anti-TNFα therapy at week 16 (P =0.011). We observed that high baseline serum soluble intercellular adhesion molecule 1 (sICAM1), associated with the myeloid phenotype, and high serum C-X-C motif chemokine 13 (CXCL13), associated with the lymphoid phenotype, had differential relationships with clinical response to anti-TNFα compared with anti-IL6R treatment. sICAM1-high/CXCL13-low patients showed the highest week 24 American College of Rheumatology (ACR) 50 response rate to anti-TNFα treatment as compared with sICAM1-low/CXCL13-high patients (42% versus 13%, respectively, P =0.05) while anti-IL-6R patients showed the opposite relationship with these biomarker subgroups (ACR50 20% versus 69%, P =0.004). CONCLUSIONS: These data demonstrate that underlying molecular and cellular heterogeneity in RA impacts clinical outcome to therapies targeting different biological pathways, with patients with the myeloid phenotype exhibiting the most robust response to anti-TNFα. These data suggest a path to identify and validate serum biomarkers that predict response to targeted therapies in rheumatoid arthritis and possibly other autoimmune diseases. TRIAL REGISTRATION: ClinicalTrials.gov NCT01119859

Critical role of activation induced cytidine deaminase in experimental autoimmune encephalomyelitis.

Multiple Sclerosis (MS) is a neurodegenerative autoimmune disorder caused by chronic inflammation and demyelination within the central nervous system (CNS). Clinical studies in MS patients have demonstrated efficacy with B cell targeted therapies such as anti-CD20. However, the exact role that B cells play in the disease process is unclear. Activation Induced cytidine deaminase (AID) is an essential enzyme for the processes of antibody affinity maturation and isotype switching. To evaluate the impact of affinity maturation and isotype switching, we have interrogated the effect of AID-deficiency in an animal model of MS. Here, we show that the severity of experimental autoimmune encephalomyelitis (EAE) induced by the extracellular domain of human myelin oligodendrocyte glycoprotein (MOG1-125) is significantly reduced in Aicda deficient mice, which, unlike wild-type mice, lack serum IgG to myelin associated antigens. MOG specific T cell responses are comparable between wild-type and Aicda knockout mice suggesting an active role for antigen experienced B cells. Thus affinity maturation and/or class switching are critical processes in the pathogenesis of EAE.

Structural basis for the dual recognition of helical cytokines IL-34 and CSF-1 by CSF-1R.

Lacking any discernible sequence similarity, interleukin-34 (IL-34) and colony stimulating factor 1 (CSF-1) signal through a common receptor CSF-1R on cells of mononuclear phagocyte lineage. Here, the crystal structure of dimeric IL-34 reveals a helical cytokine fold homologous to CSF-1, and we further show that the complex architecture of IL-34 bound to the N-terminal immunoglobulin domains of CSF-1R is similar to the CSF-1/CSF-1R assembly. However, unique conformational adaptations in the receptor domain geometry and intermolecular interface explain the cross-reactivity of CSF-1R for two such distantly related ligands. The docking adaptations of the IL-34 and CSF-1 quaternary complexes, when compared to the stem cell factor assembly, draw a common evolutionary theme for transmembrane signaling. In addition, the structure of IL-34 engaged by a Fab fragment reveals the mechanism of a neutralizing antibody that can help deconvolute IL-34 from CSF-1 biology, with implications for therapeutic intervention in diseases with myeloid pathogenic mechanisms.

Equilibrative nucleoside transporter 3 deficiency perturbs lysosome function and macrophage homeostasis.

Lysosomal storage diseases (LSDs) are a group of heterogeneous disorders caused by defects in lysosomal enzymes or transporters, resulting in accumulation of undegraded macromolecules or metabolites. Macrophage numbers are expanded in several LSDs, leading to histiocytosis of unknown pathophysiology. Here, we found that mice lacking the equilibrative nucleoside transporter 3 (ENT3) developed a spontaneous and progressive macrophage-dominated histiocytosis. In the absence of ENT3, defective apoptotic cell clearance led to lysosomal nucleoside buildup, elevated intralysosomal pH, and altered macrophage function. The macrophage accumulation was partly due to increased macrophage colony-stimulating factor and receptor expression and signaling secondary to the lysosomal defects. These studies suggest a cellular and molecular basis for the development of histiocytosis in several human syndromes associated with ENT3 mutations and potentially other LSDs.

Loss of the tumor suppressor BAP1 causes myeloid transformation.

De-ubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with increased risk of mesothelioma and uveal melanoma. Somatic BAP1 mutations occur in various malignancies. We show that mouse Bap1 gene deletion is lethal during embryogenesis, but systemic or hematopoietic-restricted deletion in adults recapitulates features of human myelodysplastic syndrome (MDS). Knockin mice expressing BAP1 with a 3xFlag tag revealed that BAP1 interacts with host cell factor-1 (HCF-1), O-linked N-acetylglucosamine transferase (OGT), and the polycomb group proteins ASXL1 and ASXL2 in vivo. OGT and HCF-1 levels were decreased by Bap1 deletion, indicating a critical role for BAP1 in stabilizing these epigenetic regulators. Human ASXL1 is mutated frequently in chronic myelomonocytic leukemia (CMML) so an ASXL/BAP1 complex may suppress CMML. A BAP1 catalytic mutation found in a MDS patient implies that BAP1 loss of function has similar consequences in mice and humans.