Difference in clinical presentation between women and men in incident primary Sjögren’s syndrome

Background: A more severe disease phenotype has been reported in men compared to women in several rheumatic diseases. However, studies have not conclusively established sex-related clinical features in primary Sjögren’s syndrome (pSS). In this study, we therefore investigated the clinical presentation of pSS in women and men at diagnosis. Methods: Incident, treatment naïve patients (n = 199) during a 5-year period in a specified area were prospectively included and examined for items of classification criteria for pSS as well as extraglandular manifestations (EGM). Serum was sampled at the time of diagnosis and anti-Ro52/SSA levels were measured by ELISA. Replication of significant findings was confirmed in an independent cohort of pSS patients (n = 377), and meta-analysis was performed. Results: An increased frequency of extraglandular manifestations in men was observed and replicated (p = 0.05, p = 0.0003, and p meta = 0.002). This related to pulmonary involvement, vasculitis, and lymphadenopathy being more common in men, for whom a lower age at diagnosis was observed in the exploratory cohort. Additionally, SSA-positive male patients had significantly higher levels of anti-Ro52 levels than their female counterparts in sera available for analysis (p = 0.02). Conclusions: Our analysis of two independent cohorts of incident pSS demonstrates that the presence and number of EGM are significantly more frequent among men with pSS than women at diagnosis. Importantly, around half of the male patients presented with more than one EGM at diagnosis, supporting the conclusion that pSS in men represents a more severe form of disease, regardless of the lower risk for men to develop pSS

Differential effects on BAFF and APRIL levels in rituximab-treated patients with systemic lupus erythematosus and rheumatoid arthritis

The objective of this study was to investigate the interaction between levels of BAFF (B-cell activation factor of the tumour necrosis factor [TNF] family) and APRIL (a proliferation-inducing ligand) and B-cell frequencies in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) treated with the B-cell-depleting agent rituximab. Ten patients with SLE were treated with rituximab in combination with cyclophosphamide and corticosteroids. They were followed longitudinally up to 6 months after B-cell repopulation. Nine patients with RA, resistant or intolerant to anti-TNF therapy, treated with rituximab plus methotrexate were investigated up to 6 months after treatment. The B-cell frequency was determined by flow cytometry, and serum levels of BAFF and APRIL were measured by enzyme-linked immunosorbent assays. BAFF levels rose significantly during B-cell depletion in both patient groups, and in patients with SLE the BAFF levels declined close to pre-treatment levels upon B-cell repopulation. Patients with SLE had normal levels of APRIL at baseline, and during depletion there was a significant decrease. In contrast, patients with RA had APRIL levels 10-fold higher than normal, which did not change during depletion. At baseline, correlations between levels of B cells and APRIL, and DAS28 (disease activity score using 28 joint counts) and BAFF were observed in patients with RA. In summary, increased BAFF levels were observed during absence of circulating B cells in our SLE and RA patient cohorts. In spite of the limited number of patients, our data suggest that BAFF and APRIL are differentially regulated in different autoimmune diseases and, in addition, differently affected by rituximab treatment

Skin infiltrating NK cells in cutaneous T-cell lymphoma are increased in number and display phenotypic alterations partially driven by the tumor

Cutaneous T-cell lymphomas (CTCL) are characterized by focal infiltration of malignant T cell clones in solitary skin lesions. Many CTCL patients experience an indolent disease, but some progress to advanced disease with high fatality. We hypothesized that natural killer (NK) cells participate in local control of tumor growth in CTCL skin. Immunohistochemistry and flow cytometry analysis of the density, localization, phenotype and function of NK cells in twenty-nine fresh or formalin-fixed skin biopsies from twenty-four CTCL patients and twenty-three biopsies from twenty healthy controls highlighted higher numbers of CD56+CD3- NK cells in CTCL skin. A reduced fraction of CTCL skin NK cells expressed the maturation marker CD57, the cytotoxic protein granzyme B and the activation marker CD69, indicating reduced tumor-killing abilities of the NK cells. Retained expression of immune checkpoint proteins or inhibitory proteins including PD1, TIM3, LAG3, CD73 and NKG2A and the activating receptors CD16 and NKp46 indicated maintained effector functions. Indeed, the capacity of NK cells to produce anti-tumor acting IFNγ upon PMA+ionomycin stimulation was similar in cells from CTCL and healthy skin. Co-cultures of primary human NK cells or the NK cell line NKL with CTCL cells resulted in reduced levels of granzyme B and CD69, indicating that close cellular interactions with CTCL cells induced the impaired functional NK cell phenotype. In conclusion, increased numbers of NK cells in CTCL skin exhibit a partially impaired phenotype in terms of activity. Enhancing NK cell activity with NK cell activating cytokines such as IL-15 or immune checkpoint blockade therefore represents a potential immunotherapeutic approach in CTCL

