Upregulation of type 1 conventional dendritic cells implicates antigen cross-presentation in multisystem inflammatory syndrome.

BACKGROUND: Multisystem inflammatory syndrome in children (MIS-C) is an acute, febrile, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-associated syndrome, often with cardiohemodynamic dysfunction. Insight into mechanism of disease is still incomplete. OBJECTIVE: Our objective was to analyze immunologic features of MIS-C patients compared to febrile controls (FC). METHODS: MIS-C patients were defined by narrow criteria, including having evidence of cardiohemodynamic involvement and no macrophage activation syndrome. Samples were collected from 8 completely treatment-naive patients with MIS-C (SARS-CoV-2 serology positive), 3 patients with unclassified MIS-C-like disease (serology negative), 14 FC, and 5 MIS-C recovery (RCV). Three healthy controls (HCs) were used for comparisons of normal range. Using spectral flow cytometry, we assessed 36 parameters in antigen-presenting cells (APCs) and 29 in T cells. We used biaxial analysis and uniform manifold approximation and projection (UMAP). RESULTS: Significant elevations in cytokines including CXCL9, M-CSF, and IL-27 were found in MIS-C compared to FC. Classic monocytes and type 2 dendritic cells (DCs) were downregulated (decreased CD86, HLA-DR) versus HCs; however, type 1 DCs (CD11c+CD141+CLEC9A+) were highly activated in MIS-C patients versus FC, expressing higher levels of CD86, CD275, and atypical conventional DC markers such as CD64, CD115, and CX3CR1. CD169 and CD38 were upregulated in multiple monocyte subtypes. CD56dim/CD57-/KLRGhi/CD161+/CD38- natural killer (NK) cells were a unique subset in MIS-C versus FC without macrophage activation syndrome. CONCLUSION: Orchestrated by complex cytokine signaling, type 1 DC activation and NK dysregulation are key features in the pathophysiology of MIS-C. NK cell findings may suggest a relationship with macrophage activation syndrome, while type 1 DC upregulation implies a role for antigen cross-presentation.

CD169/SIGLEC1 is expressed on circulating monocytes in COVID-19 and expression levels are associated with disease severity.

Coronavirus disease 2019 (COVID-19) is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Type I interferons are important in the defense of viral infections. Recently, neutralizing IgG auto-antibodies against type I interferons were found in patients with severe COVID-19 infection. Here, we analyzed expression of CD169/SIGLEC1, a well described downstream molecule in interferon signaling, and found increased monocytic CD169/SIGLEC1 expression levels in patients with mild, acute COVID-19, compared to patients with severe disease. We recommend further clinical studies to evaluate the value of CD169/SIGLEC1 expression in patients with COVID-19 with or without auto-antibodies against type I interferons.

Investigation of type I interferon responses in ANCA-associated vasculitis.

Type I interferon (IFN) dysregulation is a major contributory factor in the development of several autoimmune diseases, termed type I interferonopathies, and is thought to be the pathogenic link with chronic inflammation in these conditions. Anti-neutrophil cytoplasmic antibody (ANCA)-Associated Vasculitis (AAV) is an autoimmune disease characterised by necrotising inflammation of small blood vessels. The underlying biology of AAV is not well understood, however several studies have noted abnormalities in type I IFN responses. We hypothesised that type I IFN responses are systemically dysregulated in AAV, consistent with features of a type I interferonopathy. To investigate this, we measured the expression of seven interferon regulated genes (IRGs) (ISG15, SIGLEC1, STAT1, RSAD2, IFI27, IFI44L and IFIT1) in peripheral blood samples, as well as three type I IFN regulated proteins (CXCL10, MCP-1 and CCL19) in serum samples from AAV patients, healthy controls and disease controls. We found no difference in type I IFN regulated gene or protein expression between AAV patients and healthy controls. Furthermore, IRG and IFN regulated protein expression did not correlate with clinical measurements of disease activity in AAV patients. Thus, we conclude that systemic type I IFN responses are not key drivers of AAV pathogenesis and AAV should not be considered a type I interferonopathy.

Monocyte CD169 expression in COVID-19 patients upon intensive care unit admission.

