Opposing effects of HNP1 (α-defensin-1) on plasma cholesterol and atherogenesis.

Atherosclerosis, the predominant cause of death in well-resourced countries, may develop in the presence of plasma lipid levels within the normal range. Inflammation may contribute to lesion development in these individuals, but the underlying mechanisms are not well understood. Transgenic mice expressing α-def-1 released from activated neutrophils develop larger lipid and macrophage-rich lesions in the proximal aortae notwithstanding hypocholesterolemia caused by accelerated clearance of α-def-1/low-density lipoprotein (LDL) complexes from the plasma. The phenotype does not develop when the release of α-def-1 is prevented with colchicine. However, ApoE-/- mice crossed with α-def-1 mice or given exogenous α-def-1 develop smaller aortic lesions associated with reduced plasma cholesterol, suggesting a protective effect of accelerated LDL clearance. Experiments were performed to address this seeming paradox and to determine if α-def-1 might provide a means to lower cholesterol and thereby attenuate atherogenesis. We confirmed that exposing ApoE-/- mice to α-def-1 lowers total plasma cholesterol and decreases lesion size. However, lesion size was larger than in mice with total plasma cholesterol lowered to the same extent by inhibiting its adsorption or by ingesting a low-fat diet. Furthermore, α-def-1 levels correlated independently with lesion size in ApoE-/- mice. These studies show that α-def-1 has competing effects on atherogenesis. Although α-def-1 accelerates LDL clearance from plasma, it also stimulates deposition and retention of LDL in the vasculature, which may contribute to development of atherosclerosis in individuals with normal or even low plasma levels of cholesterol. Inhibiting α-def-1 may attenuate the impact of chronic inflammation on atherosclerotic vascular disease.

Introduction

Atherosclerosis and its thrombotic sequelae are the predominant causes of death in highly resourced countries and its incidence is increasing in “developing” parts of the world[1]. Hypercholesterolemia is a well-established risk factor for atherosclerosis and lowering plasma levels of LDL-cholesterol through a variety of approaches mitigates risk[2]. However, between 15 and 50% of all cardiovascular events occur in patients with plasma cholesterol within the normal range[1,3]. The mechanisms that drive development of atherosclerosis in such individuals are not well established.

There is compelling data that point to the contribution of inflammation to the development of atherosclerotic lesions[3-7]. More specifically, results from several groups using diverse approaches suggest that human α-defensins (α-defs) 1–4, also known as human neutrophil peptides (HNPs),[2] contribute to this process[5,6,8-11]. We recently examined the mechanism by which α-defensins released from activated neutrophils may promote formation of lipid streaks in the vasculature using HNP-1tg/tg Def+/+ (Def+/+) mice fed a high fat diet (HFD)[12]. We found that α-defensin-1 (α-def-1) forms complexes with LDL, accelerating lipoprotein clearance from the circulation by the liver, leading to hypocholesterolemia[12]. In line with this, plasma levels of total cholesterol (TCH) and LDL were lower in Def+/+ mice than in wild type control animals[12]. However, the Def+/+ mice developed more prominent lipid streaks and greater monocyte/macrophage retention and greater generation of cathepsins B and S in the proximal aorta compared with wild type controls, consistent with enhanced retention of LDL/α-def-1 complexes in the vasculature[12]. Phenotypic correction was seen when release of α-def-1 by neutrophils was blocked by colchicine or precluded by bone marrow transplantation from wild type mice[12].

More recently, formation of LDL/ α-def-1 complexes, enhanced blood clearance of LDL and low plasma LDL were also found in mice double transgenic for α-def-1 and apoE (ApoE−/−HNP-1tg/tg) and in ApoE-/- mice given exogenous α-def-1[13]. However, in this context, the lower plasma levels of LDL were associated with attenuated development of lipid streaks, showing an apparent protective effect of α-defs in this model and suggesting these peptides might serve as a pharmacological vehicle to prevent atherosclerosis[13].

Here, we asked whether the development of lipid streaks in this latter model reflects a balance between the lowering of plasma cholesterol through enhanced hepatic clearance offset by increased binding and retention of residual circulating LDL/α-def-1 complexes in the vasculature[5,12,14-17]. If so, we reasoned that the vascular lesions would be more prominent in ApoE−/−HNP-1tg/tg, or ApoE−/− mice exposed to exogenous α-def-1 than if LDL were lowered to the same extent through alternative mechanisms. We found that using cholestyramine or diet modification to lower plasma cholesterol and LDL in ApoE-/- mice to the levels seen in ApoE−/−HNP-1tg/tg leads to smaller lipid streaks than those developed in ApoE-/- mice exposed to exogenous α-def-1. The implications of our findings regarding the pathogenesis of atherosclerosis and its prevention are discussed.

