The Role of Plasma Cell-Free Mitochondrial DNA and Nuclear DNA in Systemic Lupus Erythematosus

Background : The roles of plasma cell-free (pcf) mitochondrial DNA (mtDNA pcf ) and nuclear DNA (nDNA pcf ) in the pathogenesis of systemic lupus erythematosus (SLE) remain unclear. We analyzed the relative copies of mtDNA pcf and nDNA pcf and investigated their association with the levels of plasma 8-hydroxy-2’-deoxyguanosine (8-OHdG), plasma malondialdehyde (MDA) and mRNA of leukocyte C-type lectin domain family 5 member A (CLEC5A) in SLE patients. Methods : A total of 80 SLE patients and 43 healthy controls (HCs) were enrolled. Their plasma samples were subjected to the measurements of mtDNA pcf copies, nDNA pcf copies, 8-OHdG and MDA, respectively. Their leukocytes were analyzed for CLEC5A mRNA expression. Results : SLE patients had higher nDNA pcf copies (2.84 ± 1.99 vs. 2.00 ± 0.88, p = 0.002), lower mtDNA pcf copies (4.81 ± 6.33 vs. 9.83 ± 14.20, p = 0.032), higher plasma 8-OHdG (0.227 ± 0.085 vs. 0.199 ± 0.041 ng/mL, p = 0.016), lower plasma MDA (3.02 ± 2.20 vs. 4.37 ± 2.16 µ M, p = 0.001) and similar leukocyte CLEC5A mRNA expression levels (1.21 ± 1.17 vs. 1.26 ± 1.05, p = 0.870), as compared with those of HCs. Among the HCs, SLE patients with SLE Disease Activity Index (SLEDAI) ≤ 8, and SLE patients with SLEDAI > 8, their respective mtDNA pcf copies decreased stepwisely (9.83 ± 14.20 vs. 6.28 ± 7.91 vs. 3.19 ± 3.35, p = 0.054). The nDNA pcf copies of HCs, SLE patients without nephritis, and SLE patients with nephritis were increased stepwisely (2.00 ± 0.88 vs. 2.63 ± 1.74 vs. 3.16 ± 2.34, p = 0.043). Among SLE patients, higher nDNA pcf copies were associated with higher levels of plasma 8-OHdG ( p < 0.001) but lower plasma MDA ( p = 0.019). Among HCs but not SLE patients, higher nDNA pcf copies ( p = 0.013) or lower mtDNA pcf copies ( p < 0.001) were related to higher levels of leukocyte CLEC5A mRNA expression. Conclusions : Higher nDNA pcf , lower mtDNA pcf , increased ROS-elicited oxidative DNA damage and dysregulated leukocyte CLEC5A expression might be implicated in the pathogenesis of SLE.


