Serum Complement Protein C3a Level is Associated With Anti dsDNA Ab in Systemic Lupus Erythematosus: a Brief Report

Objective. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that is characterized by complement dysfunction and a wide range of autoantibody production. However, data about the relation between C3a and anti-ds DNA Ab in SLE are scarce. Methods. Thirteen SLE patients, diagnosed on the basis of SLICC classication criteria (6 patients positive for anti-Sm Ab, 7 patients positive for anti-dsDNA Ab) were enrolled in the present study. Serum levels of C3a, C1q were quantied by Western Blotting. Clinical, biochemical, serological and other markers of disease activity (anti-SM, anti-dsDNA) were measured by standard laboratory procedure. Results. Serum C3a levels were signicantly higher in anti-dsDNA Ab (+) patients compared to anti-Sm Ab (+) patients (p < 0.01). And serum C3a levels positively correlated with SLE Disease Activity Index (SLEDAI) (p < 0.05, r = 0.6134). Interestingly, C3a was slightly correlated positively with D-Dimer, but no signicant difference was found (p = 0.0983, r = 0.4783). were performed by using GraphPad Prism (version 5.01). Mean serum level of C3a and C1q in two groups was compared by Student’s t test. Correlation of C3a with SLEDAI scores, anti-dsDNA Ab, complement components C3 and C4, IFN-a and IL-6 were analyzed by Pearson correlation test. The results were expressed as median and range. Statistical tests with two-tailed p values less than 0.05 were considered signicant.


Introduction
Complement, as an essential part of innate immunity and an evolutionary old system, is highly conserved among a wide variety of species, emphasizing its importance in immune defense throughout evolution [1]. Upon stimulation, complement can be activated within seconds via three different pathways, thereby displaying multiple immune effector functions in controlling infection and maintaining homeostasis [2,3]. The small activation production C3a and C5a, also called anaphylatoxins, are mainly involved in promoting in ammation, including release of proin ammatory cytokines, degranulation of mast cells, an increase in vascular permeability, smooth muscle cell contraction and chemotaxis of immune cells [4]. However, complement behaves as a "double edged sword". Dysfunction of complement system often disrupts homeostasis and ultimately leads to severe diseases, such as autoimmune diseases, infection, tissue damage and tumor progression [2].
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that affects all ages, sexes, ethnicities, and backgrounds [5]. It is characterized by complement dysfunction and a wide range of autoantibody production. Furthermore, complement breakdown products are factors that contribute to tissue damage in SLE[6]. The most unique and common autoantibodies are antibodies (Abs) against double-stranded DNA (ds DNA) and Smith (Sm), which are important criteria for the classi cation of SLE [7]. An important features of anti-ds DNA Ab is its quantitative variation over time, which can essentially disappear with treatment in some cases [8]. In contrast, anti-Sm Ab level is often stable over time and seems resistant to the effects of treatment [7].
Both complement C3a and anti-ds DNA Ab indicate the acute phase responses. However, data about the relation between C3a and antids DNA Ab in SLE are scarce. In this study, we investigated the correlation of serum C3a with anti-ds DNA Ab, Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) scores and D-Dimer. A signi cant association between serum C3a and anti-ds DNA Ab, SLEDAI was found, indicating C3a as an important biomarker for disease activity.

Patients And Study Design
Subjects Blood were obtained from 13 consecutive unselected SLE patients identi ed according to the Systemic Lupus International Collaborative Clinics (SLICC) classi cation criteria [9], including 6 patients (46.2%) positive for anti-Sm Ab, 7 patients (53.8%) positive for anti-dsDNA Ab (Table 2), as well as the healthy blood donors. Serum was obtained by allowing whole blood to clot at room temperature for 30 min, then leaving the whole samples stay on ice for another 60 min, followed by centrifugation at 4000 rpm at 4°C for 10 min. The supernatant was collected and stored at − 80°C until use.
Written informed consent was obtained for each participant. This study was approved by the Ethics Committee of Union Hospital at Huazhong University of Science and Technology, and the methods were applied in accordance with the approved guidelines. Disease activity was measured using the Systemic Lupus Erythematosus Disease Activity Index-2000 (SLEDAI-2K). [10] Routine laboratory investigations Routine laboratory investigations were performed, including Coagulation Tests(D-Dimer, FDP, ATIII and so on), in ammatory markers(ESR, CRP, PCT), liver and kidney function tests. Immunological examinations such as complement (C3 and C4), cytokines (IL-2,IL-4,IL-6,IL-10) and autoantibody screen were measured by standardized technique.

