Type I Interferon Response and Vascular Alteration in Chilblain-like Lesions During the COVID-19 Outbreak

L. Frumholtz; J.-D. Bouaziz; M. Battistella; J. Hadjadj; R. Chocron; D. Bengoufa; H. Le Buanec; L. Barnabei; S. Meynier; O. Schwartz; L. Grzelak; N. Smith; B. Charbit; D. Duffy; N. Yatim; A. Calugareanu; A. Philippe; C.L. Guerin; B. Joly; V. Siguret; L. Jaume; H. Bachelez; M. Bagot; F. Rieux-Laucat; S. Maylin; J. Legoff; C. Delaugerre; N. Gendron; D.M. Smadja; C. Cassius


The British Journal of Dermatology. 2021;185(6):1176-1185. 

In This Article

Patients and Methods

Study Design and Population

All patients referred to the dermatology department of Saint-Louis Hospital, Paris, France, with CLL during 9–16 April 2020 of the COVID-19 pandemic were included in this noninterventional observational study. We excluded patients with a history of chilblains or chilblain lupus. Patients provided written informed consent for the collection and analysis of their data. They were assessed at day 0 and day 14. The following controls were included: (i) patients with reverse-transcriptase polymerase chain reaction (RT-PCR)-proven mild COVID-19 without chilblains, for serological, immunological and endothelial activation assessment; (ii) patients with SC before the COVID-19 pandemic defined by typical clinical and histological presentation without antinuclear antibodies (ANA) between January 2015 and March 2019 for histological and immunological assessment; and (iii) healthy controls (HC) without COVID-19 symptoms for serological, cytokine and endothelial activation assessment. We also included HC skin samples obtained from fresh plastic surgery waste.

Human Participant Declaration

All parts of the study were approved by the appropriate institutional review boards (Cochin-Port Royal Hospital, Paris, France) and were conducted in accordance with the current ethical and legal frameworks of the Declaration of Helsinki. Informed written consent was received from participants before inclusion in this study, according to our local ethics rules.

Laboratory Parameters

Blood samples were collected in ethylenediaminetetraacetic acid (EDTA), sodium heparin or 0·11 mol L−1 trisodium citrate tubes (Greiner Bio-One, Courtaboeuf, France). The laboratory parameters recorded included whole blood count (XN3000; Sysmex, Kobe, Japan); haemostasis tests; IgA level; isotypes of IgG and IgA antineutrophil cytoplasmic antibodies (ANCA); ANA; anti-double-stranded DNA antibodies; cryoglobulinemia; cryofibrinogen; and anticardiolipin and anti-β2-glycoprotein-I IgG antibodies (IgM and IgG). The haemostasis tests (STA R Max system; Stago, Asnières sur Seine, France) included prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, D-dimers (Liatest D-Di Plus, Stago), antithrombin activity (Stachrom, Stago), protein C activity (Protein C Coag; Siemens, Munich, Germany) and protein S activity (Staclot PS, Stago), lupus anticoagulant (LA) using integrated diluted Russell viper venom time (dRVVT LAC Screen and LAC Confirm, Siemens) and LA-sensitive aPTT, as described previously.[13]

Flow Spike Assay

This assay is described in Grzelak et al.[14] and is detailed in Appendix S1 (see Supporting Information).

Cytokine Assays

Prior to protein analysis, plasma samples were treated in a P3 laboratory for viral decontamination using a protocol validated for SARS-CoV-2. Briefly, samples were treated with Triton X-100 1% (v/v) for 2 h at room temperature. IFN-α2, IFN-γ and IL-17A protein plasma concentrations were quantified by a Simoa triplex assay developed with Quanterix Homebrew kits (Quanterix, Billerica, MA, USA). Interleukin (IL)-6, tumour necrosis factor (TNF)-α, and IL-10 were measured with a commercial triplex assay (Quanterix). The limits of detection of these assays were 2 fg mL−1 for IFN-α2 and 7 fg mL−1 for IFN-γ.

Assessment of Interferon-stimulated Gene Expression in Whole Blood

Total RNA was extracted using an RNA isolation kit. Further details are provided in Appendix S1.

