Pro-angiogenic changes of T-helper lymphocytes in hereditary hemorrhagic telangiectasia

Abstract

Hereditary hemorrhagic telangiectasia (HHT) is a rare inherited disease due to heterozygous loss-of-function mutations on the BMP9/10 pathway (ENG, ACVRL1 or MADH4 mainly). HHT endothelial cells are prone to lose their quiescence, leading to progressive appearance of numerous telangiectases on skin and mucosa (complicated by epistaxis and anemia), and to larger arteriovenous malformations in lungs, liver and brain. HHT is also associated with T lymphocyte abnormalities, which are currently poorly understood. We quantified by flow-cytometry the main T lymphocyte circulating subsets in 40 HHT patients and 20 matched healthy controls. Immunostaining was done on 2 HHT skin telangiectases. Disruptions in T lymphocyte homeostasis was observed, characterized by increases in subsets known to promote angiogenesis: Th2 (1.38% vs 1.15%, p=0.021), Th17 (0.32% vs 0.22%, p=0.019 2) and Treg (4.94% vs 3.51%, p= 0.027). T angiogenic lymphocytes (Tang), defined as CD3+CD31+CXCR4+ T cells, were at similar levels in both groups, but the proportion of VEGF-A+ Tang after stimulation was higher in the HHT group compared to controls (68.2% vs 44.9%, p=0.012). The global HHT T lymphopenia predominantly affected the effector memory T-helper cells (200 vs 270 cells/mm³, p=0.017), and the lymphocytic infiltrate around HHT telangiectases consisted of memory T-helper cells. The Th17 circulating subset was positively correlated with the monthly epistaxis duration (r coefficient: +0,431, p=0.042), prospectively assessed. HHT T-helper lymphocytes are affected by several pro-angiogenic changes, potentially resulting from their recruitment by abnormal endothelial cells. They could constitute a biologically relevant source of VEGF-A and a valuable therapeutic target in HHT.

Keywords: Th17; Th2; Treg; VEGF-A; effector memory T-helper lymphocytes; hereditary hemorrhagic telangiectasia.

Figure 1

Th1, Th2, Th17, Treg and Tang polarizations. Comparisons between the HHT group (HHT: dark gray) and the healthy control group (HC: light gray): (A) T-helper polarizations after PMA/ionomycin stimulation: • Th1 (CD3+CD4+interferon γ+IL-17-, after PMA/ionomycin stimulation). • Th2 (CD3+CD4+IL-4+, after PMA/ionomycin stimulation). • Th17 (CD3+CD4+interferon γ-IL-17+, after PMA/ionomycin stimulation). • Treg (CD3+CD4+CD25+FOXP3+)*. (B) T angiogenic lymphocytes (Tang: CD3+CD31+CXCR4+): • A representative dot plot illustrating the gating strategy, according to ref (12). • Percentage of Tang in total T lymphocytes, T-helper lymphocytes and T-cytotoxic lymphocytes. • Intracytoplasmic production of pro-angiogenic cytokines by Tang after PMA/ionomycin stimulation: VEGF-A, IL-8 and IL-17. (C) VEGF-A synthesis by T lymphocytes after PMA/ionomycin stimulation: • Percentage of T lymphocytes positive for intracytoplasmic VEGF-A. • Mean Fluorescence Intensity (MFI) of intracytoplasmic VEGF-A in total T lymphocytes. Results are presented by bar charts indicating the median and aligned individual values. Comparisons were performed using the Mann-Whitney test. P-values below 0.05 are considered significant and shown in bold. * only on 19 HHT and 13 HC subjects.

Figure 2

Circulating and peri-vascular lymphocyte subsets. (A) Comparisons between the HHT group (HHT: dark gray) and the healthy control group (HC: light gray) on: • Lymphocyte subsets: B (CD19+), T (CD3+), T-helper (CD3+CD4+) and T-cytotoxic (CD3+CD8+). • T-helper subsets: naïve (T_N: CCR7+CD45RA+), effector memory (T_EM: CCR7-CD45RA-), effector memory RA+ (T_EMRA: CCR7-CD45RA+), central memory (T_CM: CCR7+CD45RA-). (B) Skin biopsies of hand telangiectases from 2 HHT patients (P1: 69 years old male with ACVRL1 mutation, P2: 40 years old male with ACVRL1 mutation): conventional hematoxylin-eosin-saffron (HES) staining, immunohistochemistry staining with anti-CD3, anti-CD4, anti-CD8 and anti-CD45RO antibodies. Results are presented by bar charts indicating the median and aligned individual values. Comparisons were performed using the Mann-Whitney test. P-values below 0.05 are considered significant and shown in bold.