A Novel Preclinical Murine Model of Systemic Lupus Erythematosus–Like Cardiovascular Disease

Abstract

Objective

Systemic lupus erythematosus (SLE) affects nine women to every man worldwide, and these patients are at greater risk for cardiovascular disease (CVD) morbidity and mortality. Clinical studies have demonstrated that patients with SLE are more likely to develop CVD, including cardiac and vascular dysfunction. Although many preclinical models of SLE are available, including treatment with Toll‐like receptor (TLR) 7/8 agonists, a consistent preclinical model of SLE‐like CVD with systemic, cardiac, renal, and cerebral endothelial activation and cardiac dysfunction has yet to be described. Here, we hypothesize that acceleration of SLE with the TLR7/8 agonist resiquimod (R848) will promote cardiac and endothelial activation with subsequent end‐stage organ damage in the SLE‐prone B6.Nba2 mouse model.

Methods

Female and male SLE‐prone B6.Nba2 mice were treated with R848 or acetone, administered topically twice weekly over a four‐week period, to accelerate the development of SLE‐like pathophysiology. Echocardiography was performed at baseline, 4 weeks, and 16 weeks. At 16 weeks, tissues were harvested, weighed, and analyzed by histology, immunofluorescence, real‐time quantitative polymerase chain reaction, and enzyme‐linked immunosorbent assays.

Results

We found that female R848‐treated mice had increased serum anti‐Smith and immunoglobulin G complex deposition in the kidney, heart, and brain consistent with SLE‐like etiology. Tissue analysis revealed significant enlargement of the spleen in both female and male R848‐treated mice, with only cardiac and renal enlargement in females compared to their respective controls. Echocardiographic imaging revealed left ventricular wall thickening by 4 weeks that was followed by a progressive increase in left ventricular internal diameters and subsequent decrease in ejection fraction over the 16‐week time course in female mice. We found that circulating levels of soluble vascular adhesion molecule‐1 and soluble intracellular adhesion molecule‐1 were increased in both female and male R848‐treated mice, whereas cardiac and renal fibrosis were significantly increased in only female R848‐treated mice.

Conclusion

Our data demonstrate that R848 treatment of SLE‐prone B6.Nba2 mice is a novel preclinical model to study the sex‐dependent pathophysiologic mechanisms of SLE‐like CVD.

INTRODUCTION

Systemic lupus erythematosus (SLE) is the most common form of lupus autoimmunity and disproportionately affects women at a higher rate than men. Patients with SLE can present with heterogenous symptomology, leading to multiple pathologies affecting the cardiovascular, renal, and central nervous system. Patients with SLE have a 5× to 50× higher incidence of cardiovascular disease (CVD), specifically myocardial infarction (~52×), hypertension (~3×), and heart failure (~5×) compared with age-matched controls.^1–4 Importantly, very limited therapeutic regimens or societal and behavior modifications have reduced the CVD risk in patients with SLE. In a retrospective cohort study of approximately 100,000 patients with SLE and 45 million controls, Kim et al demonstrated that heart failure is almost 5× higher in patients with SLE compared with the general population.³ The study demonstrated an increased risk of heart failure that is exacerbated in younger patients with SLE, >20-fold in relative risk in patients under 35, and up to 50-fold relative risk in patients under 25.³ Importantly, this extremely high relative risk of cardiac dysfunction in younger patients suggests a pathophysiologic relationship between SLE and the development of CVD. Although there are numerous murine models of SLE with CVD, specifically atherosclerosis, kidney disease, and hypertension,^5–8 few have been used to study the development of SLE-like CVD and cardiac and systemic endothelial cell activation including the heart, kidney, and brain in a sex-dependent manner. Moreover, our novel model is on the classical C57Bl/6 background, which gives it power for comparison to the expansive literature of cardiovascular studies on this mouse genetic background.

In this study, we developed a novel preclinical murine model of SLE-like CVD using the SLE-prone B6.Nba2 mouse treated with resiquimod (R848), a topical gel that modifies the immune response.⁹ Although SLE affects predominantly females, we studied both female and male mice to investigate end-stage organ and pathophysiologic complications of SLE in a sex-dependent manner, not to delineate specific sex-dependent mechanisms in disease pathogenesis. Our findings provide a unique mouse model on a classical C57Bl/6 background that promotes hallmark SLE-like complex symptomology including interferon gene signatures; autoantibody production; immunoglobulin (Ig) G complex deposition; and cardiac, vascular, and renal complications in a sex-dependent manner.