Methods

Materials

Regular rodent chow diet (RD; 6.5% fat) and HFD (15.8% fat and 1.25% cholesterol) (TD.88051, Harlan) were purchased from Harlan (Harlan, Rehovot Israel) and cholestyramine from Sigma-Aldrich. α-def-1 was purchased from Sigma-Aldrich and kindly provided by Dr. Wuyan Lu (Univ. MD School of Medicine). Each formed complexes with LDL in vitro and accelerated their clearance in vivo[12] and were used interchangeably.

Mice

ApoE-/- mice on a C57BL/6 background were bred in-house from a stock originating from Jackson Laboratories provided by M. Aviram (Rappaport Faculty of Medicine, Technion, Haifa, Israel). Animal care and experiments were conducted in accordance with protocols approved by the Animal Care Committee of the Hebrew University (approval number: MD-15-14579-4) and the University of Pennsylvania. Mice were maintained on a regular rodent chow diet, on an HFD, or a moderate high fat diet (MFD) by combining 65% RD with 35% HFD for the indicated times.

Cholesterol-lowering

Five approaches were used to modify plasma cholesterol in ApoE−/− female mice (16 per group). One set of mice was fed a HFD for 6 weeks without or with 1.5% or 3% cholestyramine[18]. A second set of mice on a HFD, received an intravenous (IV) injection, via tail vein, of α-def-1 (10 or 30 μg) or vehicle (PBS) control every other day[13,19]. A sixth group, studied in parallel, was fed a MFD for 6 weeks. Mice were monitored for adverse effects throughout the experiment. There were no statistically significant differences in body weight between mice in any of the six groups. Mice were anesthetized with an intraperitoneal injection of zolazepam (25 mg/kg) and xylazine (50 mg/kg) on the last day of the experiment and blood samples were taken by transcardiac puncture after 6 hours of fasting[12]. Serum total cholesterol (TCH) and high-density lipoprotein cholesterol (HDL) were measured by enzymatic methods using an autoanalyser (Cobas 6000; Roche, Nakakojo, Japan), and levels of low-density lipoprotein cholesterol (LDL) were calculated as reported[12].

Staining of aortic roots

After blood was withdrawn, mice were euthanized with pentobarbital and the hearts were immediately removed and transected midway between the apex and base in a plane parallel to a line defined by the tips of the atrial appendages. The basal ventricular segments in continuity with the atria and aortic roots were embedded in optimum cutting temperature compound (OCT) and frozen in liquid nitrogen. Cryostat sections were prepared at ~7 μm intervals using a CM 1900 cryotome (Leica Microsystems, Wetzlar, Germany), fixed in formalin, stained with Oil Red, and examined to assess proximity to the aortic root. Sections through the coronary ostia, coronary sinuses, and the aortic leaflets were captured. Lipid deposition was quantified in parallel sections stained with Oil Red, and quantified using Image-Pro Plus analysis software as previously reported[12]. Results are reported as the percentage of the circumference of each root that was Oil Red positive.

Statistical analysis

Group comparisons were performed using one-way ANOVA with the Newman-Keuls post hoc test[12,20]. Correlations between plasma cholesterol and the lesion score were calculated for the entire cohort and separately for each experimental group. Multivariate regression analysis was applied to assess the independent impact of α-def-1 on the size of fatty streaks, irrespective to cholesterol levels. Data is presented as means ± SD, and statistical significance was set at p <0.05.

Results

We examined the effect of exogenous α-def-1 on total plasma cholesterol (TCH) and the extent of fatty streaks formed in the aortic roots of ApoE-/- mice fed a HFD. In the first set of experiments, mice were given 10 or 30 μg α-def-1 IV (low dose and high dose, respectively) or saline vehicle, every other day. Injection of low dose and high dose α-def-1 decreased serum TCH from 1425.4 ± 177.3 to 976.5 ± 160.7 and 707.6 ± 65.3 (mg/dl), respectively (p < 0.001 vs. untreated mice) (Fig 1A), accompanied by proportional decrease in LDL, confirming previous reports from our group[12] and others[13]. The percentage of the circumferences of aortic roots occupied by fatty streaks decreased from 39.9 ± 6.6 to 34.4 ± 3.7% (p < 0.05) and 23.4 ± 2.5% (P< 0.001), respectively following injection of α-def-1 (Fig 1B & 1C), both consistent with previous findings[13].

Fig 1

Effect of intervention on plasma cholesterol and development of lipid streaks in aortic roots.

