Multicolor flow cytometric analysis of TLR2 and TLR9 expression and function in NK cells from patients with ANCA-associated vasculitis

Abstract

Background: The primary objective of this study was to provide an assessment of NK cells in patients with ANCA-associated vasculitis (AAV).

Methods: Patients were classified based on the presence or absence of ANCAs and compared with healthy controls (HCs). By multiparameter flow cytometry, we evaluated the number and proportion of NK cells (CD3-CD56+) and the CD56^dim , CD56^bright , CD56^dim CD57^bright subsets; TLR2 and TLR9 expression; intracellular IFN-γ production upon stimulation with TLR2 and TLR9 ligands; degranulation activity; serum cytokines; immunohistochemical staining of available biopsies.

Results: No differences in the number and proportion of NK cells between patients and HC were observed, except for a lower proportion of CD56^dim subset in ANCA-negative patients than in HC (P = 0.02). With respect to HC, TLR2 expression levels were reduced in NK cells from ANCA-negative patients (P = 0.03), in the CD56^dim subset of ANCA-positive (P = 0.02) and ANCA-negative patients (P = 0.01), in the CD56^bright subset of ANCA-positive patients (P = 0.007), and in the CD56^dim CD57^bright subset of ANCA-positive (P = 0.04) and ANCA-negative patients (P = 0.03). No differences between patients and HC were found concerning IFN-γ production and degranulation activity. IL-22 levels were lower in ANCA-positive patients than in HC (P = 0.01). The immunohistochemical analysis showed sporadic CD56+ cells in one renal biopsy, and a diffuse and moderate infiltrate of IL-22+ cells in all renal biopsies and in skin tissue.

Conclusions: Our data suggest a role of infectious stimuli triggering NK cells in AAV pathogenesis. Poor detection of NK cells in affected tissues suggests a marginal involvement in local inflammatory responses. © 2017 International Clinical Cytometry Society.

Keywords: ANCA; vasculitis; ANCA-associated vasculitis; NK cells; TLRs.