Platelet TLR7 is essential for the formation of platelet-neutrophil complexes and low-density neutrophils in lupus nephritis

Abstract

OBJECTIVES: Platelets and low-density neutrophils (LDNs) are major players in the immunopathogenesis of systemic lupus erythematosus (SLE). Despite evidence showing the importance of platelet neutrophil complexes (PNCs) in inflammation, little is known about the relationship between LDNs and platelets in SLE. We sought to characterize the role of LDNs and TLR7 in clinical disease. METHODS: Flow cytometry was used to immunophenotype LDNs from SLE patients and controls. The association of LDNs with organ damage was investigated in a cohort of 290 SLE patients. TLR7mRNA expression was assessed in LDNs and high-density neutrophils (HDNs) using publicly available mRNA sequencing datasets, and our own cohort using RT-PCR. The role of TLR7 in platelet binding was evaluated in platelet: HDN mixing studies using TLR7 deficient mice and Klinefelter syndrome patients. RESULTS: SLE patients with active disease have more LDNs, which are heterogeneous and more immature in patients with evidence of kidney dysfunction. LDNs are platelet bound, in contrast to HDNs. LDNs settle in the PBMC layer due to the increased buoyancy and neutrophil degranulation from platelet binding. Mixing studies demonstrated that this PNC formation was dependent on platelet-TLR7, and that the association results in increased NETosis. The neutrophil-to-platelet ratio (NPR), is a useful clinical correlate for LDNs, and a higher NPR is associated with past and current flares of lupus nephritis. CONCLUSIONS: LDNs sediment in the upper PBMC fraction due to PNC formation, which is dependent on the expression of TLR7 in platelets. Collectively, our results reveal a novel TLR7-dependent crosstalk between platelets and neutrophils, which may be an important therapeutic opportunity for lupus nephritis.