Phosphorylcholine antibodies restrict infarct size and left ventricular remodelling by attenuating the unreperfused post‐ischaemic inflammatory response

Abstract Phosphorylcholine is a pro‐inflammatory epitope exposed on apoptotic cells, and phosphorylcholine monoclonal immunoglobulin (Ig)G antibodies (PC‐mAb) have anti‐inflammatory properties. In this study, we hypothesize that PC‐mAb treatment reduces adverse cardiac remodelling and infarct size (IS) following unreperfused transmural myocardial infarction (MI). Unreperfused MI was induced by permanent ligation of the left anterior descending (LAD) coronary artery in hypercholesterolaemic APOE*3‐Leiden mice. Three weeks following MI, cardiac magnetic resonance (CMR) imaging showed a reduced LV end‐diastolic volume (EDV) by 21% and IS by 31% upon PC‐mAb treatment as compared to the vehicle control group. In addition, the LV fibrous content was decreased by 27% and LV wall thickness was better preserved by 47% as determined by histological analysis. Two days following MI, CCL2 concentrations, assessed by use of ELISA, were decreased by 81% and circulating monocytes by 64% as assessed by use of FACS analysis. Additionally, local leucocyte infiltration determined by immunohistological analysis showed a 62% decrease after three weeks. In conclusion, the local and systemic inflammatory responses are limited by PC‐mAb treatment resulting in restricted adverse cardiac remodelling and IS following unreperfused MI. This indicates that PC‐mAb holds promise as a therapeutic agent following MI limiting adverse cardiac remodelling.


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PLUIJMERT ET aL. limitations, clinical complications or simply because of unavailable facilities to provide relevant care. Especially in the less developed countries this may be a serious issue. Besides aiming for timely reperfusion and additional therapies to salvage myocardium, intervening in unreperfused transmural MI to modulate cardiac remodelling therefore remains of importance; hence, focus in this study is on unreperfused MI. Transmural MI results in adverse left ventricular (LV) remodelling, characterized by LV dilatation and reduced LV wall thickness, which successively leads to heart failure, 4 one of the leading causes of death worldwide. 5 Myocardial infarction triggers a complex inflammatory response, which helps to clear the injured myocardium from dead cardiomyocytes and matrix debris, and ultimately leads to infarct healing and mature scar formation. 6 However, when the inflammatory response is extended, it may cause viable cardiomyocytes to die. 7 Necrotic cardiomyocytes release damage-associated molecular patterns (DAMPs), like high mobility group box-1 (HMBG1), heat shock protein (HSP), interleukin (IL)-1α and extracellular RNA (eRNA), which trigger the innate immune system 7 via Toll-like receptor (TLR) activation. [8][9][10] Currently, large randomized controlled trials such as the Canakinumab Antiinflammatory Thrombosis Outcome Study (CANTOS) 11 and Colchicine Cardiovascular Outcomes (COLCOT) 12 trials reported promising therapeutic potential of anti-inflammatory therapies in decreasing cardiovascular events after MI. Additionally, the role of apoptotic cells seems to be more complicated. Uptake of apoptotic cells by macrophages might have anti-inflammatory effects 13 ; on the other hand, it has been suggested that apoptotic cells are immunogenic and pro-inflammatory. 14 In addition, effective efferocytosis of apoptotic cardiomyocytes was found to improve the resolution of inflammation after MI. 15 However, the main part of apoptotic cells in the healing injured myocardium are noncardiomyocytes. For instance, apoptotic neutrophils represent a large part of the apoptotic cells in the healing injured myocardium, and their role in inflammation resolution is yet unknown. 7 Following MI, the production of reactive oxygen species by circulating phagocytes, endothelial cells and cardiomyocytes is increased as a result of the ischaemic event. 16 These reactive oxygen species are responsible for generating oxidative damage and producing oxidation-specific epitopes on apoptotic cells, which can act as DAMPs and are recognized by innate immunity. 17 Phosphorylcholine (PC), the polar headgroup of oxidized phospholipids (oxPLs), is an important oxidation-specific epitope, present on apoptotic cells but absent on viable cells. 14 Moreover, phosphorylcholine is present on oxidized LDL (oxLDL), a key player in atherogenesis because of its pro-inflammatory properties. 18 It has been shown in mice that a specific clone of IgM autoantibodies against phosphorylcholine, termed E06 or T15 antibodies, 19 can inhibit the uptake of both apoptotic cells and oxLDL by macrophages in vitro 20,21 and in vivo 22 and has anti-inflammatory properties. 14 However, if complete cascade systems are present, E06 appears to augment efferocytosis. [23][24][25] Furthermore, B-1a and B-1b cells showed to produce oxidation-specific epitope-specific IgM antibodies, which protect against atherosclerosis, [26][27][28] and it has been found that splenic B cells display an oxidation-specific epitopeassociated atheroprotective effect, which is initiated through sterile inflammation. 29 Moreover, low levels of natural IgM phosphorylcholine antibodies are associated with an increased risk of cardiovascular events 30-34 and resulted in a worsened prognosis regarding patients with an acute coronary syndrome. 35 In addition, both active and passive immunization with antibodies against phosphorylcholine ameliorates development of atherosclerosis and is proven to be atheroprotective. [36][37][38] Altogether, these data indicate that blocking phosphorylcholine might be an interesting therapeutic approach to treat cardiovascular disease. However, compared to IgG antibodies, IgM antibodies are not optimal for therapeutic use, because of rapid elimination from plasma, and being unstable, difficult to produce and relatively expensive in addition.
We previously developed a fully human IgG1 directed against human phosphorylcholine (PC-mAb) and with anti-inflammatory properties. PC-mAb blocks oxLDL uptake by macrophages and inhibits vascular remodelling in a mouse model for accelerated atherosclerosis and preserves coronary flow reserve and attenuates atherosclerotic inflammation. 40 Above all, it attenuates the immediate inflammatory response following myocardial ischaemiareperfusion injury in hypercholesterolaemic APOE*3-Leiden mice, preserving cardiac function with an increased ejection fraction of 33%. 41 As unreperfused transmural MI yet remains a significant determinant in worldwide morbidity and mortality, pressing heavily on the healthcare system and costs, additional therapeutic effects of PC-mAb following unreperfused MI might be of interest.
Furthermore, hypercholesterolaemia causes a pro-inflammatory phenotype characterized by monocytosis, 42,43 making it an important factor to consider in experimental studies. Therefore, in the current study, the effect of PC-mAb treatment on cardiac function, LV remodelling and the inflammatory response is investigated in hypercholesterolaemic APOE*3-Leiden mice after initiating unreperfused MI.