MATERIALS AND METHODS

Animals

B6.NZB-(D1Mit47-D1Mit209)/BokJ (B6.Nba2) mice on a C57Bl/6 background were bred at Ralph H. Johnson VA Healthcare System. All animal procedures were approved by the Institutional Animal Care and Use Committee at the Ralph H. Johnson VA Healthcare System in accordance with the Guide for the Care and Use of Laboratory Animals and followed the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.¹⁰ Female and male B6.Nba2 mice were kept in a light-controlled environment with a 12-hour:12-hour light/dark cycle and given free access to standard mouse chow and water. Female and male B6.Nba2 mice ~10 to 14 weeks of age were used for the study. For the collection of tissues, all mice were anesthetized with 5% isoflurane, and the hearts, spleens, kidneys, and brains were removed. All tissues, including heart, kidneys, spleens, and brains, were harvested between 10 AM and 1 PM Eastern Standard Time to ensure consistent circadian rhythms in all groups.

R848 treatment

Female and male B6.Nba2 mice were treated with Toll-like receptor (TLR) 7/8 agonist R848 (Enzo Life Sciences; cat. no. NC9739083) 100 μg/30 μL in acetone or vehicle control (acetone; 30 μL) twice a week for four weeks on the pinnae of the right ear. After the four weeks of R848 or vehicle control treatment, mice were left untreated for 12 weeks and observed until the 16-week terminal time point.

Echocardiography

Left ventricular cardiac physiology was determined by echocardiography (Vevo 2100, VisualSonics). Mice were anesthetized with 0.5% to 3.0% isoflurane in 100% oxygen. Heart rate and body temperature were monitored during the imaging procedure for all mice. All images of the left ventricle were taken at heart rates >400 bpm for physiologic relevance. Multiple measurements for left ventricular wall thickness, end diastolic volume (EDV), end systolic volume (ESV), ejection fraction (EF), and E/e′ were taken and averaged.

Enzyme-linked immunosorbent assays

Plasma was collected at time of euthanasia, and anti–double-stranded DNA (dsDNA; Alpha Diagnostic International; cat. no. 5120), anti-Smith (anti-Sm) (Alpha Diagnostic International; cat. no. 5405), soluble vascular cell adhesion molecule-1 (sVCAM-1; Thermo Fisher Scientific; cat. no. EMVCAM1), soluble intracellular adhesion molecule-1 (sICAM-1; Thermo Fisher Scientific; cat. no. EMCICAM1ALPHA), and plasma blood urea nitrogen (BUN; Thermo Fisher Scientific; cat. no. EIABUN) were measured by enzyme-linked immunosorbent assay (ELISA). All ELISAs were performed as to manufacturer's instructions.

Real-time quantitative polymerase chain reaction

Real-time quantitative polymerase chain reaction (PCR) was used to determine gene expression of interferon regulatory factor 7 (Irf7; Thermo Fisher Scientific; Mm00516793_g1), interferon stimulating gene 15 (Isg15; Thermo Fisher Scientific; Mm01705338_s1), Icam1 (Thermo Fisher Scientific; Mm00516023_m1), Vcam1 (Thermo Fisher Scientific; Mm01320970_m1), and Gapdh (Thermo Fisher Scientific; Mm99999915_g1) as a housekeeping gene. RNA was isolated using RNeasy kits (Qiagen; cat. no. 74536), and complementary DNA (cDNA) made via high-capacity RNA-to-cDNA kit (Applied Biosystems; cat. no. 43-874-06) per manufacturer's instructions. PCRs were recorded and analyzed with QuantStudio-5 (Thermo Fisher Scientific) and the QuantStudio design & analysis software.

Western blot analysis

For Western blot analysis after R848 or acetone treatment, cell pellets were pulse sonicated with Radioimmunoprecipitation assay buffer (RIPA) (Sigma-Aldrich; R0278), Halt protease (Thermo Fisher Scientific; 186932), and phosphatase (Thermo Fisher Scientific; 78428) inhibitor for 10 seconds. The cells were pelleted at 2,000g for 5 minutes, keeping on the supernatant. Protein quantification of the cell lysate was performed using RD DC Protein Assay (Bio-Rad; 5000113-5). The cell lysate was frozen at −80°C until Western blot analysis was performed. Samples (10 μg) were subjected to gel electrophoresis and transferred onto nitrocellulose membranes (Bio-Rad; 162017) using TransBlot Turbo Transfer System (Bio-Rad). Total protein staining was performed using Ponceau S (Thermo Fisher Scientific; A40000278). Membranes were then subjected to probing with neutrophil gelatinase–associated lipocalin (NGAL) (1:1,000; R&D Systems; AF1857). All membranes were subsequently visualized by enhanced chemiluminescence (Sigma-Aldrich; WBKLS0500) on an Amersham Imager 680 (General Electric). Western blots were analyzed using ImageQuant Software (Cytiva). Bands of interested were normalized to total protein to ensure equal loading.