Panel A. Cholesterol levels. ApoE−/− mice fed a HFD for 6 weeks were divided into 5 groups (n = 16/group). One went untreated (Cont). Two groups were given two oral doses of either a low or high dose cholestyramine, 1.5% (Choles L) or 3% (Choles H), respectively. Two groups were given two IV injections of α-def-1, 10 or 30 μg, every other day (α-def-1 L) and (α-def-1 H) respectively. A sixth group was given a modified high fat diet (MFD) for 6 weeks. Blood samples were taken after 6 hours of fasting. Plasma levels of total cholesterol (TCH) were measured. Panel B. Development of lipid streaks. ApoE−/− mice on HFD or MFD for 6 weeks described in Panel A were sacrificed after blood draw. Lesion size in sections from the aortic roots (See Panel C) was measured as described in the Methods section. Results are expressed as the percent of the aortic root circumferences stained by Oil Red O. The mean ± S.D. and p < 0.05 values are shown; n = 16 per group. Panel C. Lipid streaks. Representative sections from the various experimental groups are shown. Scale bars represent 500μm.

We then compared the effect of α-def-1 with that of cholestyramine. TCH in ApoE-/- mice on HFD given a low (1.5%) or high (3%) dose of cholestyramine fell to 1108.9 ± 112.9 and 756.1 ± 62.6 mg/dl (p < 0.001 vs. untreated mice), respectively (Fig 1A), and the percentage of the aortic root circumferences occupied by fatty streaks decreased from 39.9 ± 6.6 to 20.5 ± 2.5% and14.1 ± 2.3%, respectively (p < 0.001 compared with untreated mice) (Fig 1B & 1C), also consistent with previously reported findings[18].

Of note, however, lesion sizes in mice given high dose α-def-1 were significantly larger than those in mice given the high dose of cholestyramine (p< 0.001) (Figs 1B & 1C & 2), although the two experimental groups had almost identical average plasma levels of TCH (Figs 1A & 2). Lesion size was also smaller in mice fed a modified high fat diet (MFD) (Figs 1B, 1C & 2) that had plasma TCH levels comparable to those observed in the high-dose α-def-1 group (731.9 ± 49.36 mg/dl) (Figs 1A & 2). Lesion size in MFD mice group was comparable to HFD mice given the higher dose of cholestyramine (Figs 1B, 1C & 2), with both groups having equivalent levels of plasma TCH (Figs 1A & 2).

Fig 2

Correlation between plasma cholesterol and size of lipid-rich streaks in aortic roots.

Data from the six groups of ApoE−/− mice described in Fig 1, panels A and B are compared (regression line), and separately for each experimental group (mean ± SD, n = 16 per group). A highly significant correlation is noted in between plasma cholesterol and lesion size, plotted for the entire cohort of mice (R = 0.7, p<0.0001, Pearson's correlation). The two rectangles illustrate experimental groups with comparable plasma cholesterol but significantly higher lesion size score in the α-def-1 groups (p<0.001, ANOVA).

To isolate the atherogenic effect of α-def-1 in ApoE-/- mice, we examined the relationship between plasma cholesterol and lesion size in presence and absence of α-def-1. A close correlation between serum cholesterol and lesion size was observed in all 6 cohorts combined (Fig 2) (R = 0.70, p<0.0001). However, a significantly higher proportion of the aortic roots circumferences were occupied by lesions in mice given low and high doses of α-def-1 (Fig 2, results above the regression line) compared with the other groups of mice (positioned below the regression line) having comparable levels of TCH (p<0.0001; Fig 2). These results strongly suggest an independent effect of α-def-1 on lesion size.

As a second approach to evaluate the potential atherogenic effect of α-def-1, we used multiple regression analysis with the sizes of fatty streaks defined as the dependent variable and cholesterol and α-def-1 serving as stepped predictors. As expected, the impact of plasma cholesterol alone was substantial (Beta 0.867, F-to-remove 287.9, p<0.0001). However, α-def-1 continued to show an independent effect on lesion size (Beta 0.565, F-to-remove 122.3. p<0.0001), consistent with previously reported findings[5].

Discussion

We previously reported data suggesting that α-defensins promote atherogenesis, based on studies in transgenic mice expressing α-def-1 in their neutrophils[12] (Def++ mice) that have been fed a high fat diet[12] and by the correlation in humans between tissue deposition of α-defensins and the severity of coronary artery disease[5]. The increase in the size of lipid rich lesions in the proximal aortas of Def++ mice[12] fed a HFD was observed even though α-def-1 stimulated hepatic clearance of LDL leading to hypocholesterolemia[12]. However, a decrease in lesion size was seen when ApoE-/- mice were crossed with the same Def++ mice or were exposed to exogenous α-def-1 showing an apparent protective effect, presumably as a result of accelerated LDL clearance from the plasma[13].

