Pandemic-associated pernio harbors footprints of an abortive SARS-CoV-2 infection

Abstract

Elevated pernio incidence was observed during the COVID-19 pandemic. This prospective study enrolled subjects with pandemic-associated pernio in Wisconsin and Switzerland. Because pernio is a cutaneous manifestation of the interferonopathies, and type I interferon (IFN-I) immunity is critical to COVID-19 recovery, we tested the hypothesis that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-mediated IFN-I signaling might underlie some pernio cases. Tissue-level IFN-I activity and plasmacytoid dendritic cell infiltrates were demonstrated in 100% of the Wisconsin cases. Across both cohorts, sparse SARS-CoV-2 RNA was captured in 25% (6/22) of biopsies, all with high inflammation. Affected patients lacked adaptive immunity to SARS-CoV-2. A hamster model of intranasal SARS-CoV-2 infection was used as a proof-of-principle experiment: RNA was detected in lungs and toes with IFN-I activity at both the sites, while replicating virus was found only in the lung. These data support a viral trigger for some pernio cases, where sustained local IFN-I activity can be triggered in the absence of seroconversion.

Keywords: Cell biology; Immune response; Immunology.

Graphical abstract

Figure 1

Clinical and histologic features of COVID-associated pernio (A) Workflow of the study from Wisconsin, USA, and Switzerland cohorts along with classic findings of PAP including violaceous erythema, edema, vesiculation, ulceration, and a focused superficial and deep lymphocytic infiltration on H&E-stained tissue sections. (B) Modified from Ng et al., BJD 2022, to represent the Wisconsin cohort in the current investigation: Madison, Wisconsin, PAP cases and recurrences overlayed with COVID-19 positivity and mean temperature by month. (C) Thermal photography image from an affected patient in Wisconsin: pernio lesions presented in the coldest areas of acral skin (in blue).

Figure 2

Anti-viral immune response profile in COVID-associated pernio Representative confocal images and summary quantification of MxA (A; in red), CD303 (B; in gray), CD3 (C; in green), IFNγ (D; in magenta), and HLA-DR (E; in yellow) in representative negative control pre-2019 normal skin (first row, n = 3–4), pandemic-associated pernio (PAP; second row, n = 6–11), and pre-2019 pernio lesions (third row, n = 3–4). White dotted lines indicate the dermal-epidermal junction. Atto-465p (A, B, and C) and DAPI (D and E) were used to counterstain nuclei. CD3⁺T cells and CD303⁺pDCs were counted in the entire biopsy section for each patient; positive pixel quantification was performed for MxA, IFNγ, and HLA-DR. Numbers were normalized by the total area of the tissue (in μm²). Magnification, ×40; scale bar, 50 μm. Bars in graph represent mean ± SEM. ∗p < 0.05 in Kruskal-Wallis test with Dunn’s correction for multiple comparisons. (F) Dense immune cell infiltrate consisting primarily of T cells (green) and pDCs (gray) in patients with PAP. Expression of MxA (red) by pDCs (cyan arrowhead), but rarely by T cells (orange arrowhead). Nuclei were stained with Atto-465p. (G) Presence of activated T cells in PAP tissue. Orange arrowhead shows the close proximity between HLA-DR-expressing cells (yellow) and CD3 T cells(green), indicating a possible synapse. Cyan arrowheads show activated T cells expressing IFNγ (magenta). Nuclei were stained with DAPI. (H) Both T cells (green) establishing communication with HLA-DR+ cells (yellow; white arrowhead) and T cells expressing HLA-DR are present in PAP lesions. Magnification, ×40; scale bar, 50 μm.

Figure 3

SARS-CoV-2 RNA presence in representative PAP lesions RNAscope in situ hybridization was used to detect the presence of SARS-CoV-2 viral RNA in formalin-fixed, paraffin-embedded (FFPE) tissue sections from representative PAP from Wisconsin, USA, patient cohort 1 (A) and PAP from Switzerland cohort 2 (B); autopsy lung tissue from a COVID-19 patient was used as a SARS-CoV-2 viral RNA probe positive control (C); pre-2019 pernio lesions (D) and pre-2019 acral psoriasis lesions were used as negative controls (E). 60x high-resolution images are shown in A1–E1, respectively. A probe for the housekeeping gene PPIB was used as a positive assay control (F–J), negative-control target probe DapB (K–M). Magnification x20. Scale bar, 50 μm. (N) Bar Graph showing the low and high inflammation score distribution of cases in Wisconsin, USA, and Switzerland cohorts. See also Figure S2.

Figure 4

Golden hamsters were intranasally treated with SARS-CoV-2 or PBS (mock) Lung and toe tissues from SARS-CoV-2- and mock-treated hamsters were longitudinally harvested at days 1, 3, 5, 7, 10, 14, and 30 post-infection (n = 3 per condition per time point). (A and B) Lung tissues were then assessed for the presence of (A) SARS-CoV-2 sgN and (B) Isg15 transcripts via RT-qPCR. (C and D) Toe tissues were also assessed for (C) sgN and (D) Isg15 presence via RT-qPCR. Significance was evaluated via multiple unpaired t tests performed using a two-stage step-up method to control the false discovery rate (FDR). FDR q-values less than 0.05 are displayed. (E) Toes were harvested from 3dpi hamsters inoculated with SARS-CoV-2 via either intranasal (IN) or intravenous (IV) routes and assessed for the presence of infectious virus via plaque assay. 3 dpi lung samples from SARS-CoV-2-infected hamsters were included as a positive control (n = 4 per condition). Significance was calculated using an ordinary one-way ANOVA with Tukey’s multiple comparisons test. ∗∗p < 0.01. (F and G) Toe and lung tissues were harvested at 3 days post-infection (dpi) and 30 dpi and transcriptionally profiled using RNA sequencing. SARS-CoV-2- and mock-treated datasets were compared in differential expression analysis (n = 2–4 for respective time point and infection groups). Toe differential expression data of 3dpi harvested toes was in turn analyzed using gene set enrichment analysis (GSEA) for enrichment of ontology-related gene sets (MSigDB Gene Set C5). Top enrichments from these analyses are represented in (F) as a lollipop chart, with magnitude of the stalk representative of normalized enrichment score (NES) and dot size scaled to significance. (G) RNA sequencing data for SARS-CoV-2-infected toes and lungs at 3 and 30 dpi were compared to analogous mock-treated tissues using differential expression analysis. Log2(fold change) of type I interferon-stimulated genes are presented here as a heatmap. See also Figure S3.