Introduction
Systemic lupus erythematosus (SLE) is characterized by the dysregulation of immunocompetent cells that can generate pathogenic autoantibodies after self-antigen exposure to attack systemic organs [1].The pathogenesis of SLE are multifactorial [2,3].However, few studies have discussed the potential roles of plasma cell-free DNA (DNA pcf ) [4].
Under normal circumstances, DNA molecules are located intracellularly and embedded compactly in the nucleus as nuclear DNA (nDNA) or in the mitochondrial nucleoid as mitochondrial DNA (mtDNA) [5].On the con-trary, cell-free DNA (DNA cf ) are the DNA molecules that are released into the tissue fluid from cells all over the whole body [6].They have been detected in cerebrospinal fluid [7], pleural fluid [8], saliva [9], urine [10] and plasma [11] for different purposes.The DNA cf present in blood circulation is called "plasma cell-free DNA (DNA pcf )", and most of them are originated from circulating hematopoietic cells [12].Analysis of DNA pcf has been employed for the diagnosis of acute myeloid leukemia as a non-invasive tool, which is as accurate as the invasive bone marrow aspiration or biopsy [13].Human DNA pcf was first described by Mandel and Metais in 1948 [11].From then on, the clinical usefulness of DNA pcf has been emphasized in predicting the outcomes of critical patients with myocardial infarction [14], sepsis [15] or stroke [16].High levels of DNA pcf were first found in SLE patients in 1966 [17].Later on, some studies suggested that DNA pcf might serve as an exposed self-antigen to trigger the pathogenesis of SLE [18].Because the DNA pcf is mainly derived from intracellular mtDNA or nDNA, it is of great interest and clinical relevance to explore the roles of mitochondrial DNA pcf (mtDNA pcf ) and nuclear DNA pcf (nDNA pcf ) simultaneously in SLE patients.
Dysregulated antioxidant capacity with imbalanced production of reactive oxygen species (ROS) has been speculated in the development of SLE [19,20].Abnormal ROS levels would cause oxidative damages or oxidative modifications in the intracellular or extracellular components of cells in situ, nearby, or remotely.These components may include DNA, lipid, proteins, and phospholipids.The events would trigger subsequent cellular dysfunction or autoimmune reactions in SLE patients [19][20][21].Elevated plasma 8-hydroxy-2'-deoxyguanosine (8-OHdG) representing oxidative DNA damage [21][22][23] and plasma malondialdehyde (MDA) representing lipid peroxidation [21,22,24] have been reported in SLE patients with different clinical implications.The levels of accumulated 8-OHdG and/or MDA in the plasma could be effective surrogates to represent the oxidative stress in SLE patients [25].
Viral infection causing incidental self-antigen presentation has been proposed as a possible mechanism underlying the autoimmune reaction in SLE.Similar to the immune interaction implicating high levels of interferon (INF) observed between invading virus and host pattern/pathogen recognition receptors (PRRs), a phenomenon of type I INF signature has also been recognized as a prominent feature of SLE [26].A clinical trial revealed that the treatment of anti-INF-alpha monoclonal antibody, sifalimumab, could achieve a significant improvement of symptoms/signs in moderate to severe SLE patients [27].Recently, the importance of human C-type lectin domain family 5 member A (CLEC5A, a member of PRRs) in interacting with the invading virus was reported [28].Teng et al. [29] reported that through toll-like receptor 3 (TLR, a subtype of PRRs), influenza virus infection in bone marrowderived macrophages from CLEC5A −/− mice would result in an increase of IFN-alpha.Based on these findings, we have speculated that decreased or dysregulated leukocyte CLEC5A expression might be involved in SLE pathogenesis.
In this study, we focused on the measurement of copies of mtDNA pcf and nDNA pcf to explore their association with the levels of plasma 8-OHdG and plasma MDA as well as the leukocyte CLEC5A mRNA transcripts in SLE patients.We aimed to elucidate the role of DNA pcf in the pathogenesis of SLE.

Recruitment of SLE Patients and the Healthy Controls (HCs)
According to the classification criteria for SLE and scored by the SLE Disease Activity Index (SLEDAI)-2000 [30][31][32], a total of 80 non-acute SLE patients (female/male = 67/13, mean age 45.4 ± 12.7 years) under regular follow-up and treatment in the outpatient department (OPD) of the Division of Allergy, Immunology and Rheumatology in Taipei Veterans General Hospital were recruited in this sudy.Their medications consisted of steroids, hydroxychloroquine, azathioprine, mycophenolate mofetil or NSAID according to clinical conditions.Another 43 healthy individuals, who had no evidence of systemic diseases, e.g., autoimmune diseases, diabetes, infections, cardiovascular diseases or malignancy and matched for age (within 10 years, 40.2 ± 10.4 years) and sex (female/male = 35/8), were recruited as the healthy controls (HCs) for comparative study.The demographic data of SLE patients regarding organ involvement and laboratory profiles were recorded in detail.Regarding the lupus nephritis, it was defined by a protein-to-creatinine ratio (or 24-hour urine protein) exceeding 500 mg protein/24 hours or by red blood cell casts in the urine [30].Disease activity/severity was evaluated by using the SLEDAI-2000 scoring system [32,33].
Besides, another 3 active SLE patients with severe clinical presentations were enrolled.These included Case A with nephritis, pleuritis and pericarditis, Case B with acute lupus nephritis and rapidly progressive glomerulonephritis who was undergoing dialysis, and Case C with severe pulmonary arterial hypertension.Their therapeutic regimens consisted of intravenous rituximab (Mabthera™, 500 mg) on days 1 and 14 preceded by a pre-medication with 100 mg of intravenous methylprednisolone.Blood samples were drawn before rituximab treatments on days 1 and 14.