Soluble Markers of Endothelial Activation and/or Angiogenesis

Platelet-poor plasma (PPP) was obtained from EDTA samples after centrifugation at 2500 g for 15 min at day 0 and 14. After a second centrifugation at 2500 g for 15 min, PPP was stored at −80 °C until analysis of vascular markers. Four biomarkers related to endothelial dysfunction or activation (angiopoietin-1, angiopoietin-2, endoglin and soluble E-selectin) and four biomarkers related to angiogenesis or endothelial progenitor cell mobilization [vascular endothelial growth factor (VEGF)-A, c-Kit, basic fibroblast growth factor (b-FGF) and VEGF receptor] were quantified in PPP with a Human Magnetic Luminex Assay from R&D Systems (Lille, France). Data were assessed with the Bio-Plex 200 using Bio-Plex Manager 5.0 software (Bio-Rad, Marnes-la-Coquette, France) as described previously.[10]

Circulating Endothelial Cell Counting After Immunomagnetic Separation

Peripheral venous blood samples were collected on EDTA at day 14. Circulating endothelial cells (CECs) were counted by an operator blinded to the patients' clinical features. Immunocapture of CECs from whole blood was performed at 4 °C using magnetic beads (Dynabeads M-450, Dynal; Invitrogen, Carlsbad, CA, USA) coated with S-Endo 1 (Biocytex, Marseille, France), a monoclonal antibody raised against the endothelial antigen CD146. To avoid nonspecific binding of leucocytes to CD146-coated beads, the cell suspensions were flushed vigorously through the pipette tip during the washing steps and then suspended in acridine orange (3 μg mL−1 in phosphate-buffered saline; Sigma-Aldrich, Saint-Quentin Fallavier, France) before being counted under a fluorescence microscope (λexc = 490 nm). CECs were identified according to their morphological criteria, namely > 10 beads bound to > 20-μm cells or cells with < 10 beads but with a well-preserved and recognizable morphology (clear nucleus in a well-delineated cytoplasm and a size consistent with that of endothelial cells). The number of cells in aggregates was determined from the number of cells with spherical rosette features. The endothelial nature of the isolated cells was confirmed by measuring lectin Ulex europaeus agglutinin-1 expression. The number of CECs was expressed as cells per mL of blood.[10,11]

Histological Examinations

Skin biopsies from CLL were performed in 13 patients. Histological sections were stained with haematoxylin, eosin and safran. For each sample, histological parameters (epidermal changes, basal vacuolization, inflammatory infiltrate, and type and location of affected vessel) and immunohistochemical analysis for CD61 (integrin β3, clone 2F2; Leica Biosystems, Newcastle upon Tyne, UK), identifying intravascular platelet microthrombi, CD123 (clone 6H6; Exbio, Vestec, Czech Republic) and IgA (polyclonal; Dako, Glostrup, Denmark), were performed on a BenchMark ULTRA immunostainer (Roche-Ventana, Basel, Switzerland). IgA staining was performed on paraffin-embedded tissue sections as described previously.[15]

Gene Expression

From a lesional dermoepidermal skin biopsy, 10 formalin-fixed paraffin-embedded (FFPE) sections were taken, each 10 μm thick. Total RNA was automatically extracted from tissue samples using the Maxwell RSC RNA FFPE Kit protocol (Promega, Charbonnières-les-Bains, France). We analysed 100 ng (5 μL) of total RNA from each sample using the NanoString nCounter Human Immunology V2 kit according to the manufacturer's instructions (NanoString Technologies, Seattle, WA, USA) (Appendix S1).

Statistical Analysis

GraphPad Prism v8.4.0 was used for statistical analysis (GraphPad Software, La Jolla, CA, USA). Comparisons of groups were performed using the unpaired t-test without correction, whereas correction for variance inequality was performed using Welch's correction when the F-test P-value was < 0·2. The Holm–Sidak method was used to correct for multiple testing. Correlations between quantitative variables were assessed using Spearman's correlation coefficient and the associated P-value. P-values < 0·05 were considered statistically significant.