To address these seemingly conflicting observations, the current study was designed to isolate the independent impact of α-def-1 on atherogenesis using control groups with comparable lipid profiles attained through other approaches. We extended previously published reports by including two different concentrations of α-def-1 and three control groups, i.e. two groups of mice given high or low doses of cholestyramine and a cohort of mice fed a MFD. Our data show that despite a comparable decline in plasma cholesterol, mice exposed to α-def-1 developed larger aortic lesions. This suggests that the lipid-lowering impact of α-def-1 might be offset by induction of a pro-atherogenic processes within the vasculature itself, leading to the net effect of accelerating the generation of intimal fatty streaks.

Our results confirm previous finding that α-def-1, here given exogenously to ApoE-/- mice, lowers total serum cholesterol and decreases lesion size in the aortic root[13]. Similar effects were seen when plasma cholesterol was lowered by inhibiting adsorption using cholestyramine or using a MFD. However, although total serum cholesterol was lowered to the same extent by both methods, lesion size was larger in mice given α-def-1 (Fig 1) and there was a statistically significant direct correlation between exposure to α-def-1 and lesion size that was independent of plasma cholesterol (Fig 2).

Similar conclusions can be reached from the data of Paulin[13] et al. ApoE-/- mice that had been fed a HFD and were also exposed to exogenous α-def-1 that had serum TCH levels between 1000–1500 mg/dl developed larger lesions than untreated mice with comparable serum levels (see Fig S3D[13]).

Taken together, our results suggest that α-def-1 has competing effects on atherogenesis. On the one hand, accelerated hepatic uptake and clearance of α-def-1/LDL complexes from the blood reduces their probabilities to deposit in the vasculature. On the other hand, α-def-1/LDL complexes have a greater intrinsic propensity to deposit and remain in vascular cells and matrix[5,12,14-17], where they stimulate macrophage recruitment, generation of cathepsins B and S, formation of foam cells[10] and increase endothelial permeability to LDL[12]. The seemingly paradoxical inverse relationship between low plasma LDL levels and lipid deposition in vasculature induced by α-def-1 that we observed helps to explain recent findings that colchicine, which reduces α-defensin release from neutrophils[12], increases plasma oxidized LDL and proatherogenic “small” LDL[21] but reduces the incidence of cardiovascular events[22]. It is clear that more research will be needed to fully evaluate the impact of α-defensins on atherogenesis and to explore the possibility that inhibiting their release and impact on lipid deposition might provide a novel approach to the amelioration of atherogenesis.

(DOCX)

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30 Jan 2020

PONE-D-19-31479

Opposing effects of HNP1 (α-defensin-1) on plasma cholesterol and atherogenesis

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Reviewer #1: The authors re-examine the potential role of HNP in atherosclerosis. It seems that this study is a reassessment of their own work and one other study, namely ref. 13; it is not very clear to this reviewer what the rationale behind this study is and what the added value is supposed to be.

Specific:

This study is hard to read with many typos and syntax errors. This manuscript needs much rewriting.

Basic standards in reporting are not adhered to, e.g. what is a one-way test? scale bars in images are missing; source of reagents (HNP) are missing; MFD seems like a random mix of different foods rather than a standardized diet, def-low group is shown twice in 1C, but def-hi is missing - these are just some examples but there are certainly many more.

The authors correlate plasma cholesterol levels with lesions sizes across different treatment groups and draw conclusions as to the mechanism of HNP; I am not certain this is sound as atherosclerosis develops over a long period and assessing cholesterol at just one time point likely does not reflect the timely resolution that is needed to draw the conclusions as they do.

In 1B they measure lesion size (please do not call this damage score!) and the control group is meant to range at 40%; however, looking at images in 1C, lesion size looks more like 6-10%; hence, I much question the overall robustness of their readout.

It is very unusual to see the work of another group being reinterpreted even with making reference to a supplementary figure.

Reviewer #2: Note for editor: are figure panels required to be separated before combining into one figure?

In this study Higazi et al. demonstrate in ApoE-/- mice fed a high fat diet (HFD), administration of human alpha-defensin 1 reduces plasma total cholesterol as well as lesion volume in comparison to control HFD untreated mice. In contrast to low vs high treatment with cholestyramine, the high and low alpha-def-1 treatment had larger lesion sizes, however this is still smaller than mice not receiving any alpha-def-1. The authors present this study in order to resolve the differences between their previously publication, where expression of defensins in a HFD model developed lipid streaks but formed adef:LDL complexes and the study by Paulin et al. wherein defensin expression in a HFD lowered progression of atheroprogression, enhanced LDL clearance and lowered cholesterol.

Throughout the manuscript, there are spelling errors to double check as well as punctuation, these need addressing.