Purification of Plasma Cell-Free DNA (DNA pcf ) and Extraction of the mRNA & DNA from Leukocytes
Approximately 10 mL of peripheral venous blood was drawn into a tube containing EDTA (VACUETTE® with EDTA, Greiner Bio-One).After centrifugation at 3000 g for 10 minutes at 4 °C, the supernatant plasma and leukocyte-enriched buffy coat were collected, respectively.An aliquot of 200 µL plasma was subjected to the purification of plasma cell-free DNA (DNA pcf ) using Quick-cfDNA™ Serum and Plasma Miniprepkit (Epigenetics) according to the manufacturer's instructions [34].The eluted DNA pcf was kept at -20 °C until use.
The leukocytes from 42 SLE patients and 24 HCs were isolated for RNA extraction using the TRI™ reagent (Sigma-Aldrich Chemical Co., St. Louis, MO, USA).With oligo-dT primers in a 50-µL reaction buffer, an aliquot of 2 µg of purified RNA was reverse-transcribed to cDNA (1X) by the Ready-To-Go reverse transcription -polymerase chain reaction (RT-PCR) kit (GE Healthcare, Amersham, UK ).The cDNA thus obtained was kept at -20 °C until use [20].
Genomic DNA of the leukocytes from a healthy control (HC-No.42)was purified through the standard phenolchloroform extraction and isopropanol precipitation procedure as described previously [35].The DNA pellet was dissolved in nuclease-free distilled water and kept at -20 °C until use.

Standard Curves for DNA and cDNA Quantification
Quantitative real-time PCR (Q-PCR) using Sensi-FAST SYBR® Hi-ROX Kit (BIOLINE, London, UK) was applied to determine the DNA copies and cDNA copies through a standard curve and their threshold cycle (Ct) values.To establish standard curves for calculations, genomic DNA and cDNA from the leukocytes of HC-No.42 were serially diluted by 4-fold, from 30 to 0.0018310546875 ng/µL for DNA and from 0.25 (4× dilution) to 0.00390625 (256× dilution) for cDNA, respectively, and were then subjected to Q-PCR for the determination of their Ct values.
Using these 2 equations, the mtDNA copies and nDNA copies of the analyzed sample relative to mtDNA copies and nDNA copies of the reference sample were calculated.
Using these 2 equations, the CLEC5A cDNA copies and 18S rRNA cDNA copies of the analyzed sample relative to CLEC5A cDNA copies and 18S rRNA cDNA copies of the reference sample were calculated, respectively.

