Background/Purpose: NETosis has been suggested to play a central role in several rheumatology diseases. Nevertheless, this process, and its modulation in therapeutic response has not been described yet in AS patients. Our aims were: 1) to evaluate and characterize the presence of NETosis in AS patients. 2) To explore the relationship among NETosis markers and clinical characteristics of this disease. 3) To analyze the in vivo effect of anti-TNFα therapy on NETosis.

Methods:

Thirty patients with AS and 32 healthy donors (HDs) were included in a cross-sectional study. Eight AS patients were selected for a six-month longitudinal study of anti-TNFα response. Disease activity was determined by BASDAI index and, CRP and ESR levels; in parallel, plasma inflammatory markers were determined by ELISA kits. Spinal mobility of patients was measured by the BASMI index. Ex vivo, spontaneous NETosis generation in purified neutrophils from AS patients and HDs were measured by fluorescence (n=6) and scanning electron (n=3) microscopy after 6 h of incubation. PMA, known to promote NETosis, was used as positive control. DNA extrusion was analyzed by fluorescence microscopy and fluorimetry after SYTOX staining, whereas elastase percentage (NE) was analyzed by fluorescence microscopy after staining of neutrophils with NE antibody. In vivo, mieloperoxidase (MPO) and NE protein expression were measured by flow cytometry (FACSCalibur), whereas circulating cell-free DNA and NE levels was examined using fluorimetry after SYTOX staining, and commercial kits, respectively.

Results: Compared to HDs, AS neutrophils showed spontaneous extracellular release of a meshwork of DNA nuclear and granule proteins (NETs), as demonstrated by fluorescence microscopy, fluorimetry, and scanning electron microscopy. Indeed, analysis of DNA fibers staining by SYTOX revealed that NETosis rate was above baseline levels after 6 h of ex vivo AS neutrophil incubation as compared to those from HDs (P<0.05). Furthermore, higher cell free-DNA levels were observed between AS patients and HDs at 6 h (P<0.05). Increased spontaneous NETs production in this pathology was additionally corroborated by the observation of an enhanced percentage of NE-staining cells after 6 h of incubation (P<0.05). In vivo, higher intracellular NE levels (P=0.036), and circulating cell-free DNA levels (P=0.021) were also found in AS patients as compared to HDs.

Correlation studies showed that circulating cell-free DNA levels positively correlated with inflammatory markers (i.e. CRP, ESR and TNFα), and with BASMI index. In addition, a positive correlation was found between intracellular NE levels and plasma IL-1β concentration.

Anti-TNFα treatment of selected AS patients decreased circulating cell-free DNA and NE levels (p<0.05), which paralleled the reduction of disease activity.

Conclusion: 1) NETosis is increased in AS patients. 2) Raised NETosis in AS is associated with several markers of inflammation and mobility. 3) Anti-TNFα therapy has a significant effect on NETosis inhibition. Thus, NETosis might act as a key mediator in the etiopathogenesis of AS and have potential for assessment of therapeutic effectiveness in AS patients.

Funded by JA PI-0314-2012, SER, ISCIII (RIER RD16/0012/0015).

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/induction-of-netosis-in-ankylosing-spondylitis-association-to-disease-pathogenesis-and-modulation-by-anti-tnf%ce%b1-therapy/