| Animals and diets
All animal experiments were approved by the Institutional

| Surgical myocardial infarction model and PC-mAb treatment
Myocardial infarction was induced by ligation of the LAD coronary artery at day 0 in 12-to 14-week-old female APOE*3-Leiden mice as described previously. 47 Briefly, mice were pre-anaesthetized with a gas mixture of 5% isoflurane and oxygen and placed in a supine position on a heating pad (37°C). After endotracheal intubation and ventilation (rate 160 breaths/min, stroke volume 190 μL; Harvard Apparatus), mice were kept anaesthetized with 1.5%-2% isoflurane. Subsequently, a left thoracotomy was performed in the 4th intercostal space and the LAD coronary artery was permanently ligated using a 7-0 prolene suture. Subsequently, the thorax was closed in layers with 5-0 prolene suture and mice were allowed to recover. Analgesia was obtained with buprenorphine s.c. (0.1 mg/kg) pre-operative and 12 hours post-operative. After surgery, animals were randomly grouped to receive intraperitoneal administration of 10 mg/kg PC-mAb (known as ATH3G10; Athera Biotechnologies) 39 every 3rd day or NaCl 0.9% w/v (vehicle) as a control. Sham-operated animals were operated similarly but without ligation of the LAD and received injections with NaCl 0.9% w/v (sham).
After 2 days or 3 weeks, under general anaesthesia with 1.5%-2% isoflurane, mice were euthanized by bleeding and explantation of the heart. Hearts were immersion-fixated for 24 hours in 4% paraformaldehyde and embedded in paraffin. Blood samples were collected and used for serum analysis. The heart and bodyweight were measured from all animals using a digital scale.

| Cardiac magnetic resonance imaging
Left ventricular dimensions, function and IS were assessed 2 days and 3 weeks after surgery by using 7-Tesla CMR imaging (Bruker Biospin) to obtain contrast-enhanced and cine CMR images. Mice were pre-anaesthetized with 5% isoflurane in a gas mixture of oxygen and kept anaesthetized with 1.5%-2% isoflurane. Respiratory rate was monitored by a respiration detection cushion, which was placed underneath the thorax and connected to a gating module to monitor respiratory rate (SA Instruments, Stony Brook). Image reconstruction was performed using Bruker ParaVision 5.1 software.

| Infarct size
Infarct size was determined with contrast-enhanced CMR imaging after injection of 150 μL (0.5 mmol.mL) of gadolinium-DPTA (Gd-DPTA, Dotarem, Guerbet) via the tail vein. To acquire a set of 14 contiguous 0.7 mm contrast-enhanced slices in short-axis orientation, a gradient echo sequence (FLASH) was used. Imaging parameters were as follows: echo time of 1.9 ms, repetition time of 84.16 ms, field of view of 33 mm 2 and a matrix size of 192 × 256.

| Left ventricular function
Left ventricular function was assessed with a high-resolution 2D FLASH cine sequence to acquire a set of 9 contiguous 1 mm slices in short-axis orientation covering the entire heart. Imaging parameters were as follows: echo time of 1.49 ms, repetition time of 5.16 ms, field of view of 26 mm 2 and a matrix size of 144 × 192.

| LV fibrous content and LV wall thickness
Paraffin-embedded hearts were cut into serial transverse sections of 5 µm along the entire long axis of the LV, and every 50th section was stained with Sirius Red. Collagen deposition was used as an indicator of the fibrotic area, and LV fibrous content was determined by planimetric measurements of all sections and calculated as fibrotic area divided by the total LV wall surface area.