In alpha-def-1 IV injection methodology, was 10 or 30 µg the total amount or is this µg/kg of bodyweight? This is human alpha-def-1? How much did the overall weight of the mice vary to account for the differences in relative amount of alpha-def-1 received by each individual across the treatment cohort. In addition, it is unclear if this is a tail vein injection.

Def+/+ mice need to be defined when introduced in the discussion, and rationale for using ApoE-/- mice be made more clear.

Figure legends.

1A. Clarify H & L indicate high and low, this can be included in the key as there is space. Whether the last gray bar is MFD or MHFD needs to be resolved, this and the high/low can be applied to subsequent panels. Finally, what is not significant and what is significant as indicated by the p<0.001 in the middle of the figure?

1B. The red and green asterisk description at present is unclear whether there is significant differences between low does Cholest or alpha-def 1 or whether the high and low dose treatments were compared. The Y axis labelling needs to be more clearly described in the legend.

1C. Figure legend needs to include this is representative aortic root sections... Also, there is no alpha-def high representative image.

2. Is intimal damage score expressed as a percentage similar to Figure 1B. At present, the legend is difficult for the reader to interpret that data from Figure 1 is being analyzed and needs clarification.

Overall, significant clarification in the discussion is needed. Clearly, there are multiple variables at play between alpha-def-1 and atheroprogression, especially in previous studies and the data presented in the current study, however the conclusions need to better match the data presented herein and outline what additional questions remain. First, this study attempts to control for the cholesterol-lowering effects of alpha-def-1 by including a treatment group is administered cholestyramine, and extends on previously published reports by including two different concentrations of alpha-def-1. It needs be abundantly clear in the text that while similar decreases in cholesterol were observed, alpha-def-1 treated mice had larger lesion size in comparison to cholestyramine treated mice. At present, what the authors are communicating by including the second to last paragraph of the discussion is unknown, however the correlation between the two studies overall shows a correlation between reductions in cholesterol and lesion size with the presence of alpha-def-1.

There are several key questions, would combination of cholestyramine and alpha-def-1 increase the lesion volume (due to alpha-def-1 activities?)? If alpha-def-1 was inhibited (or inactivated) in the present study, would that serve to increase both lesion size and cholesterol levels, or would lesion size be reduced in comparison to the ApoE-/- mice on the HFD, both in the presence or absence of cholestyramine? Would genetic modification to have Def+/+/ApoE-/- mice on a HFD result in mice with more atherogenesis that is reduced with something like colchicine, all these data would be helpful in supporting the authors current claims.

The data and previous studies show clearly that alpha-def-1 and neutrophils play a role in atherogenesis, one that might be a dual role, however the final sentence stating ‘inhibition of alpha-defs release or activity might help reduce the incidence or severity of atherosclerotic vascular disease and its thrombotic sequelae’ doesn’t appear to be supported currently by the data presented by the authors as in comparison to the control HFD, cholesterol and lesions are reduced.

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12 Mar 2020

Reviewer #1: The authors re-examine the potential role of HNP in atherosclerosis. It seems that this study is a reassessment of their own work and one other study, namely ref. 13; it is not very clear to this reviewer what the rationale behind this study is and what the added value is supposed to be.

We appreciate the opportunity to address the Reviewer’s concern, which is central to the merit of our paper. The reasons for submitting this paper are several fold. First, as stated succinctly by Reviewer 2, there are two peer-reviewed published manuscripts that address the role of human neutrophil alpha defensins (HNPs) in the development of atherosclerosis, one indicating these peptides are “proatherogenic”, the other concluding that these peptides inhibit the process and indeed could be employed to prevent or to reverse development of vascular lesions. We think the current paper helps to put both sets of data and both conclusions in a broader content, leading to a more nuanced conclusion that is more likely to provide insights into the human condition. Second, we also think this study provides insight into the pathogenesis of atherosclerosis in the sizable subpopulation of patients with normal or low levels of LDL for whom inhibition of neutrophil function is more likely to be salutary that focusing exclusively on lowering LDL cholesterol further. This latter hypothesis is supported by our finding that mice transgenic for alpha-defensin-1 (�-def-1) develop aortic plaques on a regular diet in the context of lower than normal plasma levels of LDL cholesterol and that colchicine prevents this from happening even when LDL cholesterol levels in the plasma are elevated by a high fat diet (J Biol Chem. 2016; 29: 2777). Our conclusions are supported by recent clinical studies that show colchicine prevents cardiovascular events in humans (N Engl J Med. 2019; 381: 2497) while increasing in the plasma, the more atherogenic LDL subspecies, small LDL and oxLDL (J Clin Lipidol. 2019; 13: 1016).

Specific:

This study is hard to read with many typos and syntax errors. This manuscript needs much rewriting.

We apologize to the Reviewer and have made a sincere effort to avoid such errors in the revised text.