Ro/SSA autoantibodies directly bind cardiomyocytes, disturb calcium homeostasis, and mediate congenital heart block

Congenital heart block develops in fetuses after placental transfer of Ro/SSA autoantibodies from rheumatic mothers. The condition is often fatal and the majority of live-born children require a pacemaker at an early age. The specific antibody that induces the heart block and the mechanism by which it mediates the pathogenic effect have not been elucidated. In this study, we define the cellular mechanism leading to the disease and show that maternal autoantibodies directed to a specific epitope within the leucine zipper amino acid sequence 200–239 (p200) of the Ro52 protein correlate with prolongation of fetal atrioventricular (AV) time and heart block. This finding was further confirmed experimentally in that pups born to rats immunized with p200 peptide developed AV block. p200-specific autoantibodies cloned from patients bound cultured cardiomyocytes and severely affected Ca2+ oscillations, leading to accumulating levels and overload of intracellular Ca2+ levels with subsequent loss of contractility and ultimately apoptosis. These findings suggest that passive transfer of maternal p200 autoantibodies causes congenital heart block by dysregulating Ca2+ homeostasis and inducing death in affected cells

Skin infiltrating NK cells in cutaneous T-cell lymphoma are increased in number and display phenotypic alterations partially driven by the tumor

Cutaneous T-cell lymphomas (CTCL) are characterized by focal infiltration of malignant T cell clones in solitary skin lesions. Many CTCL patients experience an indolent disease, but some progress to advanced disease with high fatality. We hypothesized that natural killer (NK) cells participate in local control of tumor growth in CTCL skin. Immunohistochemistry and flow cytometry analysis of the density, localization, phenotype and function of NK cells in twenty-nine fresh or formalin-fixed skin biopsies from twenty-four CTCL patients and twenty-three biopsies from twenty healthy controls highlighted higher numbers of CD56+CD3- NK cells in CTCL skin. A reduced fraction of CTCL skin NK cells expressed the maturation marker CD57, the cytotoxic protein granzyme B and the activation marker CD69, indicating reduced tumor-killing abilities of the NK cells. Retained expression of immune checkpoint proteins or inhibitory proteins including PD1, TIM3, LAG3, CD73 and NKG2A and the activating receptors CD16 and NKp46 indicated maintained effector functions. Indeed, the capacity of NK cells to produce anti-tumor acting IFNγ upon PMA+ionomycin stimulation was similar in cells from CTCL and healthy skin. Co-cultures of primary human NK cells or the NK cell line NKL with CTCL cells resulted in reduced levels of granzyme B and CD69, indicating that close cellular interactions with CTCL cells induced the impaired functional NK cell phenotype. In conclusion, increased numbers of NK cells in CTCL skin exhibit a partially impaired phenotype in terms of activity. Enhancing NK cell activity with NK cell activating cytokines such as IL-15 or immune checkpoint blockade therefore represents a potential immunotherapeutic approach in CTCL.publishedVersio

Autoantigenic properties of the aminoacyl tRNA synthetase family in idiopathic inflammatory myopathies

