A circulating cell population showing both M1 and M2 monocyte/macrophage surface markers characterizes systemic sclerosis patients with lung involvement

Abstract

Background: Systemic sclerosis (SSc) is a disorder characterized by immune system alterations, vasculopathy and fibrosis. SSc-related interstitial lung disease (ILD) represents a common and early complication, being the leading cause of mortality. Monocytes/macrophages seem to have a key role in SSc-related ILD. Interestingly, the classically (M1) and alternatively (M2) activated monocyte/macrophage phenotype categorization is currently under revision. Our aim was to evaluate if circulating monocyte/macrophage phenotype could be used as biomarker for lung involvement in SSc. To this purpose we developed a wide phenotype characterization of circulating monocyte/macrophage subsets in SSc patients and we evaluated possible relations with lung involvement parameter values.

Methods: A single centre cross-sectional study was performed in fifty-five consecutive SSc patients, during the year 2017. All clinical and instrumental tests requested for SSc follow up and in particular, lung computed tomography (CT) scan, pulmonary function tests (PFTs), Doppler echocardiography with systolic pulmonary artery pressure (sPAP) measurement, blood pro-hormone of brain natriuretic peptide (pro-BNP) evaluation, were performed in each patient in a maximum one-month period. Flow cytometry characterization of circulating cells belonging to the monocyte/macrophage lineage was performed using specific M1 (CD80, CD86, TLR2 and TLR4) and M2 surface markers (CD204, CD163 and CD206). Non-parametric tests were used for statistical analysis.

Results: A higher percentage of circulating CD204⁺CD163⁺CD206⁺TLR4⁺CD80⁺CD86⁺ and CD14⁺CD206⁺CD163⁺CD204⁺TLR4⁺CD80⁺CD86⁺ mixed M1/M2 monocyte/macrophage subsets, was identified to characterize patients affected by SSc-related ILD and higher systolic pulmonary artery pressure. Mixed M1/M2 monocyte/macrophage subset showed higher percentages in patients positive for anti-topoisomerase antibody, a known lung involvement predictor.

Conclusions: The present study shows for the first time, through a wide flow cytometry surface marker analysis, that higher circulating mixed M1/M2 monocyte/macrophage cell percentages are associated with ILD, sPAP and anti-topoisomerase antibody positivity in SSc, opening the path for research on their possible role as pathogenic or biomarker elements for SSc lung involvement.

Keywords: Anti-topoisomerase antibody; Flow cytometry; Innate immunity; Interstitial lung disease; Lung CT scan; M1; M2; Monocyte/macrophage phenotype; Pulmonary artery hypertension; Pulmonary function tests; Systemic sclerosis.

Fig. 1

Ab anti Scl70 positivity: associations with FVC%, Pro-BNP blood values and mixed M1/M2 cells percentages. a and b, clinical associations of Anti-Scl70 Ab positivity with lower FVC% and higher pro-BNP values are shown. c and d show the representative dot plots from the flow cytometry analysis of the mixed M1/M2 CD204⁺CD163⁺CD206⁺TLR4⁺CD80⁺CD86⁺ cell subset is shown in patients with positive and negative Ab anti-Scl70. Significant differences (p = 0.027) are shown between average percentages of circulating mixed M1/M2 subset CD204⁺CD163⁺CD206⁺TLR4⁺CD80⁺ over total CD204⁺ cells, in Scl70+ vs Scl70- patients (e) and between percentage of circulating mixed M1/M2 subset CD204⁺CD163⁺CD206⁺TLR4⁺CD80⁺CD86⁺ over total CD204⁺ cells, in Scl70+ vs Scl70- patients (f). Anti-Scl70 = Anti-topoisomerase; FVC = forced vital capacity; pro-BNP = prohormone of brain natriuretic peptide

Fig. 2

ILD affected SSc patients: associations with mixed M1 M2 cells percentage. a and b, representative dot plots from the flow cytometry analysis of the CD204 + CD163 + CD206 + TLR4 + CD80 + CD86+ cell subset in SSc patients affected by ILD and not affected by ILD are shown. Mixed M1/M2 cells expressing CD80 and CD86 markers, among CD204⁺CD163⁺TLR4⁺CD206⁺ cells, resulted significantly increased in percentage in the SSc-ILD group compared to the SSc-No ILD group, if calculated both over total CD204⁺ cells (c) and over total circulating leukocytes (d)

Fig. 3

DLCO%, FVC/DLCO, sPAP values associations with mixed M1 M2 phenotype cells percentages. a a linear correlation between the mixed M1/M2 phenotype subset CD14⁺CD206⁺CD163⁺CD204⁺TLR4⁺CD80⁺CD86⁺ cell percentages and DLCO% values is shown. b an FVC/DLCO ratio higher than 1.5 resulted associated with CD204⁺CD163⁺CD206⁺TLR4⁺CD86⁺ cell subset percentage. c an FVC/DLCO ratio higher than 1.5 resulted associated with CD14⁺CD206⁺CD163⁺CD86⁺ cell subset percentage. d an FVC/DLCO ratio higher than 1.5 resulted associated with CD14⁺CD206⁺CD163⁺CD204⁺TLR4⁺CD80⁺CD86⁺ cell subset. e a linear correlation between the mixed M1/M2 phenotype subset CD14⁺CD206⁺CD163⁺CD204⁺TLR4⁺CD80⁺CD86⁺ cell percentages and sPAP values is shown