Serum interferon-α2 measured by single-molecule array associates with systemic disease manifestations in Sjögren's syndrome.

OBJECTIVES: Type I IFN (IFN-I) activation is a prominent feature of primary SS (pSS), SLE and SSc. Ultrasensitive single-molecule array (Simoa) technology has facilitated the measurement of subfemtomolar concentrations of IFNs. Here we aimed to measure IFN-α2 in serum from pSS, SLE and SSc using a Simoa immunoassay and correlate these levels to blood IFN-stimulated gene (ISG) expression and disease activity. METHODS: Serum IFN-α2 was measured in patients with pSS (n = 85 and n = 110), SLE (n = 24) and SSc (n = 23) and healthy controls (HCs; n = 68) using an IFN-α Simoa assay on an HD-X analyser. IFN-I pathway activation was additionally determined from serum by an IFN-I reporter assay and paired samples of whole blood ISG expression of IFI44, IFI44L, IFIT1, IFIT3 and MxA by RT-PCR or myxovirus resistance protein 1 (MxA) protein ELISA. RESULTS: Serum IFN-α2 levels were elevated in pSS (median 61.3 fg/ml) compared with HCs (median ≤5 fg/ml, P < 0.001) and SSc (median 11.6 fg/ml, P = 0.043), lower compared with SLE (median 313.5 fg/ml, P = 0.068) and positively correlated with blood ISG expression (r = 0.66-0.94, P < 0.001). Comparable to MxA ELISA [area under the curve (AUC) 0.93], IFN-α2 measurement using Simoa identified pSS with high ISG expression (AUC 0.90) with 80-93% specificity and 71-84% sensitivity. Blinded validation in an independent pSS cohort yielded a comparable accuracy. Multiple regression indicated independent associations of autoantibodies, IgG, HCQ treatment, cutaneous disease and a history of extraglandular manifestations with serum IFN-α2 concentrations in pSS. CONCLUSION: Simoa serum IFN-α2 reflects blood ISG expression in pSS, SLE and SSc. In light of IFN-targeting treatments, Simoa could potentially be applied for patient stratification or retrospective analysis of historical cohorts.

Hyperresponsive cytosolic DNA-sensing pathway in monocytes from primary Sjögren's syndrome.

OBJECTIVES: Cytosolic DNA-sensing pathway stimulation prompts type I IFN (IFN-I) production, but its role in systemic IFN-I pathway activation in primary SS (pSS) is poorly studied. Here we investigate the responsiveness of pSS monocytes and plasmacytoid dendritic cells (pDCs) to stimulator of interferon genes (STING) activation in relation to systemic IFN-I pathway activation and compare this with SLE. METHODS: Expression of DNA-sensing receptors cGAS, IFI16, ZBP-1 and DDX41, signalling molecules STING, TBK1 and IRF3, positive and negative STING regulators, and IFN-I-stimulated genes MxA, IFI44, IFI44L, IFIT1 and IFIT3 was analysed in whole blood, CD14+ monocytes, pDCs, and salivary glands by RT-PCR, monocyte RNA sequencing data, flow cytometry and immunohistochemical staining. Peripheral blood mononuclear cells (PBMCs) from pSS, SLE and healthy controls (HCs) were stimulated with STING agonist 2'3'-cGAMP. STING phosphorylation (pSTING) and intracellular IFNα were evaluated using flow cytometry. RESULTS: STING activation induced a significantly higher proportion of IFNα-producing monocytes, but not pDCs, in both IFN-low and IFN-high pSS compared with HC PBMCs. Additionally, a trend towards more pSTING+ monocytes was observed in pSS and SLE, most pronounced in IFN-high patients. Positive STING regulators TRIM38, TRIM56, USP18 and SENP7 were significantly higher expression in pSS than HC monocytes, while the dual-function STING regulator RNF26 was downregulated in pSS monocytes. STING was expressed in mononuclear infiltrates and ductal epithelium in pSS salivary glands. STING stimulation induced pSTING and IFNα in pSS and SLE pDCs. CONCLUSION: pSS monocytes and pDCs are hyperresponsive to stimulation of the STING pathway, which was not restricted to patients with IFN-I pathway activation.

