Background: The typically inflammatory environment present in SLE and its associated tissue damage promotes cell death and the consequent increase in circulating levels of cell-free DNA. Additionally, the increased capacity of SLE low density granulocytes (LDG) to induce NETosis play a key role in the release of cell-free circulating DNA (cirDNA) and contributes to the formation of anti-dsDNA antibodies.

Objectives: The present study aims to analyze the role of DNase I, responsible for cirDNA degradation, in relation to LDG subsets and cirDNA levels in SLE.

Methods: DNase I, anti-DNase I and and pGSN levels were determined by ELISA techniques and DNase I activity by a fluorescence-based assay in 42 controls and 144 SLE patients. The frequency of total blood LDGs, as well as the CD16 ^neg CD14 ^neg (nLDG) and CD16 ^pos CD14 ^low (pLDG) subsets, was quantified by flow cytometry. Total cirDNA and relative amounts of mitochondrial (mtDNA) and nuclear (nDNA) cell-free DNA were measured by fluorometry or qPCR in plasma.

Results: DNase-I serum levels were increased in SLE patients compared with controls (p=0.040), especially in those presenting anti-dsDNA antibodies (p=0.005) the patient group with the highest levels of cirDNA. Remarkably, DNase I levels in controls correlated with mtDNA (r=0,438, p=0.012), a main component in the backbone structure of NETs. However, the DNase I/cirDNA ratio was reduced in patients compared with controls, mainly in those with active disease (p=0.017), suggesting that the raise of DNase I in SLE was unable to counteract cirDNA. Furthermore, SLE patients displayed increased levels of anti-DNase I antibodies (p=0.040), that were directly associated to the DNase I/cirDNA ratio (r=0.247, p=0.007). Thus, the upregulated levels of DNase I in SLE, probably induced by the elevated cirDNA amounts, could not be fully functional and might promote the generation of anti-DNase I antibodies. Further analysis of DNase I activity revealed that was unrelated to DNase I levels and does not shown significant differences between patients and controls. However, the DNase I activity rate (DNase I activity relative to DNase I levels) was reduced in SLE patients, especially in those presenting anti-dsDNA antibodies (p=0.025) and correlated negatively with the amount of mtDNA in controls (r=-0.356, p=0.045) and non-active patients (r=-0.213, p=0.040). As expected, blood levels of LDGs were increased in SLE, and associated directly with cirDNA, but only mtDNA amount was linked to pLDG. DNase I activity was negatively correlated with all LDG subpopulations. Finally, pGSN levels were inversely correlated with DNase I in healthy controls (r=-0.492, p=0.032) and SLE patients (r=-0.176, p=0.043) but positively associated with DNase I activity rate (HC: r = 0.454, p = 0.051; SLE: r = 0.155, p = 0.073). In fact, elevated pGSN levels attenuated the DNase I upregulation observed in SLE patients with anti-dsDNA antibodies without impairing DNase I activity, leading to a significant increase in the “DNase I activity rate”.

Conclusion: Present results highlight the relevance of the “cirDNA-NETosis-DNase I” axis as a pathogenic mechanism directly involved in the etiopathogenia of SLE. mtDNA could upregulate DNase-I levels and activity in physiological conditions as part of a homeostatic mechanism aimed to clear cell-free circulating DNA, thus controlling the DNase-I/cirDNA balance. However, unknown factors upregulate DNase-I levels in SLE, but without a parallel increase in DNase-I activity. Therefore, DNA clearance was limited and the DNase-I/cirDNA ratio increased, thus fueling a vicious cycle conducting to immune activation and anti-dsDNA production. Additionally, given the suggested role of mtDNA in the generation of pLDG, this subset could be a pathogenic factor in the dysregulated balance among mtDNA-Dnase I-DNA clearance in SLE. In this scenario, pGSN appears to modulate DNase I activity and exert a protective role against nLDG expansion, thus suggesting a therapeutic role of pGSN to mitigate inflammation in SLE patients.

REFERENCES: NIL.

Acknowledgements: The authors acknowledge the SLE patients and the “Asociación Lúpicos de Asturias” for their continuous encouragement.

Disclosure of Interests: None declared.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.