Neutrophil Extracellular Traps Profiles in Patients with Incident Systemic Lupus Erythematosus and Lupus Nephritis

Abstract

Objective: Neutrophil extracellular traps (NET) expose modified antigens for autoantibodies in vasculitis. Little is known about levels and removal pathways of NET in systemic lupus erythematosus (SLE), especially in lupus nephritis (LN). We determined circulating levels and defined NET removal in large subsets of patients with incident SLE (iSLE), some of whom had new-onset nephritis.

Methods: Serum levels of NET (ELISA), DNase1/DNase1L3 (ELISA), and DNase activity (functional assay) were determined in 216 patients with iSLE [103 had incident LN (iLN)], in 50 patients with other primary glomerulonephritis, and in healthy controls. Ex vivo NET production by neutrophils purified from a random selection of patients was quantified as elastase/DNA release and by immunofluorescence techniques.

Results: Serum NET levels were very high in iSLE/iLN compared to all groups of controls and correlated with anti-dsDNA, C3-C4, and proteinuria; iLN had the highest levels. DNase activity was decreased in iLN compared to SLE (20% had one-half DNase activity) despite similar serum levels of DNase1/DNase1L3. In these cases, pretreatment of serum with protein A restored DNase efficiency; 1 patient was homozygous for a c.289_290delAC variant of DNASE1L3. Ex vivo NET production by neutrophils purified from LN, SLE, and normal controls was similar in all cases.

Conclusion: Patients with iLN have increased circulating NET and reduced DNase activity, the latter being explained by the presence of inhibitory substances in circulation and/or by rare DNase1L3 mutations. Accumulation of NET derives from a multifactorial mechanism, and is associated and may contribute to disease severity in SLE, in particular to renal lesions. (Clinical trial registration: The Zeus study was registered at ClinicalTrials.gov, study number NCT02403115).

Keywords: DNASE ACTIVITY; DNASE LEVEL; DNASE MUTATIONS; NEUTROPHIL EXTRACELLULAR TRAPS.

Conflict of interest statement

Competing interests. Authors declare no conflicts of interest. A patent on the use of anti-enolase antibodies in diagnosis of LN is pending.

Figures

Figure 1.. Circulating Neutrophil Extracellular Traps (NETs) Remnants.

(A) Serum NETs were determined using an ELISA measuring the DNA-MPO complex. Results are Relative Unit/ml given as median and interquartile range. The dotted line indicates the upper limit of normality (0.89). Here, it is reported serum NETs in all SLE, in all LN and in normal people. It is also, in parallel, shown NETs levels in LN patients split in the two subgroups divided according to the indications given in Materials and Methods (i.e. LN as onset, LN after 1year from the SLE onset). (B) ROC curves showing specificity and sensitivity of the DNA-MPO assay for LN and SLE patients.

Figure 2.. Serum DNase activity.

(A) DNase activity was determined with a one-step assay based on fluorescence decrease of degrading Picogreen DNA dye/double-stranded DNA (dsDNA). In patients indicated with a square serum DNase activity was re-tested after treatment with Protein A/G. The triangle indicates a boy who presented a c.289_290delAC homozygous variant in DNASE1L3. Here, it is also in parallel reported DNase activity in LN patients split in the two subgroups divided according to the indications given in Materials and Methods (i.e. LN as onset, LN after 1year from the SLE onset). (B) patients with LN were subdivided according to their serum levels of NETs remnants (higher and lower than the normal level of 0.5 RU/ml). (C) DNase activity in patients with high and low NETs remnants. All patients with DNase activity < 30% were patients in the high NETs group.

Figure 3.. DNASE1 and DNASE1L3 levels.

(A) DNASE1 serum levels. A homemade ELISA assay has been utilized to test DNASE1. Results are expressed as ng/ml and represented as median and interquartile range. (B) DNASE1L3 serum levels. For DNASE1L3 we utilized a commercial ELISA (LSBio kit, Seattle, USA). Results expressed as ng/ml are given as median and interquartile range. In this case, those LN patients who presented maximal variability in DNase activity were chosen for testing DNASE1L3 levels here including patients with low and patients with high DNase activity. For the broad distribution of values, ROC and normal limits were not calculated.

Figure 4.. NETs production and protein composition.

(A)Ex vivo NETs formation was evaluated as elastase and DNA release by resting neutrophils purified from patients with SLE (n=5), lupus nephritis (n=9) and from healthy controls (n=8). More cells (15 SLE, 18 LN and 27 controls) were utilized for stimulation with phorbol-12-myristate-13-acetate (PMA). Kinetics of NETs formation was analyzed in all supernatants utilizing the elastase method(13). (B) In all experiments the release of elastase from stimulated neutrophils was highly correlated with the release of DNA.