Determination of mtDNA pcf
Copies, nDNA pcf Copies and the Leukocyte CLEC5A mRNA Expression Levels Q-PCR was used for the quantification of mtDNA pcf , nDNA pcf , leukocyte CLEC5A cDNA and leukocyte 18S rRNA cDNA copies, respectively.For each Q-PCR tube, 1 µL of DNA pcf /or 1 µL of cDNA (16× dilution) of analyzed sample was amplified in a 10-µL reaction mixture that contained 0.25 µL of each primer (20 µM), 5 µL of SensiFAST SYBR® Hi-ROX premix, and 3.5 µL of PCRgrade water.Simultaneously, 1 µL of HC-No.42 leukocyte DNA (1 ng/µL)/or cDNA (16× dilution) and PCR-grade water were used as the reference (positive) and negative controls, respectively.The Q-PCR protocol contained a hot start at 95 °C for 10 minutes followed by 45 cycles of amplifications at 95 °C for 15 seconds and 60 °C for 60 seconds.By equations established above, the mtDNA pcf copies, nDNA pcf copies, leukocyte CLEC5A cDNA copies and leukocyte 18S rRNA cDNA copies of analyzed samples relative to those of HC-No.42 leukocyte, were calculated, respectively.We defined the CLEC5A mRNA expression level as total CLEC5A cDNA copies divided by total 18S rRNA cDNA copies in the present study.Finally, relative mtDNA pcf copies, nDNA pcf copies and CLEC5A mRNA expression levels of analyzed samples were calculated after adjusting with the mtDNA pcf copies, nDNA pcf copies and leukocyte CLEC5A mRNA expression level of HC-No.42 as 1.000.Each reaction was done in duplicate to get the average of data.

Determination of Plasma 8-OHdG
Part of the results of plasma 8-OHdG in the present analyses had been reported in other studies for different purposes [20,38].An aliquot of 200 µL of plasma, filtered by an ultra-filter (CENTRICON®, Ultracel YM-10 membrane, Millipore, Amicon, USA) with a cut-off molecular weight of 10 kDa, was subjected to centrifugation at 10,000 g at 4 °C for 2 hours to get rid of the interfering substances.Then, an aliquot of 50 µL of filtered plasma was employed for the measurement of 8-OHdG content by using the highly sensitive 8-OHdG Check ELISA kit (Japan Institute for the Control of Aging, Nikken SEIL Co., Ltd.Haruoka, Fukuroi, Shizuoka, Japan) according to the manufacturer's instructions.Each reaction was done in duplicate to get the average for data presentation [20,38].

Determination of Plasma MDA
In the present study, we measured the plasma MDA, without the hydrolysis of plasma sample, by a spectrophotometric assay kit (MDA-586, OxisResearch, Inc. Portland, OR, USA) according to the procedure recommended by the manufacturer, which involved the reaction with a chromogenic reagent N-methyl-2-phenylindole (NMPI) to form an intensely colored carbocyanine dye with a maximum absorption at 586 nm.A standard curve was established by using the referenced MDA samples at the concentration range of 0-4 µM and the plasma MDA levels in clinical samples were then calculated [39].

Statistical Analysis
The results are presented as the mean ± standard deviation (mean ± S.D.).The continuous variables between HCs and SLE patients were compared using the t-test or Mann-Whitney U test when appropriate.The continuous variables among HCs, SLE patients with SLEDAI ≤8 and SLE patients with SLEDAI >8 or among HCs, SLE patients without clinical manifestations and SLE patients with clinical manifestations were compared using Jonckheere-Terpstra trend test to demonstrate their trends of distributions.Alterations of continuous variables before and after the treatment of rituximab were compared by Wilcoxon signed-ranks test.Relationships between two continuous variables were evaluated by using Pearson's or Spearman's rho correlation and are presented with the correlation coefficient (CC).Differences and associations were considered significant when p-values were less than 0.05.

The Distributions of mtDNA pcf and nDNA pcf Copies and Their Association with Plasma 8-OHdG, Plasma MDA and Leukocyte CLEC5A mRNA Expression Levels in 43 HCs and 80 SLE Patients
As shown in Table 4, among 80 SLE patients, their distributions of mtDNA pcf copies were not related to the distri-bution of plasma 8-OHdG (p = 0.471) nor plasma MDA (p = 0.132).However, it is interesting that their distributions of nDNA pcf copies were positively correlated with plasma 8-OHdG (p < 0.001; CC = 0.457) and negatively correlated with plasma MDA (p = 0.019; CC = -0.262),respectively.Among the 43 HCs, their distributions of mtDNA pcf copies (p = 0.009, CC = 0.392) and nDNA pcf copies (p = 0.016, CC = 0.366) were positively correlated with the levels of plasma 8-OHdG, significantly.On the contrary, their distributions of mtDNA pcf copies (p = 0.167) and nDNA pcf copies (p = 0.849) were not related to the levels of plasma MDA (Table 4).

