Background Neutrophils are one of the first cell types to be recruited to sites of inflammation or infections, yet have been implicated in a wide range of non-infectious inflammatory conditions. Low-density granulocytes (LDGs) are a distinct subset of neutrophils recently documented in a number of systemic autoimmune diseases such as Systemic Lupus Erythematosus (SLE) (1). LDGs show increased ability to undergo neutrophil extracellular trap (NET) formation, leading to potential autoantigen presentation, and subsequent inflammation (2,3). Citrullination of histones in neutrophils, a post-translational histone modification, is a critical step in NET formation (4). Juvenile idiopathic arthritis (JIA) is a heterogeneous autoimmune disease characterized by chronic joint inflammation. We have previously shown increased LDG expression in JSLE patients; however histone citrullination in JSLE and JIA, and LDG populations in JIA has yet to be characterized.
To compare LDG expression in JIA peripheral venous blood to healthy controls.
To investigate the presence of histone citrullination in JSLE and JIA patients compared to healthy controls.
Methods While mature neutrophils typically sediment with the red-blood cell fraction in a Ficoll-density gradient, LDGs co-purify in the PMBC fraction. LDGs and neutrophils were isolated from whole blood using HetaSep and magnetic bead separation. The percentage populations of LDGs were analysed using flow cytometry in JIA and healthy paediatric controls. Where possible, protein was extracted from the neutrophils of each patient group. Citrullinated histone 3 relative to the expression of histone 3 was measured using Western Blot analysis in JIA and JSLE patients compared to controls. Results are presented as mean ± SEM.
Results Preliminary data suggests the expression of LDGs in the peripheral blood of JSLE patients (n=11; 8.31%±2.5) and JIA patients (n=3; 3.6%±2.4) is increased compared to healthy paediatric controls (n=4; 1.62%±0.95). Overall, the ratio of citrullination of histone 3 to pan histone 3 was higher in JSLE neutrophils (0.75±0.15) compared to healthy controls (0.38±0.12; p<0.05). However there was no significant difference between JIA and control neutrophils.
Conclusions Increased expression of LDGs was observed in JIA and JSLE patients compared to controls. This supports previous research from our group that has shown increased expression of LDGs in JSLE patients correlates with disease activity. LDGs may therefore play an integral role in triggering an autoimmune inflammatory response. Notably, citrullination of histone 3 was increased in JSLE neutrophils compared to controls, but not JIA neutrophils. This may indicate that JSLE neutrophils are intrinsically more primed to undergo NETosis compared to JIA patients. Further investigations are being carried out to increase patient size and to characterize LDG phenotype and function.
Carmona-Rivera C, Kaplan MJ. Semin Immunopathol, 2013. 35(4): p. 455-63
Villanueva E, et al. The Journal of Immunology 2011 July 01;187(1):538-552.
Denny MF, et al. The Journal of Immunology 2010 March 15;184(6):3284-3297.
Wang, Y, et al. J Cell Biol, 2009. 184(2): p. 205-13
Disclosure of Interest None declared