Immunofluorescent staining

Immunofluorescent staining was performed to evaluate renal, cardiac, and cerebral IgG complex deposition. Female and male B6.Nba2 mouse hearts, kidneys, and brains were excised and fixed in 10% neutral buffered formalin, routinely processed and paraffin embedded, and cut into 5-μm sections. To expose antigen epitopes, heat-mediated antigen retrieval was performed in 10 mM sodium citrate buffer + 0.05% Tween 20 (pH 6.0) at 96°C for 45 minutes. Slides were then rinsed, blocked in serum (10% Triton X and Goat Serum in PBS), and incubated for 45 minutes at room temperature. Heart, kidneys, and brain were probed overnight at 4°C for IgG (1:50, Alexa Fluor 546 goat anti-mouse; Invitrogen). Slides were washed and treated with 1× TrueBlack (Thermo Fisher Scientific) to minimize autofluorescence within the heart, kidney, and brain. DAPI Pro Long mounting media (Thermo Fisher Scientific) was applied to coverslips, placed on slides, and sealed with clear nail polish. Heart, kidney, and brain slides were imaged for fluorescence intensities using an Olympus 20× UPLAN X Pro air objective on an Echo Revolve Microscope (Discover Echo, San Diego, CA, USA).

Immunofluorescent staining was performed to evaluate infiltration cardiac macrophages. Female and male B6.Nba2 hearts were excised and fixed in 10% neutral buffered formalin, routinely processed and paraffin embedded, and cut into 5-μm sections. To expose antigen epitopes, heat-mediated antigen retrieval was performed in 10 mM sodium citrate buffer + 0.05% Tween 20 (pH 6.0) at 96°C for 45 minutes. Slides were then rinsed, blocked in serum (10% Triton X and Goat Serum in PBS), and incubated for 45 minutes at room temperature. Hearts were probed overnight at 4°C for anti-Lysosome-associated membrane protein 2 (LAMP2) (1:50; Thermo Fisher Scientific; PA1-655). Slides were washed at room temperature and fluorescent secondary antibody corresponding to LAMP2 (1:500; Alexa Fluor 546 goat anti-rabbit; Invitrogen; A11010) was added and incubated for 30 minutes at room temperature. Slides were treated with 1× TrueBlack (Thermo Fisher Scientific; NC1125051) to minimize autofluorescence within the heart. DAPI Pro Long mounting media (Thermo Fisher Scientific) was applied to coverslips, placed on slides, and sealed with clear nail polish. Hearts were imaged for fluorescence intensities using an Olympus 40× UPLAN X Pro air objective on an Echo Revolve Microscope (Discover Echo).

Immunohistochemistry

Slides were stained with Masson's trichrome stain and used to detect glomerular injury. A glomerular injury was estimated by masked observers’ assigned scores (on a scale of 1–4) for representative examples (120 glomeruli per group) within groups (n ≥ 6 for each group).The morphometric analysis is based on the following score criteria: 1, no damage; 2, mesangial proliferation of any degree; 3, no more than 50% of mesangial proliferative lupus nephritis with the presence of immune cell depositions; 4, global glomerular lesion with significant glomerular hypertrophy and damage. The score distributions and statistical analyses were performed as described previously.¹¹ Briefly, a cumulative distribution plot was generated (OriginPro 9.0) using glomerular injury scores obtained by morphometric analysis, and the probability for a corresponding score interval was calculated.

Picrosirius Red staining was performed on heart tissue to visualize collagen I and III fibers. Paraffin-embedded tissue sections were deparaffinized and dehydrated before a three-minute exposure to phosphomolybdic acid, 0.01% Sirius Red in saturated picric acid (Electron Microscopy Sciences) for 90 minutes, and 0.01% HCl for two minutes. Heart and kidney slides were imaged for collagen deposition using an Olympus 20× UPLAN X Pro air objective on an Echo Revolve Microscope (Discover Echo).