Contrasting expression pattern of RNA-sensing receptors TLR7, RIG-I and MDA5 in interferon-positive and interferon-negative patients with primary Sjögren's syndrome.

OBJECTIVE: The interferon (IFN) type I signature is present in over half of patients with primary Sjögren's syndrome (pSS) and associated with higher disease-activity and autoantibody presence. Plasmacytoid dendritic cells (pDCs) are considered as the main source of enhanced IFN type I expression. The objective of this study was to unravel the molecular pathways underlying IFN type I bioactivity in pDCs of patients with pSS. METHODS: Blood samples from 42 healthy controls (HC) and 115 patients with pSS were stratified according to their IFN type I signature. CD123+BDCA4+ pDCs and CD14+ monocytes were isolated from peripheral blood mononuclear cells (PBMCs). Genome-wide microarray analysis was conducted on sorted pDCs in a small sample set, followed by validation of differentially expressed genes of interest in pDCs and monocytes. RESULTS: We found an upregulation of endosomal toll-like receptor (TLR) 7, but not TLR9, in IFN-positive (IFNpos) pDCs (p<0.05) and monocytes (p=0.024). Additionally, the downstream signalling molecules MyD88, RSAD2 and IRF7 were upregulated, as were the cytoplasmic RNA-sensing receptors DDX58/retinoic acid inducible gene-I (RIG-I) and IFIH1/melanoma differentiation associated gene-5 (MDA5). In vitro triggering of the TLR7-pathway in HC PBMCs induced upregulation of DDX58/RIG-I and IFIH1/MDA5, and downregulated TLR9. The upregulation of TLR7, its downstream signalling pathway, DDX58/RIG-I and IFIH1/MDA5 were confined to patients with IFN-positive pSS. IFN-negative patients had a contrasting expression pattern-TLR7 normal, and decreased TLR9, RIG-I and MDA5. CONCLUSIONS: Here we conclude a contrasting expression pattern of the RNA-sensing receptors TLR7, RIG-I and MDA5 in pDCs and monocytes of patients with IFNpos pSS. This profile could explain the pathogenic IFN production and might reveal novel therapeutic targets in these patients.

The interferon type I signature is present in systemic sclerosis before overt fibrosis and might contribute to its pathogenesis through high BAFF gene expression and high collagen synthesis.

BACKGROUND: Interferon (IFN) signature has been reported in definite systemic sclerosis (SSc) but it has not been characterised in early SSc (EaSSc). We aim at characterising IFN type I signature in SSc before overt skin fibrosis develops. METHODS: The expression of 11 IFN type I inducible genes was tested in whole-blood samples from 30 healthy controls (HCs), 12 subjects with primary Raynaud's phenomenon (RP), 19 patients with EaSSc, 7 patients with definite SSc without cutaneous fibrosis, 21 limited cutaneous SSc and 10 diffuse cutaneous SSc subjects. The correlation between IFN activity in monocytes, B cell activating factor (BAFF) mRNA expression and type III procollagen N-terminal propeptide (PIIINP) serum levels was tested. RESULTS: In all the SSc groups, higher IFN scores were observed compared with HC. An IFN score ≥7.09 discriminated HCs from patients with SSc (sensitivity=0.7, specificity=0.88, area under receiving operating characteristic (AUROC)=0.82); the prevalence of an elevated IFN score was: HC=3.3%; RP=33.3%, EaSSc=78.9%, definite SSc=100%, limited cutaneous SSc=42.9%, diffuse cutaneous SSc=70.0%. In monocytes an IFN score ≥4.12 distinguished HCs from patients with fibrotic SSc (sensitivity=0.62, specificity=0.85, AUROC=0.76). Compared with IFN-negative subjects, IFN-positive subjects had higher monocyte BAFF mRNA levels (19.7±5.2 vs 15.20±4.0, p=2.1×10(-5)) and serum PIIINP levels (median=6.0 (IQR 5.4-8.9) vs median=3.9 (IQR 3.3-4.7), p=0.0004). CONCLUSIONS: An IFN type I signature is observed in patients with SSc from the earliest phases of the disease, even before overt skin fibrosis. The presence of IFN type I signature in monocytes is correlated with BAFF mRNA expression and serum PIIINP levels, supporting a contribution in the pathogenesis and progression of SSc.