Objectives: Autoantibodies are thought to play a key role in the pathogenesis of idiopathic inflammatory myopathies (IIM). However, up to 40% of IIM patients, even those with clinical manifestations of anti-synthetase syndrome (ASSD), test seronegative to known myositis-specific autoantibodies. We hypothesized the existence of new potential autoantigens among human cytoplasmic aminoacyl tRNA synthetases (aaRS) in patients with IIM. Methods: Plasma samples from 217 patients with IIM according to 2017 EULAR/ACR criteria, including 50 patients with ASSD, 165 without, and two with unknown ASSD status were identified retrospectively, as well as age and gender-matched sera from 156 population controls, and 219 disease controls. Patients with previously documented ASSD had to test positive for at least one of the five most common anti-aaRS autoantibodies (anti-Jo1, -PL7, -PL12, -EJ, and -OJ) and present with one or more of the following clinical manifestations: interstitial lung disease, myositis, arthritis, Raynaud's phenomenon, fever, or mechanic's hands. Demographics, laboratory, and clinical data of the IIM cohort (ASSD and non-ASSD) were compared. Samples were screened using a multiplex bead array assay for presence of autoantibodies against a panel of 117 recombinant protein variants, representing 33 myositis-related proteins, including all nineteen cytoplasmic aaRS. Prospectively collected clinical data for the IIM cohort were retrieved and compared between groups within the IIM cohort and correlated with the results of the autoantibody screening. Principal component analysis was used to analyze clinical manifestations between ASSD, non-ASSD groups, and individuals with novel anti-aaRS autoantibodies. Results: We identified reactivity towards 16 aaRS in 72 of the 217 IIM patients. Twelve patients displayed reactivity against nine novel aaRS. The novel autoantibody specificities were detected in four previously seronegative patients for myositis-specific autoantibodies and eight with previously detected myositis-specific autoantibodies. IIM individuals with novel anti-aaRS autoantibodies (n = 12) all had signs of myositis, and they had either muscle weakness and/or muscle enzyme elevation, 2/12 had mechanic's hands, 3/12 had interstitial lung disease, and 2/12 had arthritis. The individuals with novel anti-aaRS and a pathological muscle biopsy all presented widespread up-regulation of major histocompatibility complex class I. The reactivities against novel aaRS could be confirmed in ELISA and western blot. Using the multiplex bead array assay, we could confirm previously known reactivities to four of the most common aaRS (Jo1, PL12, PL7, and EJ (n = 45)) and identified patients positive for anti-Zo, -KS, and -HA (n = 10) that were not previously tested. A low frequency of anti-aaRS autoantibodies was also detected in controls. Conclusion: Our results suggest that most, if not all, cytoplasmic aaRS may become autoantigenic. Autoantibodies against new aaRS may be found in plasma of patients previously classified as seronegative with potential high clinical relevance.publishedVersio

SOCS3 Expression by Thymic Stromal Cells Is Required for Normal T Cell Development

The suppressor of cytokine signaling 3 (SOCS3) is a major regulator of immune responses and inflammation as it negatively regulates cytokine signaling. Here, the role of SOCS3 in thymic T cell formation was studied in Socs3fl/fl Actin-creER mice (Δsocs3) with a tamoxifen inducible and ubiquitous Socs3 deficiency. Δsocs3 thymi showed a 90% loss of cellularity and altered cortico-medullary organization. Thymocyte differentiation and proliferation was impaired at the early double negative (CD4-CD8-) cell stage and apoptosis was increased during the double positive (CD4+CD8+) cell stage, resulting in the reduction of recent thymic emigrants in peripheral organs. Using bone marrow chimeras, transplanting thymic organoids and using mice deficient of SOCS3 in thymocytes we found that expression in thymic stromal cells rather than in thymocytes was critical for T cell development. We found that SOCS3 in thymic epithelial cells (TECs) binds to the E3 ubiquitin ligase TRIM 21 and that Trim21−/− mice showed increased thymic cellularity. Δsocs3 TECs showed alterations in the expression of genes involved in positive and negative selection and lympho-stromal interactions. SOCS3-dependent signal inhibition of the common gp130 subunit of the IL-6 receptor family was redundant for T cell formation. Together, SOCS3 expression in thymic stroma cells is critical for T cell development and for maintenance of thymus architecture.publishedVersio

Enhanced interferon regulatory factor 3 binding to the interleukin-23p19 promoter correlates with enhanced interleukin-23 expression in systemic lupus erythematosus.