Basic standards in reporting are not adhered to, e.g. what is a one-way test?

One-way ANOVA means performing between-group comparisons or within-group comparisons of repeated measurements, but not their combination (an assessment termed two-way ANOVA). Thank you for your remark. In the revised version we substituted "one-way ANOVA" with "between-group ANOVA" for simplicity.

scale bars in images are missing;

We have added the scale bars per the Reviewer.

source of reagents (HNP) are missing;

We have added details concerning the sources of HNPs per the Reviewer (Please page 4, Materials section lines 3- 5).

MFD seems like a random mix of different foods rather than a standardized diet,

We appreciate the Reviewer’s concern and we apologize for any confusion that we created. The diet we used was actually tightly controlled. The diet was composed of 35 parts high fat diet (See attached) and 65 parts regular diet (see attached), a conventional approach used by commercial companies to prepare specific diets. This composition was chosen based on empirical evidence that it generated plasma levels of total cholesterol and LDL cholesterol comparable to plasma levels in animals treated with low dose of cholestyramine and because it led to formation of lipid streaks in the aortas of comparable size. We used this as a second independent control to animals exposed to a low dose of HNPs.

def-low group is shown twice in 1C, but def-hi is missing –

Thank you for noticing the mistake, we have corrected the error in labeling.

The authors correlate plasma cholesterol levels with lesions sizes across different treatment groups and draw conclusions as to the mechanism of HNP; I am not certain this is sound as atherosclerosis develops over a long period and assessing cholesterol at just one-time point likely does not reflect the timely resolution that is needed to draw the conclusions as they do.

We appreciate and, of course, agree with the Reviewer’s point as to the duration required to develop vascular disease. In experiments not shown, we measured plasma LDL and cholesterol weekly. Differences between Def++ and WT mice were already evident by the end of the first week and these differences remained constant throughout the experimental period. The data shown in this paper is representative of many such determinations, all leading to the same conclusion.

In 1B they measure lesion size (please do not call this damage score!) and the control group is meant to range at 40%; however, looking at images in 1C, lesion size looks more like 6-10%; hence, I much question the overall robustness of their readout.

The Reviewer is correct. We expressed our data as the % of aortic root circumference occupied by the lesion. We provide a figure typical of those that we used to make these calculations. The data in the manuscript are presented as the ratio between the length of the red color line and the blue color line (See left figure below), which correlates very well with the size of the lesion (See right figures below). The Methods section of the paper has been amended (Please see page 5, line 6-7) and we changed the label of the Y axis in the figures 1B and 2 from "lesion size" to “damage score".

It is very unusual to see the work of another group being reinterpreted even with making reference to a supplementary figure.

We appreciate and concur with the Reviewer’s concern. We simply could not figure out how NHPs could be both pro-atherogenic and anti-atherogenic. We trust that we have been scrupulously fair to Paulin and colleagues and that our new findings help to reconcile what otherwise would appear to be opposing conclusions.

Reviewer #2: Note for editor: are figure panels required to be separated before combining into one figure?

In this study Higazi et al. demonstrate in ApoE-/- mice fed a high fat diet (HFD), administration of human alpha-defensin 1 reduces plasma total cholesterol as well as lesion volume in comparison to control HFD untreated mice. In contrast to low vs high treatment with cholestyramine, the high and low alpha-def-1 treatment had larger lesion sizes, however this is still smaller than mice not receiving any alpha-def-1. The authors present this study in order to resolve the differences between their previously publication, where expression of defensins in a HFD model developed lipid streaks but formed adef:LDL complexes and the study by Paulin et al. wherein defensin expression in a HFD lowered progression of atheroprogression, enhanced LDL clearance and lowered cholesterol.

Throughout the manuscript, there are spelling errors to double check as well as punctuation, these need addressing.

We apologize to the Reviewer. We have made a sincere attempt to eliminate such errors in the revised text.

In alpha-def-1 IV injection methodology, was 10 or 30 µg the total amount or is this µg/kg of bodyweight?

10 or 30 µg are the total amount. The average weight of the mice was ~25 g. Thus, the dose of �-def-1 was 0.4 mg/kg (the low dose) and 1.2 mg/kg (the high dose).

This is human alpha-def-1? How much did the overall weight of the mice vary to account for the differences in relative amount of alpha-def-1 received by each individual across the treatment cohort. In addition, it is unclear if this is a tail vein injection.