Prevalence of distal renal tubular acidosis in primary Sjögren's syndrome.

OBJECTIVES: Our objectives were to analyse the prevalence of distal renal tubular acidosis (dRTA) in primary SS (pSS) and to compare a novel urinary acidification test with furosemide and fludrocortisone (FF) with the gold standard ammonium chloride (NH4Cl) to detect dRTA. METHODS: Urinary acidification was assessed in 57 pSS patients using NH4Cl and FF. A urinary acidification defect was defined as an inability to reach a urinary pH of <5.3 after NH4Cl. RESULTS: The prevalence of complete dRTA (urinary acidification defect with acidosis) was 5% (3/57). All three patients had positive SSA/Ro and SSB/La autoantibodies and impaired kidney function. The prevalence of incomplete dRTA (urinary acidification defect without acidosis) was 25% (14/57). Compared with patients without dRTA, patients with incomplete dRTA had significantly lower venous pH and serum bicarbonate and higher urinary pH. SSB/La antibodies were more prevalent in the dRTA groups (P < 0.05). Compared with NH4Cl, the positive and negative predictive values of FF were 46% and 82%, respectively. Vomiting occurred more often during the urinary acidification test with NH4Cl than with FF (9 vs 0, P < 0.05). CONCLUSION: Incomplete dRTA is common in pSS and causes mild acidaemia and higher urinary pH, which may contribute to bone demineralization and kidney stone formation. FF cannot replace NH4Cl in testing urinary acidification in pSS, but may be considered as a screening tool, given its reasonable negative predictive value and better tolerability.

MxA as a clinically applicable biomarker for identifying systemic interferon type I in primary Sjogren's syndrome.

OBJECTIVE: To establish an easy and practical assay for identifying systemic interferon (IFN) type I bioactivity in patients with primary Sjögren's syndrome (pSS). The IFN type I signature is present in over half of the pSS patients and identifies a subgroup with a higher disease activity. This signature is currently assessed via laborious expression profiles of multiple IFN type I-inducible genes. METHODS: In a cohort of 35 pSS patients, myxovirus-resistance protein A (MxA) was assessed as a potential biomarker for type I IFN activity, using an enzyme immunoassay (EIA) on whole-blood and flow cytometric analyses (fluorescence-activated cell sorting, FACS) of isolated CD14 monocytes. In addition, potential biomarkers such as CD64, CD169 and B cell-activating factor (BAFF) were simultaneously analysed in CD14 monocytes using FACS. The IFNscore, a measure for total type I IFN bioactivity, was calculated using expression values of the IFN type I signature genes--IFI44, IFI44L, IFIT3, LY6E and MX1--in CD14 monocytes, determined by real-time quantitative PCR. RESULTS: IFNscores correlated the strongest with monocyte MxA protein (r=0.741, p<0.001) and whole-blood MxA levels (r=0.764, p<0.001), weaker with CD169 (r=0.495, p<0.001) and CD64 (r=0.436, p=0.007), and not at all with BAFF protein. In particular, whole blood MxA levels correlated with EULAR Sjögren's Syndrome Disease Activity Index scores and numerous clinical pSS parameters. Interestingly, patients on hydroxychloroquine showed reduced MxA levels (EIA, p=0.04; FACS p=0.001). CONCLUSIONS: The MxA assays were excellent tools to assess IFN type I activity in pSS, MxA-EIA being the most practical. MxA levels associate with features of active disease and are reduced in hydroxychloroquine-treated patients, suggesting the clinical applicability of MxA in stratifying patients according to IFN positivity.

Prevalence of interferon type I signature in CD14 monocytes of patients with Sjogren's syndrome and association with disease activity and BAFF gene expression.