Left ventricular wall thickness was analysed in five different
sections centralized in the infarct area. Per section wall thickness was measured at three places in the infarct area, both border zones, and at two places in the intraventricular septum. All measurements were performed using the ImageJ 1.47v software program (NIH).

| Local inflammatory response
For analysis of the cardiac inflammatory response, a subpopulation was selected, and sections were stained using antibodies against leucocytes (anti-CD45, 550539; BD Pharmingen). The number of leucocytes was expressed as a number per 0.25 mm 2 in the septum (2 areas), border zones (2 areas) and infarcted myocardium (3 areas).

| FACS analysis
To examine the effect of PC-mAb therapy on the acute inflammatory response, mice were killed and blood samples were collected after 2 days. To study the systemic effects, whole blood was analysed for monocytosis. Total circulating leucocytes were determined using a semi-automatic haematology analyser F-820 (Sysmex Corporation).
For FACS analysis, 35 μL of whole blood was incubated for

| CCL2 and PC-mAb ELISA
A PC-mAb ELISA kit (Athera Biotechnologies) was used to determine serum PC-mAb concentrations, with a secondary antibody detecting human IgG. To study the effects of PC-mAb on systemic inflammation, inflammatory cytokine concentration of chemokine (C-C motif) ligand 2 (CCL2) was determined using an ELISA kit (Cat. No. 555260, BD Biosciences).

| Phosphorylcholine and TLR4 co-localization
The presence of Toll-like receptor 4 (TLR4) and phosphorylcholine co-localization in the infarct area was investigated by immunohistochemistry. TLR4 was stained using specific antibodies against TLR4 (anti-CD284, AHP1822, Bio-Rad Laboratories Inc).
Phosphorylcholine was stained using the same antibody (Athera Biotechnologies) as was used for treatment.

| Statistical analysis
Values were expressed as mean ± SEM. Comparisons of parameters between the sham, PC-mAb and vehicle groups were made using 1-or 2-way analysis of variance (ANOVA) with repeated measures and Tukey's post hoc correction for multiple pairwise comparisons.
Comparisons were made between PC-mAb and vehicle using un-

| PC-mAb concentrations, cellular mechanisms and phosphorylcholine-TLR4 colocalization
To confirm the observed effects to be the result of PC-mAb treatment, circulating PC-mAb serum concentrations were determined using ELISA. PC-mAb levels were detectable only in the PC-mAb group after 2 days (32 ± 8 µg/ml) and 3 weeks (36 ± 6 µg/ml), confirming the absence of a native immune response against PC-mAb, and were not observed in the vehicle or sham-operated groups ( Figure S1). In addition, it was shown before that PC-mAb binds to late apoptotic cells with strong affinity. 39,41 Moreover, treatment with oxidized low-density lipoprotein of cultured peripheral blood mononuclear cells isolated from human blood showed suppressed CCL2 levels following concomitant PC-mAb treatment ( Figure S2).
As the rationale for using PC-mAb to treat adverse cardiac remodelling following unreperfused MI was to inhibit the pro-inflammatory response, TLR4, as a triggering factor of the inflammatory response, 8 and phosphorylcholine co-localization was investigated. As can be appreciated from Figure 1, TLR4 is indeed localized at comparable areas in the infarct area as is phosphorylcholine.

| PC-mAb reduces contrast-enhanced CMR assessed LV infarct size
Baseline IS was assessed using contrast-enhanced CMR two days following unreperfused MI. No differences in IS could be observed between the PC-mAb-treated group and the vehicle group at baseline (30.9 ± 3.2% vs 36.7 ± 2.7%, P = .175). However, after 3 weeks, PC-mAb treatment compared to vehicle treatment showed a smaller IS (19.7 ± 2.4% vs 28.6 ± 3.3%, P = .042; Figure 2A). Interestingly, IS following unreperfused MI was significantly smaller after 3 weeks compared to 2 days in both vehicle and PC-mAb group. This may indicate that after initial transitory infarct oedema as may occur after 2 days, some degree of favourable infarct remodelling occurs as observed after 3 weeks.

| PC-mAb limits the local inflammatory response
In addition, a striking decrease in local leucocyte infiltration in all areas was observed in the PC-mAb-treated group compared to the