Thank you for the opportunity to address these concerns. First, all studies were performed with humans α-def-1. The two sources of α-def-1 are specified in more detail in revised manuscript (Please page 4, Materials section lines 3- 5). Both proteins had the same effect formation of complexes with LDL in vitro and acceleration of LDL clearance in vivo (J Biol Chem. 2016; 29: 2777). Second, there was no significant difference in the weights of apoE-/- mice that did or did not receive α-def-1or cholestyramine. This has been added to the revised text (Please see page 4, Cholesterol-lowering section lines 6-7). Third, we now specify that the injections of alpha-def-1 were made by injection into the tail vein (Please see page 4, Cholesterol-lowering section lines 3-4).

Def++ mice need to be defined when introduced in the discussion, and rationale for using ApoE-/- mice be made more clear.

The mice mentioned in this paper are the progeny of those previously described in detail and reported by us. They are the same mice provided by one of us (KB) to Paulin et al. We have noted this is the revised Discussion (Please see page 6, line 2 in the Discussion).

Figure legends.

1A. Clarify H & L indicate high and low, this can be included in the key as there is space. Whether the last gray bar is MFD or MHFD needs to be resolved, this and the high/low can be applied to subsequent panels. Finally, what is not significant and what is significant as indicated by the p<0.001 in the middle of the figure?

The terms “H” and “L” have now been clarified in the legend to Figure 1A (Please see page 8, legend figure 1 A line 3-4). We now use the term MFD throughout. We have revised Figure 1A for clarity. There was no statistically significant difference between results in mice receiving a low dose of cholestyramine and a low dose of �-def-1. There were also no statistically significant differences between outcomes in mice receiving a high dose of cholestyramine, a high dose of α-def-1 or on MFD. Significant differences (p < 0.001) were found between the two former groups and the last three groups (now shown in the figure) and between the control HFD and all other groups (as stated in the text in Results).

1B. The red and green asterisk description at present is unclear whether there is significant differences between low does Cholest or alpha-def 1 or whether the high and low dose treatments were compared. The Y axis labelling needs to be more clearly described in the legend.

We revised Figure 1B for clarity. In doing so, we deleted the red and green asterisks. Lesion diameters in mice given a low dose of cholestyramine (Cholest L) were compared with those in mice given a low dose of �-def (�-Def L). Lesion diameters in mice given a high dose of cholestyramine (Cholest H) were compared with outcomes in mice given a high dose of �-def (� -Def H). Mice given �-Def H were compared to mice given a modified fat diet (MFD). In each case, the differences were statistically significant with a P value of <0.001.

1C. Figure legend needs to include this is representative aortic root sections... Also, there is no alpha-def high representative image.

We have corrected the error in the label.

Is intimal damage score expressed as a percentage similar to Figure 1B. At present, the legend is difficult for the reader to interpret that data from Figure 1 is being analyzed and needs clarification.

We have revised the legend to state the section shown is representative of the 16 lesions analyzed in Panel B.

Overall, significant clarification in the discussion is needed. Clearly, there are multiple variables at play between alpha-def-1 and atheroprogression, especially in previous studies and the data presented in the current study, however the conclusions need to better match the data presented herein and outline what additional questions remain. First, this study attempts to control for the cholesterol-lowering effects of alpha-def-1 by including a treatment group is administered cholestyramine, and extends on previously published reports by including two different concentrations of alpha-def-1. It needs be abundantly clear in the text that while similar decreases in cholesterol were observed, alpha-def-1 treated mice had larger lesion size in comparison to cholestyramine treated mice. At present, what the authors are communicating by including the second to last paragraph of the discussion is unknown, however the correlation between the two studies overall shows a correlation between reductions in cholesterol and lesion size with the presence of alpha-def-1.

The Discussion has been revised to include the suggestions of the Reviewer (See page 7, 3nd paragraph lines 4-7 and last paragraph beginning in lines 6).

There are several key questions, would combination of cholestyramine and alpha-def-1 increase the lesion volume (due to alpha-def-1 activities?)?

As suggested by the Reviewer, we performed a pilot study in which we combined low doses of α-def-1 and low doses of cholestyramine in ApoE-/- mice on a high fat diet. Lesion size was larger in mice given both reagents than in mice given cholestyramine alone. We did not add this data to the manuscript because it does not change our conclusions but we are happy to do so.

If alpha-def-1 was inhibited (or inactivated) in the present study, would that serve to increase both lesion size and cholesterol levels, or would lesion size be reduced in comparison to the ApoE-/- mice on the HFD, both in the presence or absence of cholestyramine?