OBJECTIVE: To determine the prevalence of upregulation of interferon (IFN) type I inducible genes, the so called 'IFN type I signature', in CD14 monocytes in 69 patients with primary Sjögren's syndrome (pSS) and 44 healthy controls (HC) and correlate it with disease manifestations and expression of B cell activating factor (BAFF). METHODS: Expression of IFI44L, IFI44, IFIT3, LY6E and MX1 was measured using real time quantitative PCR in monocytes. Expression values were used to calculate IFN type I scores for each subject. pSS patients positive for the IFN type I signature (IFN score≥10) and patients negative for the signature (IFN score<10) were then compared for clinical disease manifestations and BAFF expression. A bioassay using a monocytic cell line was performed to study whether BAFF mRNA expression was inducible by IFN type I activity in serum of patients with pSS. RESULTS: An IFN type I signature was present in 55% of patients with pSS compared with 4.5% of HC. Patients with the IFN type I signature showed: (a) higher EULAR Sjögren's Syndrome Disease Activity Index scores; higher anti-Ro52, anti-Ro60 and anti-La autoantibodies; higher rheumatoid factor; higher serum IgG; lower C3, lower absolute lymphocyte and neutrophil counts; (b)higher BAFF gene expression in monocytes. In addition, serum of signature-positive patients induced BAFF gene expression in monocytes. CONCLUSIONS: The monocyte IFN type I signature identifies a subgroup of patients with pSS with a higher clinical disease activity together with higher BAFF mRNA expression. Such patients might benefit from treatment blocking IFN type I production or activity.

Extranodal marginal zone lymphoma clonotypes are detectable prior to eMZL diagnosis in tissue biopsies and peripheral blood of Sjögren's syndrome patients through immunogenetics.

Introduction: Activated B cells play a key role in the pathogenesis of primary Sjögren's syndrome (pSS) through the production of autoantibodies and the development of ectopic germinal centers in the salivary glands and other affected sites. Around 5-10% of pSS patients develop B-cell lymphoma, usually extranodal marginal zone lymphomas (eMZL) of the mucosa-associated lymphoid tissue (MALT). The aim of the current study is to investigate if the eMZL clonotype is detectable in prediagnostic blood and tissue biopsies of pSS patients. Methods/Results: We studied prediagnostic tissue biopsies of three pSS patients diagnosed with eMZL and four pSS controls through immunoglobulin (IG) gene repertoire sequencing. In all three cases, we observed the eMZL clonotype in prediagnostic tissue biopsies. Among controls, we observed transient elevation of clonotypes in two pSS patients. To evaluate if eMZL clonotypes may also be detected in the circulation, we sequenced a peripheral blood mononuclear cell (PBMC) sample drawn at eMZL diagnosis and two years prior to eMZL relapse in two pSS patients. The eMZL clonotype was detected in the peripheral blood prior to diagnosis in both cases. Next, we selected three pSS patients who developed eMZL lymphoma and five additional pSS patients who remained lymphoma-free. We sequenced the IG heavy chain (IGH) gene repertoire in PBMC samples taken a median of three years before eMZL diagnosis. In two out of three eMZL patients, the dominant clonotype in the prediagnostic PBMC samples matched the eMZL clonotype in the diagnostic biopsy. The eMZL clonotypes observed consisted of stereotypic IGHV gene combinations (IGHV1-69/IGHJ4 and IGHV4-59/IGHJ5) associated with rheumatoid factor activity, a previously reported feature of eMZL in pSS. Discussion: In conclusion, our results indicate that eMZL clonotypes in pSS patients are detectable prior to overt eMZL diagnosis in both tissue biopsies and peripheral blood through immunogenetic sequencing, paving the way for the development of improved methods of early detection of eMZL.

Trained Immunity in Primary Sjögren's Syndrome: Linking Type I Interferons to a Pro-Atherogenic Phenotype.