Alterations of mtDNA pcf Copies, nDNA pcf Copies and Plasma 8-OHdG, Plasma MDA and Leukocyte CLEC5A mRNA Expression Levels in 3 SLE Patients Undergoing Rituximab Treatment
In Table 5, changes of mtDNA pcf copies, nDNA pcf copies and plasma 8-OHdG, plasma MDA and leukocyte CLEC5A mRNA expression levels in 3 SLE patients un- dergoing rituximab treatment are listed.Because only the data of 3 patients were analyzed, the clinical relevance was limited and insufficient to draw a conclusion.Nevertheless, regarding the alterations of mtDNA pcf copies and leukocyte CLEC5A mRNA expression levels, the changes were universally increased although not statistically significant (p = 0.109).

Discussion
In summary, we have demonstrated that: (1) SLE patients tended to have lower mtDNA pcf copies, higher nDNA pcf copies, higher levels of plasma 8-OHdG, and lower levels of plasma MDA than did HCs, but they had similar leukocyte CLEC5A mRNA levels (Table 2).( 2) SLE patients with higher SLEDAI tended to have lower mtDNA pcf copies, and SLE patients with nephritis had higher nDNA pcf copies but lower mtDNA pcf copies (Table 3).(3) In SLE patients, higher nDNA pcf copies were correlated with higher levels of plasma 8-OHdG but lower levels of plasma MDA (Table 4).(4) In HCs, higher nDNA pcf or lower mtDNA pcf copies were associated with higher leukocyte CLEC5A mRNA expression levels, which showed no associations in SLE patients (Table 4) and (5) In active SLE patients, increases in mtDNA pcf copies and leukocyte CLEC5A mRNA levels were found after rituximab treatment (Table 5).We have thus speculated that lower mtDNA pcf copies, higher nDNA pcf copies, oxidative damages/stress as well as dysregulated leukocyte CLEC5A expression might be implicated in the pathogenesis of SLE.A proposed mechanism is illustrated in Fig. 1.
Higher levels of DNA pcf in SLE patients was first described by Tan et al. in 1966 [17].Up to now, DNA pcf has been regarded as an important biomarker in autoimmune rheumatic diseases [40].Nevertheless, DNA pcf including mtDNA pcf and nDNA pcf and their potential implications in disease development were less explored in SLE.Giaglis et al. [41] reported that a high level of DNA pcf is resulted from mtDNA pcf rather than from nDNA pcf in SLE.However, Truszewska et al. [42] found that high DNA pcf is due to high nDNA pcf in SLE.They also failed to find differences in mtDNA pcf copies between SLE patients and HCs.The present investigation has revealed higher nDNA pcf but lower mtDNA pcf copies in SLE patients compared with those of HCs (Table 2).Similar to the Q-PCR protocol described by Giaglis and Truszewska and those reported in the literature [41][42][43], we measured the mtDNA pcf and nDNA pcf copies in a given volume through the established standard curves.There are small differences between the absolute copies reported by Giaglis and Truszewska and the relative copies detected in the present investigation.Although the data by Giaglis and Truszewska were generated in more precise manner, the results of the present study could still provide useful information.So to speak, our patients exhibited higher SLEDAI of 8 in median than the cohort in Truszewska's of 7.42 in average or in Giaglis' of 2 in median, indicating more severe clinical manifestations in our patients.These similarities and disparities suggest that the mechanism underlying the release of intracellular DNA into plasma to become nDNA pcf or mtDNA pcf in SLE patients needs re-appraisal.Through cell death, degradation or damage and extrusion of intracellular molecules, partial or complete genomes from in situ or nearby tissue cells that may or may not contain invading viruses or microbes, would be continuously shed into human body fluids to form DNA cf [44].Different from the other types of DNA cf , most DNA pcf are originated from hemopoietic cells [12].As a result, necrosis, apoptosis, degradation or damages of dysregulated SLE-immunocytes might be the major contributors to the release of SLE-DNA pcf [40,45].Among the different types of immunocytes, neutrophil is the most abundant one with plenty of DNA molecules and has been implicated in the trigger and perpetuation of SLE [46].DNA pcf from dysregulated neutrophils might be involved in the pathogenesis of SLE.