The Reviewer suggests what in theory would be an important experiment. Unfortunately, no antagonists of �-defs or means to inactive �-defs have been developed to the best of our knowledge. This is why in prior studies, we (1) compared results in HNP1-expressing transgenic mice with syngeneic wild type controls that do not express �-defs, (2) showed outcomes after bone marrow transplantation of �-def mice into wild type and wild type marrow into the transgenic, and (3) inhibited release of �-defs from neutrophils in vitro and in vivo with colchicine – all with the same result. Data using all three approaches show that decreasing plasma concentrations of �-defs tracks with smaller atherosclerotic changes in the aortic roots even though plasma LDL levels increase (J Biol Chem. 2016; 29: 2777). As underscored in the revised Discussion, these results are in line with recent clinical observations that colchicine reduces cardiac endpoints (N Engl J Med. 2019; 381: 2497) although it increase plasma concentrations of atherosclerotic lipoproteins (J Clin Lipidol. 2019; 13: 1016).

Would genetic modification to have Def+/+/ApoE-/- mice on a HFD result in mice with more atherogenesis that is reduced with something like colchicine, all these data would be helpful in supporting the authors current claims.

The Reviewer makes an interesting suggestion, but one that could take considerable effort and time to address and might or might not affect the central conclusions of our paper. In a simpler experimental system, Def+/+ mice on a regular or a high fat diet developed larger aortic lesions (J Biol Chem. 2016; 29: 2777) and colchicine reduced the effect (J Biol Chem. 2016; 29: 2777). A similar anti-atherogenic effect of colchicine was recently reported in humans (N Engl J Med. 2019; 381: 2497). Therefore, we would hypothesize that colchicine would reduce atherogenesis in Def+/+/ApoE-/- mice. On the other hand, double transgenic mice would create a less physiological system with extraordinarily high LDL and triglyceride levels and thus may generate novel interactions that are not relevant to physiological conditions and may be hard to interpret. Therefore, the proposed scheme might will not provide new compelling insights into our central thesis, i.e. that ��defs may contribute to the development of atherosclerosis in the clinical setting of normal levels of LDL. We hope the Reviewer will see this as a formidable and potentially unrevealing task.

The data and previous studies show clearly that alpha-def-1 and neutrophils play a role in atherogenesis, one that might be a dual role, however the final sentence stating ‘inhibition of alpha-defs release or activity might help reduce the incidence or severity of atherosclerotic vascular disease and its thrombotic sequelae’ doesn’t appear to be supported currently by the data presented by the authors as in comparison to the control HFD, cholesterol and lesions are reduced.

We appreciate the Reviewer’s insight. We changed the sentence accordingly (Please see last 8 lines of Discussion section). As mentioned, in support of our hypothesis, newly published data by others show a dissociation between the rise in atherogenic LDL in patients on colchicine (J Clin Lipidol. 2019; 13: 1016) and a reduction in cardiovascular events (N Engl J Med. 2019; 381: 2497).

Submitted filename: Response to Reviewers Sub F.docx

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20 Mar 2020

PONE-D-19-31479R1

Opposing effects of HNP1 (α-defensin-1) on plasma cholesterol and atherogenesis

PLOS ONE

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Reviewer #1: (No Response)

Reviewer #2: Higazi et al. have sufficiently revised their manuscript. There are several minor copyedits to be made by the authors, which hopefully will help. I apologize as a reviewer if I missed any others.

1. Second introduction paragraph – comma needed ‘… liver, leading to hypercholesterolemia.’ Following sentence also needs to be broken up, it is five lines long with only one comma.

2. Methods: correct to Sigma-Aldrich in materials.

3. 4th results paragraph – ‘However, a significantly higher proportion [of] the aortic roots circumferences…’ Add of please.

4. Last results paragraph, first sentence: ‘As [a] second approach…’ Add a please.

5. Figure 1 legend: Fix parentheses for Choles L and H, several closed parentheses appear to be missing.

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25 Mar 2020

Dr. Michael Bader

Academic Editor

PLOS ONE

Dear Dr. Bader:

We are writing in reference to MS: PONE-D-19-31479 “Opposing effects of HNP1 (α-defensin-1) on plasma cholesterol and atherogenesis” that we revised and are re-submitting to your journal.

We appreciate the efforts taken by the reviewers and the editor to examine our study in detail, consider it of merit for publication in PLOS ONE, and inviting us to submit a revised version of the manuscript.

In the revised version of our manuscript we addressed all the concerns raised by reviewer 2. We have corrected the errors noted by the reviewer and have made a sincere effort by a thorough re-reading to avoid any errors, including copyediting, in the revised text.

We hope that the revised manuscript is now suitable for publication in PLOS ONE.

Submitted filename: Ansawring the revwieres 24-3-2020.docx

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27 Mar 2020

Opposing effects of HNP1 (α-defensin-1) on plasma cholesterol and atherogenesis

PONE-D-19-31479R2

Dear Dr. Higazi,

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31 Mar 2020

PONE-D-19-31479R2

Opposing effects of HNP1 (α-defensin-1) on plasma cholesterol and atherogenesis

Dear Dr. Higazi:

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