During the second surge of COVID-19 in France (fall 2020), we assessed the expression of monocyte CD169 (i.e., Siglec-1, one of the numerous IFN-stimulated genes) upon admission to intensive care units of 45 patients with RT-PCR-confirmed SARS-CoV2 pulmonary infection. Overall, CD169 expression was strongly induced on circulating monocytes of COVID-19 patients compared with healthy donors and patients with bacterial sepsis. Beyond its contribution at the emergency department, CD169 testing may be also helpful for patients' triage at the ICU to rapidly reinforce suspicion of COVID-19 etiology in patients with acute respiratory failure awaiting for PCR results for definitive diagnosis.

CD169 and CD64 could help differentiate bacterial from CoVID-19 or other viral infections in the Emergency Department.

The identification of a bacterial, viral, or even noninfectious cause is essential in the management of febrile syndrome in the emergency department (ED), especially in epidemic contexts such as flu or CoVID-19. The aim was to assess discriminative performances of two biomarkers, CD64 on neutrophils (nCD64) and CD169 on monocytes (mCD169), using a new flow cytometry procedure, in patients presenting with fever to the ED during epidemics. Eighty five adult patients presenting with potential infection were included during the 2019 flu season in the ED of La Timone Hospital. They were divided into four diagnostic outcomes according to their clinical records: no-infection, bacterial infection, viral infection and co-infection. Seventy six patients with confirmed SARS-CoV-2 infection were also compared to 48 healthy volunteers. For the first cohort, 38 (45%) patients were diagnosed with bacterial infections, 11 (13%) with viral infections and 29 (34%) with co-infections. mCD169 was elevated in patients with viral infections, with a majority of Flu A virus or Respiratory Syncytial Virus, while nCD64 was elevated in subjects with bacterial infections, with a majority of Streptococcus pneumoniae and Escherichia coli. nCD64 and mCD169 showed 90% and 80% sensitivity, and 78% and 91% specificity, respectively, for identifying patients with bacterial or viral infections. When studied in a second cohort, mCD169 was elevated in 95% of patients with SARS-CoV-2 infections and remained at normal level in 100% of healthy volunteers. nCD64 and mCD169 have potential for accurately distinguishing bacterial and acute viral infections. Combined in an easy and rapid flow cytometry procedure, they constitute a potential improvement for infection management in the ED, and could even help for triage of patients during emerging epidemics.

Flow cytometry evaluation of infection-related biomarkers in febrile subjects in the emergency department.

Aim: In an Emergency Department (ED), the etiological identification of infected subjects is essential. 13 infection-related biomarkers were assessed using a new flow cytometry procedure. Materials & methods: If subjects presented with febrile symptoms at the ED, 13 biomarkers' levels, including CD64 on neutrophils (nCD64) and CD169 on monocytes (mCD169), were tested and compared with clinical records. Results: Among 50 subjects, 78% had bacterial infections and 8% had viral infections. nCD64 showed 82% sensitivity and 91% specificity for identifying subjects with bacterial infections. mCD169, HLA-ABC ratio and HLA-DR on monocytes had high values in subjects with viral infections. Conclusion: Biomarkers showed promising performances to improve the ED's infectious stratification.

The plasma biomarker soluble SIGLEC-1 is associated with the type I interferon transcriptional signature, ethnic background and renal disease in systemic lupus erythematosus.

BACKGROUND: The molecular heterogeneity of autoimmune and inflammatory diseases has been one of the main obstacles to the development of safe and specific therapeutic options. Here, we evaluated the diagnostic and clinical value of a robust, inexpensive, immunoassay detecting the circulating soluble form of the monocyte-specific surface receptor sialic acid binding Ig-like lectin 1 (sSIGLEC-1). METHODS: We developed an immunoassay to measure sSIGLEC-1 in small volumes of plasma/serum from systemic lupus erythematosus (SLE) patients (n = 75) and healthy donors (n = 504). Samples from systemic sclerosis patients (n = 99) were studied as an autoimmune control. We investigated the correlation between sSIGLEC-1 and both monocyte surface SIGLEC-1 and type I interferon-regulated gene (IRG) expression. Associations of sSIGLEC-1 with clinical features were evaluated in an independent cohort of SLE patients (n = 656). RESULTS: Plasma concentrations of sSIGLEC-1 strongly correlated with expression of SIGLEC-1 on the surface of blood monocytes and with IRG expression in SLE patients. We found ancestry-related differences in sSIGLEC-1 concentrations in SLE patients, with patients of non-European ancestry showing higher levels compared to patients of European ancestry. Higher sSIGLEC-1 concentrations were associated with lower serum complement component 3 and increased frequency of renal complications in European patients, but not with the SLE Disease Activity Index clinical score. CONCLUSIONS: Our sSIGLEC-1 immunoassay provides a specific and easily assayed marker for monocyte-macrophage activation, and interferonopathy in SLE and other diseases. Further studies can extend its clinical associations and its potential use to stratify patients and as a secondary endpoint in clinical trials.