Similar to necrosis, apoptosis or exocytosis of general cells, neutrophils are able to undergo a unique mechanism of metamorphosis resulting in neutrophil extracellular trap (NET), which is accordingly called "NETosis", to deal with the invading pathogens or sterile inflammation (Fig. 1).Stimulated by ROS and extruding web-like NET, which is composed of a nucleus-derived decondensed DNA coated with histones, granular proteins, and cytoplasmic proteins, into the extracellular space, some neutrophils can entangle the pathogens and trigger immune reactions to result in a lysis of their own cell membrane and the death of invading microbes, termed lytic NETosis (Fig. 1a) [47].Different from the lytic NETosis, some "immortal neutrophils" may undergo vital NETosis to keep intact cell membranes, in which they only extrude a small amount of mtDNA into NET, allowing themselves to remain alive and continue to exert antimicrobial activity, even if they become anucleated (Fig. 1b) [48,49].Through the release of neutrophil nDNA pcf and mtDNA pcf , NETosis might lead to a self-antigen exposure and participate in the pathogenesis of SLE (Fig. 1c) [40,50,51].
As shown in Fig. 1, both lytic and vital NTEosis in neutrophils could contribute to the release of nDNA molecules, which could account for our results of higher nDNA pcf in SLE patients than in HCs (Table 2).In our cohort, SLE patients tended to present lower levels of mtDNA pcf (Table 2) and such a decrease was highly correlated with the high SLEDAI (Table 3).This implies that mtDNA are captured in the NET without releasing into the plasma, which is a possible result of vital NETosis.The mtDNA-enriched NET may triggered a vicious cycle of immune response because the high CpG motif in mtDNA that would propagate immunogenicity cascade in innate immunity [52].Therefore, based on the findings of low mtDNA pcf copies and higher median SLEDAI of 8 as demonstrated in our cohort, similar mtDNA pcf copies and less higher mean SLEDAI of 7.42 in Truszewska's cohort [42] or high mtDNA pcf copies but low median SLEDAI of 2 in Giaglis' cohort [41], we speculated that the different results of mtDNA pcf or nDNA pcf might be hybrid results from diverse proportions of lethal (lytic) and vital NETosis (Fig. 1a-c) regulated by oxidative stress (Fig. 1d).To validate this speculation, we need again to evaluate the NETosis activity and the differences among intracellular DNA, cell membrane-bound DNA and DNA pcf of SLE patients in the future.
Lupus nephritis is an important clinical manifestation in SLE, and the role of oxidative DNA damage has been evaluated [53].However, the role of DNA pcf remained unclear.Because 8-OHdG is a stabilized product after oxidative DNA damage, it is an optimal marker to reflect the ROS levels.Our preliminary results showed that SLE patients harbored higher levels of plasma 8-OHdG and higher nDNA pcf copies than did HCs' (Table 2), and the high plasma 8-OHdG levels were related to high nDNA pcf copies among SLE patients (Table 4).It is suggested that high oxidative stress might cause an abundant release of nDNAfrom impaired leukocytes during NETosis.We also found that SLE patients with nephritis had higher nDNA pcf copies (Table 3), suggesting that damaged kidney might be related to impaied clearance of NET remnant or nDNA pcf .As a result, higher nDNA pcf copies were detected and the vicious cycle sustained.On the contrary, we did not find any association between the levels of plasma 8-OHdG and mtDNA pcf copies in SLE patients, but SLE patients with nephritis tended to have a lower mtDNA pcf copies (p = 0.082) (Table 3).However, Fernandez et al. [54], reported that an elevation in mtDNA pcf would define a subgroup of SLE patients with membranous lupus nephritis.Due to the dynamic changes of intracellular and extracellular mtDNA molecules and the inconsistency in reported literature, Truszewska normalized their ratios between intra to extra cellular mtDNA copy number to correlate the occurrence of nephritis, and it showed good association [42].Besides the mtDNA pcf , the high levels of urine mtDNA contents from damaged kidney (i.e., a kind of mtDNA cf in urine) have been advocated to correlate severity of nephritis [55,56].The precise role of mtDNA either intracellular or extracellular to reflect lupus nephritis needs further investigation.
On the other hand, we failed to observe a higher level of plasma MDA in SLE patients (Table 2) and the levels of plasma MDA were inversely correlated with that of nDNA pcf (Table 4).Unlike the stabilized 8-OHdG, MDA is the peroxidized product of lipid with highly reactive activity, the unstable free form could conjugate with proteins through covalent alterations of amide and amine groups of peptides to form MAD adducts at a post-translational level, giving rise to neo-epitopes that can elicit autoantibody responses [52].The literature has demonstrated that high levels of MDA adducts would trigger obvious autoimmune reactivity in SLE patients [57].Similar to oxidized low density lipoprotein, MDA adducts could induce the formation of