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. 2020 Mar;79(3):379-386.
doi: 10.1136/annrheumdis-2019-215894. Epub 2019 Nov 25.

Global skin gene expression analysis of early diffuse cutaneous systemic sclerosis shows a prominent innate and adaptive inflammatory profile

Brian Skaug  1 , Dinesh Khanna  2 , William R Swindell  3   4 , Monique E Hinchcliff  5 , Tracy M Frech  6 , Virginia D Steen  7 , Faye N Hant  8 , Jessica K Gordon  9 , Ami A Shah  10 , Lisha Zhu  11 , W Jim Zheng  11 , Jeffrey L Browning  12 , Alexander M S Barron  12 , Minghua Wu  13 , Sudha Visvanathan  14 , Patrick Baum  15 , Jennifer M Franks  16   17 , Michael L Whitfield  16   17 , Victoria K Shanmugam  18 , Robyn T Domsic  19 , Flavia V Castelino  20 , Elana J Bernstein  21 , Nancy Wareing  13 , Marka A Lyons  13 , Jun Ying  13 , Julio Charles  13 , Maureen D Mayes  13 , Shervin Assassi  13
Affiliations

Global skin gene expression analysis of early diffuse cutaneous systemic sclerosis shows a prominent innate and adaptive inflammatory profile

Brian Skaug et al. Ann Rheum Dis. 2020 Mar.

Abstract

Objectives: Determine global skin transcriptome patterns of early diffuse systemic sclerosis (SSc) and how they differ from later disease.

Methods: Skin biopsy RNA from 48 patients in the Prospective Registry for Early Systemic Sclerosis (PRESS) cohort (mean disease duration 1.3 years) and 33 matched healthy controls was examined by next-generation RNA sequencing. Data were analysed for cell type-specific signatures and compared with similarly obtained data from 55 previously biopsied patients in Genetics versus Environment in Scleroderma Outcomes Study cohort with longer disease duration (mean 7.4 years) and their matched controls. Correlations with histological features and clinical course were also evaluated.

Results: SSc patients in PRESS had a high prevalence of M2 (96%) and M1 (94%) macrophage and CD8 T cell (65%), CD4 T cell (60%) and B cell (69%) signatures. Immunohistochemical staining of immune cell markers correlated with the gene expression-based immune cell signatures. The prevalence of immune cell signatures in early diffuse SSc patients was higher than in patients with longer disease duration. In the multivariable model, adaptive immune cell signatures were significantly associated with shorter disease duration, while fibroblast and macrophage cell type signatures were associated with higher modified Rodnan Skin Score (mRSS). Immune cell signatures also correlated with skin thickness progression rate prior to biopsy, but did not predict subsequent mRSS progression.

Conclusions: Skin in early diffuse SSc has prominent innate and adaptive immune cell signatures. As a prominently affected end organ, these signatures reflect the preceding rate of disease progression. These findings could have implications in understanding SSc pathogenesis and clinical trial design.

Keywords: autoimmunity; inflammation; systemic Sclerosis.

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Conflict of interest statement

Competing interests: DK reports consultancy fees from Acceleron, Actelion, Bayer, Blade Therapeutics, BMS, Galapagos, Genentech/Roche, GSK, Mitsubishi Tanabi, Sanofi-A ventis/Genzyme, and UCB Pharma, and reports grants from Bayer, BMS, and Genentech/Roche outside the submitted work, and reports ownership interest in Eicos Sciences, Inc. WRS reports consulting fees from UT Health Science Center at Houston. JKG reports grants from Corbus Pharmaceuticals, Cumberland Pharmaceuticals and Eicos Pharmaceuticals outside the submitted work. AAS reports support from Sanofi outside the submitted work. MLW reports grants and personal fees from Celdara Medical, LLC and personal fees from Abbvie, Acceleron, BMS, Corbus Pharmaceuticals and Boehringer Ingelheim outside the submitted work. JLB reports grants from Hoffman La Roche and the Scleroderma Foundation outside the submitted work. SB and PB are employees of Boehringer Ingelheim. FVC reports personal fees from Boehringer Ingelheim outside the submitted work. EJB reports a grant from Pfizer and support from Bohringer Ingelheim, Corbus Pharmaceuticals and Eicos outside the submitted work. MDM reports personal fees from Medtelligence, Actelion Pharma, Astellas, Mitsubishi-Tanabe, and Boehringer Ingelheim and grants from Bayer, Reata, Sanofi, Corbus, Eicos and Boehringer Ingelheim outside the submitted work. SA reports personal fees from Boehringer Ingelheim and grants from Boehringer Ingelheim, Bayer and Momenta outside the submitted work.

Figures

Figure 1
Figure 1
Differentially expressed transcripts and pathways in Prospective Registry for Early Systemic Sclerosis systemic sclerosis (SSc) patients compared with healthy controls (HCs). (A) Heatmap of differentially expressed transcripts, represented by z-score normalised count values. Unsupervised hierarchical clustering is shown at the top, with HCs represented by purple squares and SSc patients represented by red squares. (B) Top 10 over-represented pathways in SSc compared with HC as determined by Ingenuity Pathway Analysis of differentially expressed transcripts (fold change >1.5 or <0.67 in SSc vs HC, with false discovery rate <0.05). (C) Top 10 predicted upstream transcriptional regulators in SSc compared with HC. (D) Top 10 predicted upstream cytokines/growth factors.
Figure 2
Figure 2
Cell type signatures in skin of PRESS SSc patients compared with healthy controls and compared with GENISOS SSc patients. (A) Cell type signature scores for each SSc sample (n=48). Scores represent the average fold-change (SSc/HC) for 125 cell type-specific signature genes (see online supplementary methods). Up-triangles indicate significantly higher scores for signature genes compared with non-signature genes (p<0.05, Wilcoxon rank-sum test). Down-triangles indicate significantly lower scores for signature genes compared with non-signature genes (p<0.05, Wilcoxon rank-sum test). Bottom margin values indicate the percentage of up-triangles (red) and down-triangles (blue), respectively. Patients were clustered based on signature scores (average linkage, Euclidean distance). The coloured boxes to the left of the cell type signature scores indicate the mRSS (left), local skin score at the site of the biopsy (middle) and the intrinsic subset classification, with legends at the right of the figure. White boxes (n=3) indicate no skin scores recorded at the time of the biopsy. (B) Signature scores for PRESS patients (n=48) were compared with those of GENISOS patients (n=55). The mean PRESS score is represented by round symbols with error bars spanning ±1 SD. The mean GENISOS score is represented by the midline for each grey box with boxes spanning ±1 SD. Right margin p values were obtained from a two-sample t-test of PRESS versus GENISOS scores (red: PRESS>GENISOS, FDR<0.05; blue: PRESS
Figure 3
Figure 3
Associations between preceding skin thickness progression rate and skin immune cell type signatures in PRESS SSc patients. Skin thickness progression rate (mRSS at the time of biopsy/years since first skin thickening or puffy fingers) preceding the skin biopsy is plotted on the x-axis. Cell type signature scores for (A) M1 macrophages, (B) M2 macrophages, (C) CD4 cells, (D) CD8 T cells or (E) B cells are plotted on the y-axis. mRSS, modified Rodnan Skin Score; PRESS, Prospective Registry for Early Systemic Sclerosis; SSc, systemic sclerosis; ST, skin thickness.
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