Sialic Acid-Binding Immunoglobulin-Like Lectin1 as a Novel Predictive Biomarker for Relapse in Graves' Disease: A Multicenter Study.

BACKGROUND: There are currently no reliable biomarkers to predict relapse in Graves' disease (GD). In the present study, we investigated novel diagnostic biomarkers to predict the long-term remission of or relapse in GD. METHODS: A DNA microarray analysis was performed to examine gene expression in the peripheral leukocytes of a frequently relapsing patient with GD and a patient in long-term remission after the discontinuation of antithyroid drugs (ATDs). Based on the DNA microarray analysis, we focused on Sialic acid-binding immunoglobulin-like lectin1 (SIGLEC1) as a candidate novel biomarker to predict GD relapse. Three hundred and fifty-eight patients with GD in the thyroid clinics of four different hospitals in Japan were included in a cross-sectional study to establish whether SIGLEC1 mRNA levels distinguish GD relapse experience from long-term remission. An additional 55 patients with GD were enrolled in a prospective study to clarify whether SIGLEC1 mRNA levels at ATD discontinuation predict GD relapse. RESULTS: SIGLEC1 mRNA levels were significantly higher in patients with GD relapse experience than in those in long-term remission. Based on the receiver operating characteristic analysis, we found that high SIGLEC1 mRNA levels (≥258.9 copies) significantly distinguished GD relapse experience from long-term remission (p < 0.0001; sensitivity 66.7%, specificity 70.1%). In the prospective study, when the optimal cutoff value from the receiver operating characteristic curve analysis was applied to SIGLEC1 mRNA positivity at ATD discontinuation, SIGLEC1-positive patients (≥258.9 copies) showed a significantly higher cumulative risk of relapse than SIGLEC1-negative patients (<258.9 copies) (p = 0.022, the log-rank test). CONCLUSIONS: SIGLEC1 mRNA levels have potential as a novel predictive biomarker for GD relapse.

Are interferon-related biomarkers advantageous for monitoring disease activity in systemic lupus erythematosus? A longitudinal benchmark study.

Objective: To determine the clinical value of six traditional and three IFN-related biomarkers in monitoring disease activity (DA) in SLE. Methods: Prospective longitudinal study of IFNα, IFNγ-inducible protein 10 (IP-10) and sialic acid-binding Ig-like lectin 1 (SIGLEC1) vs antibodies against dsDNA (ELISA and Farr radioimmunoassay), dsDNA-complexed nucleosomes (anti-dsDNA-NcX: ELISA), nucleosomes (ANuA: ELISA) and complement C3/C4 for correlation with DA (measured by BILAG 2004 index) in 26 SLE patients (77 visits). Optimal upper and lower longitudinal thresholds for the biomarkers and their accuracies for reflecting clinically relevant changes in DA (flares and remission) were determined by receiver operating characteristic and Youden index analysis. Results: Increases in IP-10, SIGLEC1 and ANuA to + 101.6 pg/ml, +5.01 relative mean fluorescence intensity and +16.20 IU/ml above the calculated upper longitudinal threshold significantly reflected lupus flares, with a sensitivity and specificity of 50 and 95% for IP-10, 83 and 90% for SIGLEC1 and 58 and 95% for ANuA. Decreases in anti-dsDNA (ELISA), IFNα and anti-dsDNA (Farr assay) to - 64.7 IU/ml, -16.69 pg/ml and -3.3 IU/ml below lower longitudinal thresholds, respectively, best reflected remission, with sensitivity and specificity of 75 and 95%, 62 and 90%, and 75 and 90%, respectively. Conclusion: IP-10, SIGLEC1 and ANuA emerged as advantageous biomarkers for monitoring disease activity. This is the first study in SLE that provides longitudinal biomarker thresholds and test accuracies for SLE flares and remitting disease. In the context of IFN-directed therapies, chemokines and fluorescence-activated cell sorting-based IFN biomarkers for monitoring SLE activity should be further studied.

