B Cell Signatures Distinguish Cutaneous Lupus Erythematosus Subtypes and the Presence of Systemic Disease Activity

Abstract

Cutaneous lupus erythematosus (CLE) is a chronic inflammatory skin disease characterized by a diverse cadre of clinical presentations. CLE commonly occurs in patients with systemic lupus erythematosus (SLE), and CLE can also develop in the absence of systemic disease. Although CLE is a complex and heterogeneous disease, several studies have identified common signaling pathways, including those of type I interferons (IFNs), that play a key role in driving cutaneous inflammation across all CLE subsets. However, discriminating factors that drive different phenotypes of skin lesions remain to be determined. Thus, we sought to understand the skin-associated cellular and transcriptional differences in CLE subsets and how the different types of cutaneous inflammation relate to the presence of systemic lupus disease. In this study, we utilized two distinct cohorts comprising a total of 150 CLE lesional biopsies to compare discoid lupus erythematosus (DLE), subacute cutaneous lupus erythematosus (SCLE), and acute cutaneous lupus erythematosus (ACLE) in patients with and without associated SLE. Using an unbiased approach, we demonstrated a CLE subtype-dependent gradient of B cell enrichment in the skin, with DLE lesions harboring a more dominant skin B cell transcriptional signature and enrichment of B cells on immunostaining compared to ACLE and SCLE. Additionally, we observed a significant increase in B cell signatures in the lesional skin from patients with isolated CLE compared with similar lesions from patients with systemic lupus. This trend was driven primarily by differences in the DLE subgroup. Our work thus shows that skin-associated B cell responses distinguish CLE subtypes in patients with and without associated SLE, suggesting that B cell function in skin may be an important link between cutaneous lupus and systemic disease activity.

Keywords: B cells; autoantibodies; cutaneous lupus; discoid; lupus; transcriptomic.

Figure 1

Weighted gene correlation network analysis identifies an immunoglobulin signature associated with DLE skin lesion status, independent of IFN score. (A) Module-trait relationship heatmap. Each module eigengene was correlated with the indicated clinical parameter. For categorical parameters, non-systemic/systemic disease and DLE/SCLE, numerical values were assigned to each categorical group. The scale bar on the right represents the correlation coefficient with green for negative correlation and red for positive correlation, p-values for each correlation are presented on the heatmap. The yellow module was the module with the strongest positive correlation with IFN score and the cyan module was the module that had the strongest negative correlation with the DLE versus SCLE lesion status. (B) The cyan module eigengene from the SCLE and DLE lesions encompasses a 32-gene, primarily immunoglobulin signature. (C) The yellow module eigengene was significantly correlated with IFN score (r = 0.85, p = 3E-28). The data in each panel represent 47 DLE patients and 43 SCLE patients.

Figure 2

Cell type enrichment analysis using xCell tool reveals a B cell signature higher in DLE lesional skin compared to SCLE and ACLE lesions. (A) Heatmap of the B cell subtypes representing average xCell score for each skin lesion type compared to normal healthy controls (N). *p-value < 0.05 in SCLE versus DLE. (B) xCell enrichment score for B cells, naïve B cells and memory B cells in lesional skin from patients with DLE, ACLE, SCLE as well as normal healthy controls (N) in both the discovery and the validation cohort. Comparisons were made via unpaired Students’ t-test.

Figure 3

B cell subset enrichment score in lesional skin from CLE patients with and without systemic lupus. (A) Heatmap of the B cell subtypes representing the xCell enrichment score for each patient from the discovery cohort. (B) xCell enrichment score for B cell subtypes in normal healthy controls (N) (n = 13) and all CLE patients with and without systemic lupus (n = 46 and n = 44, respectively). (C) xCell enrichment score for B cell subtypes in DLE patients with and without systemic lupus (n = 22 and n = 25, respectively) and SCLE patients with and without systemic lupus (n = 24 and n = 19, respectively). Comparisons were made via unpaired Students’ t-test.

Figure 4

The relationship between skin B cell enrichment and the presence of circulating SLE autoantibodies in patients with DLE or SCLE. Patients with active DLE or SCLE skin lesions were stratified by the presence (positive) or absence (negative) of SLE autoantibodies at the time of biopsy. The patients in which the status of a particular autoantibody was not known at the time of biopsy were classified as “unknown”. (A) ANA and anti-dsDNA. (B) Anti-Smith and anti-Ro. (C) Anti-phospholipid. Comparisons were made via unpaired Students’ t-test.

Figure 5

B cell quantification by tissue CyTOF and gene expression in lesional skin from ACLE, SCLE, and DLE patients. (A) The number of B cells numbers per millimeter of skin were quantified in SCLE (n = 8), ACLE (n = 8), and DLE (n = 8) lesions. Normalized gene expression of (B) CD20, and (C) Bank1 from SCLE (n = 20), ACLE (n = 20), and DLE (n = 20) lesional skin and normal healthy control skin (N) (n = 4).

Figure 6

Immunohistochemistry staining for total immune cells and B cell subsets in lesional skin from healthy controls or patients with DLE, ACLE, or SCLE. Formalin-fixed paraffin embedded tissue sections from skin were stained for CD45+ total leukocytes, CD20+ B cells, and CD27+ mature B cells. Representative images from 3-5 patients of each subtype are shown at 100X magnification with a scale bar of 200 μm.