Background: Trained immunity - or innate immune memory - can be described as the long-term reprogramming of innate immune cells towards a hyperresponsive state which involves intracellular metabolic changes. Trained immunity has been linked to atherosclerosis. A subgroup of patients with primary Sjögren's syndrome (pSS) exhibits systemic type I interferon (IFN) pathway activation, indicating innate immune hyperactivation. Here, we studied the link between type I IFNs and trained immunity in an in vitro monocytic cell model and peripheral blood mononuclear cells (PBMCs) from pSS patients. Methods: The training stimuli heat killed Candida albicans, muramyl dipeptide, IFNß, and patient serum were added to THP-1 cells for 24 hours, after which the cells were washed, rested for 48 hours and subsequently re-stimulated with LPS, Pam3Cys, poly I:C, IFNß or oxLDL for 4-24 hours. PBMCs from pSS patients and healthy controls were stimulated with LPS, Pam3Cys, poly I:C or IFNß for 0.5-24 hours. Results: Training with IFNß induced elevated production of pro-atherogenic cytokines IL-6, TNFα and CCL2, differential cholesterol- and glycolysis-related gene expression, and increased glucose consumption and oxLDL uptake upon re-stimulation. Type I IFN production was increased in Candida albicans- and IFNß-trained cells after LPS re-stimulation, but was reduced after poly I:C re-stimulation. Training with muramyl dipeptide and IFNß, but not Candida albicans, affected the IFN-stimulated gene expression response to IFNß re-stimulation. PBMCs from pSS patients consumed more glucose compared with healthy control PBMCs and tended to produce more TNFα and type I IFNs upon LPS stimulation, but less type I IFNs upon poly I:C stimulation. Conclusions: Type I IFN is a trainer inducing a trained immunity phenotype with pro-atherogenic properties in monocytes. Conversely, trained immunity also affects the production of type I IFNs and transcriptional response to type I IFN receptor re-stimulation. The phenotype of pSS PBMCs is consistent with trained immunity. This connection between type I IFN, trained immunity and cholesterol metabolism may have important implications for pSS and the pathogenesis of (subclinical) atherosclerosis in these patients.

Characteristics of COVID-19 infection and antibody formation in patients known at a tertiary immunology department.

BACKGROUND: Knowledge about COVID-19 infections is expanding, although knowledge about the disease course and antibody formation in patients with an auto-immune disease or immunodeficiency is not fully unraveled yet. It could be hypothesized that immunodeficient patients, due to immunosuppressive drugs or their disease, have a more severe disease course due to their immunocompromised state. However, it could also be hypothesized that some of the immunosuppressive drugs protect against a hyperinflammatory state. METHODS: We collected data on the incidence of COVID-19, disease course and SARS-CoV-2 antibody formation in COVID-19 positive patients in a cohort of patients (n â€‹= â€‹4497) known at the Clinical Immunology outpatient clinic in a tertiary care hospital in the Netherlands. RESULTS: In the first six months of the pandemic, 16 patients were identified with COVID-19, 14 by nasal swab PCR, and 2 patients by SARS-CoV-2 antibodies. Eight patients were admitted to the hospital. SARS-CoV-2 antibodies were measured in 8 patients and were detectable in all, including one patient on B-cell ablative therapy and one patient with Common Variable Immunodeficiency Disorder. CONCLUSION: This study indicates that the disease course differs among immunocompromised patients, independently of (dis)continuation of immunosuppressive drugs. Antibody production for SARS-CoV-2 in immunocompromised patients was shown. More research needs to be conducted to confirm these observations and guidelines regarding (dis)continuation of immunosuppressive drugs in COVID-19 positive immunocompromised patients should be developed.

Association of Increased Treg Cell Levels With Elevated Indoleamine 2,3-Dioxygenase Activity and an Imbalanced Kynurenine Pathway in Interferon-Positive Primary Sjögren's Syndrome.