OBJECTIVE: To examine the role of interferon regulatory factor 3 (IRF-3) in the regulation of interleukin-23 (IL-23) production in patients with systemic lupus erythematosus (SLE). METHODS: Bone marrow-derived macrophages were isolated from both wild-type and IRF3(-/-) C57BL/6 mice. These cells were stimulated with the Toll-like receptor 3 (TLR-3) agonist poly(I-C), and IL-23p19 cytokine levels were analyzed by enzyme-linked immunosorbent assay. IRF-3 binding to the IL-23p19 gene promoter region in monocytes from patients with SLE and healthy control subjects was analyzed by chromatin immunoprecipitation (ChIP) assay. Luciferase reporter gene assays were performed to identify key drivers of IL-23p19 promoter activity. TANK-binding kinase 1 (TBK-1) protein levels were determined by Western blotting. RESULTS: ChIP assays demonstrated that IRF-3 was stably bound to the human IL-23p19 promoter in monocytes; this association increased following TLR-3 stimulation. Patients with SLE demonstrated increased levels of IRF-3 bound to the IL-23p19 promoter compared with control subjects, which correlated with enhanced IL-23p19 production in monocytes from patients with SLE. Investigations of the TLR-3-driven responses in monocytes from patients with SLE revealed that TBK-1, which is critical for regulating IRF-3 activity, was hyperactivated in both resting and TLR-3-stimulated cells. CONCLUSION: Our results demonstrate for the first time that patients with SLE display enhanced IL-23p19 expression as a result of hyperactivation of TBK-1, resulting in increased binding of IRF-3 to the promoter. These findings provide novel insights into the molecular pathogenesis of SLE and the potential role for TLR-3 in driving this response

Loss of the Lupus Autoantigen Ro52/Trim21 Induces Tissue Inflammation and Systemic Autoimmunity by Disregulating the IL-23–Th17 Pathway

Ro52/Trim21 is targeted as an autoantigen in systemic lupus erythematosus and Sjögren's syndrome. Polymorphisms in the Ro52 gene have been linked to these autoimmune conditions, but the molecular mechanism by which Ro52 may promote development of systemic autoimmune diseases has not been explored. To address this issue, we generated Ro52-null mice ($Ro52^{−/−}$), which appear phenotypically normal if left unmanipulated. However, $Ro52^{−/−}$ mice develop severe dermatitis extending from the site of tissue injury induced by ear tags. The affected mice further develop several signs of systemic lupus with hypergammaglobulinemia, autoantibodies to DNA, proteinuria, and kidney pathology. Ro52, which was recently identified as an E3 ligase, mediates ubiquitination of several members of the interferon regulatory factor (IRF) family, and the Ro52-deficient mice have an enhanced production of proinflammatory cytokines that are regulated by the IRF transcription factors, including cytokines involved in the Th17 pathway (interleukin [IL] 6, IL-12/IL-23p40, and IL-17). Loss of IL-23/IL-17 by genetic deletion of IL-23/p19 in the $Ro52^{−/−}$ mice conferred protection from skin disease and systemic autoimmunity. These data reveal that the lupus-associated Ro52 protein is an important negative regulator of proinflammatory cytokine production, and they provide a mechanism by which a defective Ro52 function can lead to tissue inflammation and systemic autoimmunity through the IL-23–Th17 pathway

Polymorphisms of the ITGAM Gene Confer Higher Risk of Discoid Cutaneous than of Systemic Lupus Erythematosus

Background Lupus erythematosus (LE) is a heterogeneous disease ranging from mainly skin-restricted manifestations (discoid LE [DLE] and subacute cutaneous LE) to a progressive multisystem disease (systemic LE [SLE]). Genetic association studies have recently identified several strong susceptibility genes for SLE, including integrin alpha M (ITGAM), also known as CD11b, whereas the genetic background of DLE is less clear. Principal findings To specifically investigate whether ITGAM is a susceptibility gene not only for SLE, but also for cutaneous DLE, we genotyped 177 patients with DLE, 85 patients with sporadic SLE, 190 index cases from SLE families and 395 population control individuals from Finland for nine genetic markers at the ITGAM locus. SLE patients were further subdivided by the presence or absence of discoid rash and renal involvement. In addition, 235 Finnish and Swedish patients positive for Ro/SSA-autoantibodies were included in a subphenotype analysis. Analysis of the ITGAM coding variant rs1143679 showed highly significant association to DLE in patients without signs of systemic disease (P-value = 4.73x10-11, OR = 3.20, 95% CI = 2.23-4.57). Significant association was also detected to SLE patients (P-value = 8.29x10-6, OR = 2.14, 95% CI = 1.52-3.00), and even stronger association was found when stratifying SLE patients by presence of discoid rash (P-value = 3.59x10-8, OR = 3.76, 95% CI = 2.29-6.18). Significance We propose ITGAM as a novel susceptibility gene for cutaneous DLE. The risk effect is independent of systemic involvement and has an even stronger genetic influence on the risk of DLE than of SLE.Peer reviewe