NET and propagate immune reaction [58].More precisely speaking, MDA could exist in free and conjugated (adduct) forms, and a free form MDA could positively extrapolate total MDA to predict oxidative stress in exhaled breath condensate as well as urine.But the relevant role of free MDA in plasma/serum, nasal fluid, or saliva to predict total MDA remained controversial and dismal.Different proportions of free MDA and conjugated MDA (MDA-adducts) in different clinical samples might account for these discrepancies [59,60].In the present study, what we measured was the free plasma MDA rather than MDA adducts and lower levels of plasma MDA were inversely correlated with high nDNA pcf copies in SLE patients (Table 4).It is suggested that large amounts of MDA were underestimated because of the presence of high proportion of MDA-protein adducts in SLE patients.
CLEC5A can drive human immune response to defense viral infections [29,37].Recent studies showed that CLEC5A is a critical receptor in innate immune system, causing NETosis to deal with Dangue virus [28,37] or Japanese encephalitis virus [61].Among the 24 HCs, we found that higher levels of leukocyte CLEC5A mRNA level were associated with higher nDNA pcf copies or a lower mtDNA pcf copies, suggesting a possible role of DNA pcf in CLEC5A related immune response (Table 4).High nDNA in the NET is required for neutrophils to trigger suicidal NETosis and to defend against invading pathogens [62].The present results have indicated that the leukocytes in healthy subjects hold functional CLEC5A to trigger proper immune reaction.In contrast, such function may vanish or be dampened in SLE patients, because the above association becomes absent among SLE leukocyte CLEC5A mRNA transcripts and nDNA pcf copies, suggesting an impaired leukocytes/neutrophils in SLE [46].Influenza infection in bone marrow-derived macrophages of CLEC5A −/− mice (dampened CLEC5A) would induce an increase in the secretion of IFN-α [29], which is similar to the IFN signature in SLE [26].Of note, although the added cohort of 3 SLE patients was small in number, the present preliminary results showed an increase of leukocyte CLEC5A mRNA level after the treatment with rituximab (Table 5).Taken together, we propose that an aberrant expression and impaired function of CLEC5A may be implicated in the pathogenesis of SLE.Similar to CLEC5A, cyclic guanosine monophosphate (GMP) -adenosine monophosphate (AMP) synthase (cGAS) is also a member of PRR to act as an additional first-line host immune defense that can induce type I IFN response through its interaction with downstream stimulator of interferon genes (STING), i.e., the cGAS-STING pathway [63].Recently, many immunological studies have focused on the cGAS-STING pathway as another player in the pathogenesis of autoimmune diseases, including SLE [64].It is speculated that cGAS could sense and interact with free plasma intrinsic or extrinsic DNA fragments derived from engulfed NETs to trigger immune cascade [65].This process would be more potent if the cGAS is stimulated by free mtDNA fragments [66,67].Intriguingly, in the present investigation, we observed that SLE patients with higher SLEDAI tended to have lower mtDNA pcf copies extracellularly.But we have also demonstrated a lower mtDNA copy number intracellularly in our previous report [35].In addition, the mtDNA pcf copies was increased after rituximab treatment in severe SLE patients (Table 5).These dynamic alterations of mtDNA strengthen the possibility of the interaction among mtDNA pcf molecules and consumption of mtDNA fragments through PRR, cGAS or CLEC5A, which might contribute to the pathogenesis of SLE.
Viral infection has been proposed to play a role in the SLE pathogenesis.A meta-analysis demonstrated that SLE patients express higher IgG titers to EBV viral capsid antigen (VCA) and early antigen (EA), and exhibit higher positive rates for EBV DNA [68].Furthermore, thanks to the new computational methods to account for the genetics of human diseases, it has been found that nearly half of SLE risk loci are occupied by the Epstein-Barr nuclear antigen 2 (EBNA2) protein and many co-cluster with human transcription factors [69].As to the lupus nephritis, some studies have revealed that a higher serum EBV load is associated with renal damage, and the involved kidneys can express higher EBV-latent membrane protein-1 (EBV-LMP1) and EBV-encoded RNA 1 (EBER-1) [70,71].Notably, higher EBV loads were correlated with higher DNA cf levels in SLE patients [70].The measurement of DNA pcf copies to dissect the interrelations between EBV infection and selfantigen presentation in SLE requires further investigations.

