Title page Title: PTX3 activates classic complement pathway and mediates phagocytosis of cholesterol-induced apoptotic macrophages in atherosclerosis Running Title: PTX3 activates classic complement pathway

Backgrounds/Aims: Apoptotic macrophages are removed by neighboring phagocytes (efferocytosis), which is an important event in advanced atherosclerosis. We reported the long pentraxin 3 (PTX3) located at the membrane of late apoptotic macrophages, mediates efferocytosis in a cell model of advanced atherosclerosis. However, the mechanism underlying PTX3-mediated apoptotic cell clearance in atherogenesis is unclear. Methods: We modeled macrophage apoptosis in advanced plaques by incubating macrophages (peritoneal macrophages isolated from C57 mice) with free cholesterol (free cholesterol-induced apoptotic macrophages, FC-AMs). FC-AMs were added to a monolayer of fresh phagocytes°Cmacrophages°C to study the engulfment response. The percentage of phagocytes that ingested FC-AMs was quantified by using confocal microscopy. C1q and C3b were detected by using fluorescence-activated cell sorter (FACS) and the confocal microscopy. Results: We found that PTX3 significantly enhances complement C1q binding on FC-AMs and was correlated with improved activating of classic complement pathway. C3b was the


PTX3;
C1q; classic complement pathway; efferocytosis; atherosclerosis Background Immune responses participate in several phases of atherosclerosis [1]. Both adaptive immunity and innate immunity are believed to tightly regulate atherogenesis. PTX3, an essential component of the humoral arm of innate immunity, is a superfamily of acute-phase proteins highly conserved during evolution and involves the development of atherosclerosis [2]. PTX3 is rapidly produced and released by several cell types, particularly mononuclear phagocytes, dendritic cells, fibroblasts, osteoblasts 4 / 27 and endothelial cells [3,4]. PTX3 is produced obviously by macrophages and endothelial cells in advanced atherosclerotic lesions [2,5]. Mice lacking PTX3 showed more pronounced inflammatory profile in the vascular wall, an increased macrophage accumulation within the plaque, a greater no-reflow area in a model of acute myocardial infarction, however, all of which could be reversed by exogenous PTX3 [6,7]. The above data support the cardio-protective function of PTX3.
However, the mechanism underlying PTX3-mediated cardioprotective function is also unclear. Our previous studies modeled macrophage apoptosis in advanced plaques by incubating macrophages (peritoneal macrophages isolated from C57 mice) with free cholesterol (free cholesterol-induced apoptotic macrophages, FC-AMs) [8]. Our results showed PTX3 was located at the membrane of late apoptotic macrophages and mediates the phagocytosis of apoptotic macrophages(efferocytosis) [8]. However, the components interacting with PTX3 and mediating phagocytosis are also unknown till now.
Complement plays a key role in atherosclerosis [9]. Cholesterol crystals can activate the complement system and induce an inflammatory response in the development of atherosclerosis [10].
In atherosclerotic lesions, expression of C1q occurs during 5 / 27 differentiation of monocytes to DCs and macrophages and found be important in playing a protective role in early lesion formation in LDL receptor deficient mice [11]. C1q promotes macrophage survival during ingestion of excess cholesterol, as well as improves foam cell efferocytic function [12]. Surface-immobilized monomer Creactive protein was able to initiate classical pathway activation and lead to C3 turnover [13]. A cell surface receptor for C3b is also expressed by macrophages in human atherosclerosis, which is involved in the clearance of circulating opsonized particles by hepatic Kupffer cells from the circulation [14,15]. If PTX3 interacts with complement and mediates phagocytosis are also unknown till now.
In this study, we would explore the mechanism of PTX3 activates C1q-mediated classic complement pathway and mediates phagocytosis of cholesterol-induced apoptotic macrophages, which may be important in providing insight into the protective role of PTX3 in atherosclerosis.

Detection of C1q
The binding of C1q to FC-AMs was analyzed in the presence of C1q, PTX3 or anti-PTX3 mAbs. To harvest these FC-AMs, a nonenzymatic cell dissociation solution was used to treat FC-AMs on FC-AMs was analyzed using FACS.

Detection of C1q and C3b
Immunofluorescence assay was analyzed according to the manufacturer's instructions. FC-AMs were fixed with 4% paraformaldehyde for 15 min at room temperature. After incubation with goat serum, FC-AMs were incubated with anti-C1q mAbs or anti-C3b mAbs for 12 h at 4℃. After washing, FC-AMs were incubated with Alexa Fluor-labeled or Cy3-labeled IgG at room temperature for 60 min. FC-AMs were washed with PBS for three times. Confocal microscopy was used to analyze the fluorescence.