OBJECTIVE: Indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme that converts tryptophan to kynurenine, is driven in part by type I and type II interferons (IFNs). Naive T cells are polarized into FoxP3+ Treg cells upon exposure to either IDO+ cells or kynurenine. Recent studies have suggested that the kynurenine pathway reflects a crucial interface between the immune and nervous system. The aims of the present study were to evaluate whether Treg cell levels are elevated, in conjunction with increased IDO activity, in patients with primary Sjögren's syndrome (SS) who are positive for the IFN gene expression signature, and to investigate the downstream kynurenine pathway in these patients. METHODS: Serum from 71 healthy controls, 58 IFN-negative patients with primary SS, and 66 IFN-positive patients with primary SS was analyzed using high-performance liquid chromatography to measure the levels of tryptophan and kynurenine. Expression levels of messenger RNA (mRNA) for IDO and downstream enzymes in the kynurenine pathway were assessed in CD14+ monocytes using real-time quantitative polymerase chain reaction. CD4+CD45RO+ T helper memory cell populations were analyzed by flow cytometry. RESULTS: Significantly increased levels of IDO activity (assessed as the kynurenine:tryptophan ratio) (P = 0.0054) and percentages of CD25(high) FoxP3+ Treg cells (P = 0.039) were observed in the serum from IFN-positive patients with primary SS, and these parameters were significantly correlated with one another (r = 0.511, P = 0.002). In circulating monocytes from IFN-positive patients with primary SS, the expression of IDO1 mRNA was up-regulated (P < 0.0001), and this was correlated with the IFN gene expression score (r = 0.816, P < 0.0001). Interestingly, the proapoptotic and neurotoxic downstream enzyme kynurenine 3-monooxygenase was up-regulated (P = 0.0057), whereas kynurenine aminotransferase I (KATI) (P = 0.0003), KATIII (P = 0.016), and KATIV (P = 0.04) were down-regulated in IFN-positive patients with primary SS compared to healthy controls. CONCLUSION: These findings demonstrate enhanced IDO activity in conjunction with increased percentages of CD25(high) FoxP3+ Treg cells in primary SS patients who carry the IFN signature. In addition, IFN-positive patients with primary SS exhibit an imbalanced kynurenine pathway, with evidence of a shift toward potentially more proapoptotic and neurotoxic metabolites. Intervening in these IFN- and IDO-induced immune system imbalances may offer a new array of possibilities for therapeutic interventions in patients with primary SS.

Type I IFN signature in primary Sjögren's syndrome patients.

Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease characterized by lymphocytic infiltrates in salivary and lacrimal glands. Clinical manifestations range from ocular and oral dryness to vasculitis and severe fatigue. pSS is a disease with heterogeneous symptoms and a variable response to the available treatment. Recently, a key role for Interferon (IFN) type I has been implicated in the pathogenesis of pSS. As type I IFN consists of 17 different subtypes, it cannot be easily assessed using a conventional ELISA. Therefore the expression of type I IFN inducible genes--the so-called type I IFN signature--is assessed in salivary gland tissue and blood from patients as a readout for type I IFN activity. In this review we discuss the potential of type I IFN as a novel biomarker for disease activity, subclassification of patients, prediction of therapy response and most importantly as a target for therapeutic intervention.

T-helper 17 cell cytokines and interferon type I: partners in crime in systemic lupus erythematosus?

INTRODUCTION: A hallmark of systemic autoimmune diseases like systemic lupus erythematosus (SLE) is the increased expression of interferon (IFN) type I inducible genes, so-called IFN type I signature. Recently, T-helper 17 subset (Th17 cells), which produces IL-17A, IL-17F, IL-21, and IL-22, has been implicated in SLE. As CCR6 enriches for Th17 cells, we used this approach to investigate whether CCR6⁺ memory T-helper cells producing IL-17A, IL-17F, IL-21, and/or IL-22 are increased in SLE patients and whether this increase is related to the presence of IFN type I signature. METHODS: In total, 25 SLE patients and 15 healthy controls (HCs) were included. SLE patients were divided into IFN type I signature-positive (IFN⁺) (n = 16) and negative (IFN⁻) (n = 9) patients, as assessed by mRNA expression of IFN-inducible genes (IFIGs) in monocytes. Expression of IL-17A, IL-17F, IL-21, and IL-22 by CD4⁺CD45RO⁺CCR6⁺ T cells (CCR6⁺ cells) was measured with flow cytometry and compared between IFN⁺, IFN⁻ patients and HCs. RESULTS: Increased percentages of IL-17A and IL-17A/IL-17F double-producing CCR6⁺ cells were observed in IFN⁺ patients compared with IFN⁻ patients and HCs. IL-17A and IL-17F expression within CCR6⁺ cells correlated significantly with IFIG expression. In addition, we found significant correlation between B-cell activating factor of the tumor necrosis family (BAFF)-a factor strongly correlating with IFN type I - and IL-21 producing CCR6⁺ cells. CONCLUSIONS: We show for the first time higher percentages of IL-17A and IL-17A/IL-17F double-producing CCR6⁺ memory T-helper cells in IFN⁺ SLE patients, supporting the hypothesis that IFN type I co-acts with Th17 cytokines in SLE pathogenesis.