Conclusions
In conclusion, we have demonstrated that higher nDNA pcf copies, lower mtDNA pcf copies, increased ROSelicited oxidative DNA damages and dysregulated leukocyte CLEC5A expression might participate in the pathogenesis of SLE.Further evaluation of the underlying mechanisms in this regard is necessary in the future.

Fig. 1 .
Fig. 1.Illustration is a proposed mechanism of DNA pcf released from immunocytes/neutrophils under pathogen invasion or stress stimulations, which is involved in the self-antigen presentation in SLE pathogenesis.(a) Lytic NETosis, DNA molecules, including nDNA and mtDNA, as well as other proteins in extruded NET can entangle the pathogens and trigger immune reactions to result in a subsequent lysis of their own cell membrane and the death of invading microbes.(b) Vital NETosis, different from the cell death during lytic NETosis, some immune cells secrete only mtDNA, harboring high immunogenicity, into NET, which enable them to maintain alive even if they become anucleate.(c) Both lytic and vital NETosis could cause the release of DNA into blood circulation and involve the presentation of self-antigen.(d) ROS plays an important regulator in adjusting NETois, and the degrees of ROS-elicited modifications could be reflected by the abundance of 8-OHdG and MDA.(e) CLEC5A can drive human immune response to defend viral infections and is a critical receptor in innate immune system causing NETosis.

Table 3 ,
Left) and among HCs, SLE patients without nephritis and

Table 3 . Fluctuation of the distributions in mtDNA pcf copies and nDNA pcf copies from 43 HCs to 80 SLE patients with the absence or presence of different clinical manifestations.
pcf copies, plasma cell-free mtDNA copies; nDNA, nuclear DNA; nDNA pcf copies, plasma cellfree nDNA copies; HC, healthy control; SLE, systemic lupus erythematosus; SLEDAI, SLE disease activity index; CNS, central nervous system; a Comparison among HCs, SLE patients with SLEDAI ≤8 and SLE patients with SLEDAI >8 or among HCs, SLE patients without clinical manifestations and SLE patients with clinical manifestations (e.g., abnormal anti-dsDNA antibody, CNS involvement, nephritis, skin rash, alopecia, oral ulcer or complement decrease), using Jonckheere-Terpstra trend test to demonstrate their trends of distributions.