Phagocytosis
FC-AMs were labeled with the membrane probe DiL at room temperature for 15 min. Then, they were added to a monolayer of 9 / 27 fresh phagocytes (macrophages). After incubation with the phagocytes at 37℃ for 30 min, non-ingested FC-AMs were removed by vigorous washing. The phagocytes were labeled with DAPI and fixed with 4% paraformaldehyde and viewed by using the confocal microscopy. The percentage of phagocytes that ingested FC-AMs was quantified by confocal microscopy (blind counting).

Statistical Analysis
The data were leveled as the mean ± standard deviation and the comparisons between the groups were performed using the independent sample't tests. The statistical significance was defined as P<0.05.

Binding of C1q to apoptotic cells
The binding of C1q to apoptotic cells was analyzed in the absence or presence of C1q with FACS and Confocal Microscopy analysis.
We used the anti-C1q-mAbs to bind the membrane surface protein C1q. These antibodies were recognized using goat anti-mouse-Cy3labeled IgG (red-orange). The mean optical density representing C1q levels were higher with pre-incubation with C1q as shown in mAbs, an obvious decrease in C3b deposition was observed (Fig. 3). mAbs, leading to the reduced C3b deposition (Fig. 4C). The statistical results of mean optical density presented as mean ± SD were shown in Fig. 4E.

The activating of classic complement pathway mediates phagocytosis of FC-AMs
We studied the role of the classical complement activation component C3b on the phagocytosis of FC-AMs by employing the macrophage. Pre-incubation with anti-C3b mAbs resulted in phagocytosis decline of FC-AMs (Fig. 5A, B). In control groups, the percentage of phagocytosis was 94%±1%. Compared to the control groups, the percentage of phagocytosis in anti-C3b mAbs groups was lower significantly (83% ± 2%) (mean ± SE, n=2, P<0.05) (Fig.   5B). epidermal growth factor 8 (mfge8) and trans-glutaminase 2 (tg2) [20]. Complement C1q, the first component of the classical pathway, initiates the activation of the classic pathway [21]. Then, pyrolysis components, C3b and C4b, produced from the complement activation accumulate on the apoptotic cells, mediate the efferocytosis of apoptotic cells [22]. C1q gene-targeted mice confirmed that the classic complement pathway plays a role in apoptotic cell clearance in atherosclerosis [23]. Meanwhile, the defective apoptotic cell clearance increases the lesion development [23]. PTX3 and C1q belong to the humoral arm of the innate immune system. PTX3 enhances C1q deposition and triggers the subsequent complement activation on apoptotic cells [24].
These are consistent with our study that showed that PTX3 enhanced the C1q binding on FC-AMs in atherosclerotic cell model.
In atherosclerotic cell model, we also observed the C1q involved activation of C3. We found that the activated complement with a monoclonal antibody to CR1 [7,26,27]. We also observed that C3b played an important part in efferocytosis of FC-AMs, however, which was inhibited by a monoclonal antibody of C3b. Taken together, there is convincing evidence to suggest that PTX3 mediates phagocytosis of apoptotic macrophages by activating classic complement pathway in an atherosclerotic cell model.

Conclusion
Efferocytosis has tremendous potential as both a therapeutic and diagnostic target [28]. The processes of macrophage apoptosis and defective efferocytic clearance of these apoptotic cells may play a critical role in plaque progression of coronary artery disease (CAD) [20]. Our previous study confirmed that PTX3 is located at the membrane of late apoptotic macrophages and mediates 15 / 27 phagocytosis of apoptotic macrophages [8], which is associated with activating classic complement pathway in an atherosclerotic cell model. Understanding of the indepth molecular and cellular biology of efferocytosis would be beneficial to the open up novel opportunities for mechanism-based therapy. Our another published study showed that PTX3 promoter methylation is associated with the PTX3 plasma levels in CAD [29]. Compared to neutrophil to lymphocyte ratio, matrix metalloprotein 9, and interleukin-6, PTX3 displayed greater area under the receiver operating characteristic curve and association with CAD. PTX3 may become a potentially powerful inflammatory biomarkers for the CAD [30,31]. It may provide a possibility for early screening of CAD patients independently of all classical risk pathways [32]. PTX3, a novel therapeutic and diagnostic target may provide an actionable opportunity for the precision cardiovascular medicine [33].

Consent for publication
All authors have seen the manuscript and approved to submit to your journal.

Funding
This work was supported by the grants from the National Natural Science Foundation of P. R. China (No.81701376).
All authors read and approved the final manuscript.

Acknowledgements
We sincerely acknowledge Director Yong Zhou for writing assistance.

Authors' information
Prof. Bei Cheng, MD PhD    The statistical results presented as mean ± SD were shown in G.