Flow Cytometric Analysis of Myeloid Cells in Human Blood, Bronchoalveolar Lavage, and Lung Tissues.

Clear identification of specific cell populations by flow cytometry is important to understand functional roles. A well-defined flow cytometry panel for myeloid cells in human bronchoalveolar lavage (BAL) and lung tissue is currently lacking. The objective of this study was to develop a flow cytometry-based panel for human BAL and lung tissue. We obtained and performed flow cytometry/sorting on human BAL cells and lung tissue. Confocal images were obtained from lung tissue using antibodies for cluster of differentiation (CD)206, CD169, and E cadherin. We defined a multicolor flow panel for human BAL and lung tissue that identifies major leukocyte populations. These include macrophage (CD206(+)) subsets and other CD206(-) leukocytes. The CD206(-) cells include: (1) three monocyte (CD14(+)) subsets, (2) CD11c(+) dendritic cells (CD14(-), CD11c(+), HLA-DR(+)), (3) plasmacytoid dendritic cells (CD14(-), CD11c(-), HLA-DR(+), CD123(+)), and (4) other granulocytes (neutrophils, mast cells, eosinophils, and basophils). Using this panel on human lung tissue, we defined two populations of pulmonary macrophages: CD169(+) and CD169(-) macrophages. In lung tissue, CD169(-) macrophages were a prominent cell type. Using confocal microscopy, CD169(+) macrophages were located in the alveolar space/airway, defining them as alveolar macrophages. In contrast, CD169(-) macrophages were associated with airway/alveolar epithelium, consistent with interstitial-associated macrophages. We defined a flow cytometry panel in human BAL and lung tissue that allows identification of multiple immune cell types and delineates alveolar from interstitial-associated macrophages. This study has important implications for defining myeloid cells in human lung samples.

IFNα and its response proteins, IP-10 and SIGLEC-1, are biomarkers of disease activity in systemic lupus erythematosus.

OBJECTIVES: To evaluate and compare the clinical efficacy of three biomarkers for interferon (IFN) activity (measured directly and indirectly) and six traditional biomarkers in indicating current and prospective disease activity (DA) in systemic lupus erythematosus (SLE). METHODS: IFNα (dissociation-enhanced lanthanide fluorescent immunoassay), IFNγ-inducible protein 10 (IP-10) (ELISA) and sialic acid-binding Ig-like lectin 1 (SIGLEC-1) (flow cytometry) were measured in 79 accurately characterised patients with lupus and compared with serum titres of Anti-dsDNA (ELISA and radioimmunoassay), Anti-dsDNA-NcX ELISA, Anti-Nuc ELISA, and complement C3 and C4. DA was evaluated using the British Isles Lupus Assessment Group 2004 Index (BILAG-2004) and a modified SLE Disease Activity Index-2000 (mSLEDAI-2K). In addition, 31 clinically quiescent patients were monitored for flares over the course of 180 days. RESULTS: Increased levels of IFNα, IP-10 and SIGLEC-1 were found in 32%, 50% and 86%, respectively, of 66 patients with active SLE. IFNα (r=0.45; p<0.0001) and SIGLEC-1 (r=0.54; p<0.0001) correlated better with BILAG-2004 than did IP-10 (r=0.38; p=0.0002), Farr assay (r=0.40; p=0.0001), Anti-dsDNA-NcX ELISA (r=0.28; p=0.0061), Anti-dsDNA ELISA (r=0.31; p=0.0025), Anti-Nuc ELISA (r=0.25; p=0.0121), C3 (r=-0.43; p<0.0001) and C4 (r=-0.33; p=0.0013). Predictors of SLE flares were disease duration ≤92 months, mild clinical activity (in contrast with no activity), complement C3≤89 mg/dl and IFNα≥20 pg/ml, while only lymphocyte count and age were independent predictors in multivariate analysis. CONCLUSIONS: IFNα, IP-10 and SIGLEC-1 emerged as beneficial biomarkers of DA in patients with SLE. Therefore the implementation of IFN biomarkers in standard lupus diagnostics should be reappraised, especially in view of emerging anti-IFN-directed therapies.