Measurement of the Complement proteins
The Serum C3a and C1q were detected by western blot. Brie y, serum (0.5ul/sample) was premixed with PBS (7.5ul/sample) and rotiload 1 (2ul/sample), then cooked for 10min at 98°C. The mixed samples were then separated by 12% SDS-PAGE and transferred onto polyvinylidene uoride membranes. The membranes were blocked in 4% skim milk powder and 1% BSA (supplied with 0.05% Tween-

Statistical analyses
Statistical analyses were performed by using GraphPad Prism (version 5.01). Mean serum level of C3a and C1q in two groups was compared by Student's t test. Correlation of C3a with SLEDAI scores, anti-dsDNA Ab, complement components C3 and C4, IFN-a and IL-6 were analyzed by Pearson correlation test. The results were expressed as median and range. Statistical tests with two-tailed p values less than 0.05 were considered signi cant.

Clinical characteristics of SLE patients
The comparison of clinical characteristics of SLE patients with anti-Sm Ab (+) and anti-dsDNA Ab (+) is shown in Table 1. A total of 13 SLE patients were enrolled in the present investigation. The mean age (range) of anti-dsDNA Ab and anti-Sm Ab positive patients were 38.5 (23-57) and 48 (16-68) years, respectively. The mean duration of disease (range) were 2.5 (0-13) and 1 (0-18) years, respectively. The distributions of the demographic and laboratory features and scores on the SLEDAI were not statistically different between the two groups.  AS: anti-Sm antibodies (+) AD: anti-dsDNA antibodies (+) Higher serum C3a levels were identi ed in anti-dsDNA Ab positive SLE patients Serum levels of C3a and C1q in SLE patients (anti-Sm Ab (+) (n=6); anti-dsDNA Ab (+) (n=7)) were quanti ed by western blot (Figure   1(a) and 1(b)). As shown in Figure 1(c), anti-dsDNA Ab (+) patients displayed a signi cantly higher level of serum C3a integrated density (mean=1.24, SD=0.561) compared to anti-Sm Ab (+) patients (mean=2.59, SD=0.79) (P < 0.01; n = 13). while the level of serum C1q don't show differences between two groups (P = 0.1812; n = 13) (Figure 1 (d)), indicating the appearance of anti-dsDNA closely correlates with complement overactivation.
Serum C3a correlates positively with SLEDAI score Correlation between serum C3a and SLEDAI scores was analyzed by Spearman rank correlation coe cient and the result is shown in Figure 2(a). We observed a signi cant positive correlation of serum C3a levels with SLEDAI score (p < 0.05, r = 0.6134), while C3a was slightly correlated positively with D-Dimer, but no signi cant difference was found (Figure 2(b)) (p = 0.0983, r = 0.4783). These data indicate C3a level, to some extent might be used for predicting the SLE disease severity.

Discussion
SLE is an autoimmune disease characterized mainly by in ammation. After onset, the clinical manifestations of patients are relatively complex, the condition is unstable, and the recurrence rate is high [11]. Complement system is a central immune surveillance system, which can be activated through classical, lectin and alternative pathways. Dysfunction of complement leads to many diseases' progression, such as SLE, a-HUS, PNH, DIC and so on [12]. Currently, C3 and C4 have become the mainly biomarkers for monitoring SLE disease activity.But several limitations exist as low levels of C3, C4 are found, even more infrequently in very early and milder disease, instead of acute phase. [13].
The present study observed elevated C3a levels in anti-dsDNA Ab positive SLE patients, but not in anti-Sm Ab positive patient. Further C3a level positively correlates with disease activity scores. C3a and C5a are fragments, immediately generated upon activation of the complement cascades, which re ects complement activation more accurately than the levels of the individual intact proteins [14].
Previously, it was shown that increased levels of complement split products are associated with disease activity, however, they were not used to study the relationship between serum C3a and Disease Activity considering extremely short half-lives of C3a [15].
In addition, the current investigation also identi ed a slightly association between serum C3a levels and D-Dimer. It was shown that complement anaphylatoxins C3a and C5a can activate macrophages and neutrophils, release more vasoactive substances, leading to local vascular in ammation. Therefore, for normal people, the levels of complement C3a and C5a are relatively low, but when the complement is overactivated, its expression level will increase rapidly. Those indicates that injury of the vascular endothelium caused by complement overactivation and immune complex formation might contribute to the vasculopathy in SLE. Observations in the current study indicates C3a shows promise as a biomarker for monitoring SLE disease activity and thrombophilia.
However, a possible limitation to the use of C3a for evaluation of SLE disease activity is that not all increase of C3a is relative to SLE disease activity, many other stimuli, like pathogenic microbes, tumors and other autoimmune diseases can also cause complement overactivation. In addition, how anti-dsDNA positively correlates with complement overaction and what is the mechanism behind are still unclear. These aspects can be an interesting senarios for future research.
Declarations Figure 1 See image above for gure legend.

Supplementary Files
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