PC-mAb is a human phosphorylcholine monoclonal IgG1 antibody
with anti-inflammatory properties. 39 In our study, post-ischaemic administration of PC-mAb attenuated the immediate systemic inflammatory response after 2 days and the late local inflammatory response after 3 weeks. As a result, IS and LV dilatation were restricted with concomitant preservation of LV wall thickness. Adverse left ventricular remodelling is one of the mechanisms responsible for development of heart failure, 4 known for its high morbidity and mortality rates worldwide, 5 especially in the absence of reperfusion. PC-mAb therapy compared to control F I G U R E 2 Quantification of infarct size using contrast-enhanced CMR imaging. Infarct size is quantified as percentage of the LV mass (A; n = 14-16 per group). Representative contrastenhanced CMR images 2 d following unreperfused MI after vehicle (B) and PC-mAb (C) treatment. Epicardial borders are indicated by red lines, endocardial borders by green lines and infarct area by yellow lines. Data are mean ± SEM. # P < .05 vs vehicle limited adverse cardiac remodelling as observed by restricted LV dilatation. Additionally, PC-mAb treatment compared to control reduced HW and HW/BW ratio, indicating reduced compensatory cardiac hypertrophy, which is another hallmark of adverse cardiac remodelling and heart failure. 48 Moreover, PC-mAb treatment compared to control causes a decreased IS, which has been directly linked to heart failure and mortality following MI. 49 These results suggest PC-mAb treatment to be a potential therapeu- wall thickness probably is a result of the dampened inflammatory response, although it cannot be excluded that it partially is a direct result of the decreased LV dilatation and limited adverse cardiac remodelling. 55 CCL2 is a chemoattractant known for its ability to attract inflammatory leucocytes to sites of tissue injury, 56 for example after myocardial ischaemia-reperfusion injury. 57 Although these attracted leucocytes promote removal of dead tissue and infarct healing, it has been shown that CCL2-deficient mice show decreased recruitment of macrophages into the infarcted myocardium that coincides with decreased LV remodelling following myocardial ischaemiareperfusion injury. 57 This agrees with our finding of decreased CCL2 serum concentration and restricted adverse LV remodelling upon PC-mAb treatment. Previously, PC-mAb was shown to reduce CCL2 levels produced by human monocytes stimulated with oxLDL in vitro and regarding accelerated atherosclerosis local expression of CCL2 in the vessel wall was inhibited. 39 Furthermore, it is known that blood CCL2 levels are increased in hypercholesterolaemic APOE*3-Leiden mice. 58 Therefore, PC-mAb treatment is suggested to reduce the systemic inflammatory response by binding phosphorylcholine on apoptotic cells and/or oxLDL, which contributes to the restricted adverse LV remodelling as a result of reduced serum CCL2 concentrations.
Hypercholesterolaemia causes a pro-inflammatory phenotype, which is characterized by monocytosis, 42 mainly caused by an increase in the pro-inflammatory Ly-6C hi subset. It has been shown that following unreperfused MI in hypercholesterolaemic APOE -/mice, more Ly-6C hi monocytes are recruited into the infarct area, which resulted in decreased LV function 59 and impaired infarct healing. 60 Additionally, myocardial ischaemia-reperfusion injury in hypercholesterolaemic APOE*3-Leiden mice preceded by a preischaemic Ly-6C hi monocytosis resulted in a decreased LV function as well, but was paradoxically coinciding with a reduced IS. 43 This underscores the complex interplay between different mechanisms of ischaemia and concomitant luxating cardiovascular risk factors and the necessity of selecting appropriate experimental models to investigate hypotheses correctly. 50 Following unreperfused MI, we showed that PC-mAb therapy compared to control reduces circulating monocytes, accompanied by a reduced IS and restricted LV dilatation.
In conclusion, PC-mAb treatment following unreperfused transmural MI limits adverse cardiac remodelling and IS as compared to control, likely by ameliorating the immediate inflammatory response upon myocardial ischaemia. Interestingly, PC-mAb seems to mitigate both the atherosclerotic as the ischaemic inflammatory process related to MI. Until now, phase 1 studies showed good safety and tolerability and an additional phase 2a, randomized, placebo-controlled, double-blind, multicentre pilot study is currently running in patients suffering an acute MI. Therefore, PC-mAb treatment might be a potential valuable novel therapeutic strategy to restrict inflammation and adverse cardiac remodelling, improving outcome in ischaemic heart disease when immediate reperfusion is unavailable or not possible.

CO N FLI C T S O F I NTE R E S T
KP is a named inventor on patent and minor shareholder in Athera Biotechnologies. The remaining authors have nothing to disclose.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets generated and/or analysed during the current study are available from the corresponding author upon reasonable request.

O RCI D
Niek J.