Statistics

All samples were given a random identifier so that analysis was performed masked. GraphPad Prism version 10 was used to perform all statistical analysis. To test values for normality, the Shapiro–Wilk normality test was performed with an α set to 0.05. To ensure that values were not outliers, a Grubbs test was employed to identify outliers with an α set to 0.05. If outliers were identified, they were removed from their respective data set. All data sets were expressed as a mean ± SEM. All studies were compared within sex (female vs female and male vs male). For studies comparing the effect of R848 treatment on probability of survival, body weight, EF, left ventricular wall thickness, ESV, and EDV, a two-way repeated measures analysis of variance was employed, followed by a Bonferroni post hoc test when significance indicated. For studies comparing the effect of R848 treatment on plasma levels of anti-dsDNA, anti-Sm, sICAM-1, and sVCAM-1, a two-tailed Student's t-test or Mann–Whitney test were employed depending on normality. For the effect of R848 treatment of Ifr7, Isg15, Vcam1, and Icam1 gene expression and all anatomic organ weights, a two-tailed Student's t-test or Mann–Whitney test were employed. For cardiac Picrosirus red histologic analysis and E/e′, a two-tailed Student's t-test or Mann–Whitney test was performed. For renal glomerular damage histologic analysis, the statistical significance among the groups was measured using a chi-square test (Cramer's V) in IBM SPSS software (version 27). P values were considered significant when <0.05. All data that support the findings will be available upon request.

RESULTS

Induction of SLE-like disease by R848 treatment

We found that four-week R848 treatment promoted death in female mice with a ~70% survival rate, whereas male mice had a 100% survival rate (Figure 1A). We found no significant difference in body weight for vehicle or treatment female groups, whereas R848-treated male mice weighed significantly less than vehicle controls (Figure 1B). We found no significantly different anti-dsDNA plasma levels in response to R848 treatment at the 16-week time point. It should be noted that female mice had a trend for increased anti-dsDNA plasma levels, indicating disease exacerbation to some extent. Moreover, R848-treated female mice had elevated plasma anti-Sm, whereas treated male mice were unchanged compared with controls (Figure 1C and D). Renal, cardiac, and cerebral IgG complex deposition within the vascular system were exacerbated in R848 treatment compared with controls in both female and male mice (Figure 1E). Lastly, key interferon genes Irf7 and Isg15 were unchanged at the 16-week time point in all three end-stage organs, except Isg15 in the kidneys of R848-treated female mice (Figure 1F).

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Induction of SLE-like cardiac dysfunction by R848 treatment

We found that both female and male R848-treated mice had a significant increase in spleen-to-body-weight ratio (Figure 2A). However, the other organs indicate strong sexual dimorphism in body weight ratio changes after the treatment. Female mice had a significant increase in kidney-to-body-weight ratio, whereas treated male mice exhibited a significant reduction compared to controls (Figure 2B), indicating a sexual dichotomy to the resultant renal injury likely due to renal hypertrophy, usually seen in models of chronic kidney disease and hypertension. R848-treated female mice had a profound increase in heart-weight-to-body-weight ratio compared to vehicle controls, indicative of cardiomegaly, which was absent in treated male mice (Figure 2C). Lastly, we found no change in lung wet-to-dry weight ratio in the females and a decrease in R848-treated male mice (Figure 2D).

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When longitudinal echocardiography was analyzed, we found that R848-treated female mice had a profound reduction in EF with subsequent increase in E/e′ compared with acetone controls, indicative of cardiac dysfunction, which was absent in the male mice independent of treatment (Figure 2E). R848-treated female mice had a significant increase in left ventricular wall thickness at the 4-week time point, but it was normalized by the 16-week time point. Moreover, R848-treated male mice had no significant change in left ventricular wall thickness compared with acetone controls over the 16-week time course (Figure 2F). The significant reduction in EF in the females was coupled with increases in both ESV and EDV, with no significant difference in the male mice independent of treatment (Figure 2G).

Induction of systemic cardiovascular complications by R848 treatment

Our data indicate that female R848-treated mice had a significant elevation in plasma sVCAM-1 levels (Figure 3A). Interestingly, both R848-treated female and male groups had a significant increase in plasma sICAM-1 levels compared with their respective vehicle controls (Figure 3A). In R848-treated females, tissue level of cardiac Vcam1 was elevated, whereas cardiac Icam1 was not. The effect was sex and organ specific because neither renal or cerebral Vcam1 or Icam1 were changed at the 16-week time point (Figure 3C–E). The male mice show significant decrease only in cerebral Vcam1 (Figure 3E). Histologic analysis of cardiac fibrosis demonstrated that R848-treated female mice had a significant increase in perivascular fibrosis compared to controls, whereas male mice were unchanged independent of treatment (Figure 3F). In accordance with cardiac dysfunction and fibrosis, we set out to determine whether R848-treated mice had elevated cardiac macrophages. Immunofluorescence analysis of cardiac macrophages demonstrated that R848 treatment increased Mac3⁺ cells in both female and male mice compared to their respective acetone controls (Figure 3G).

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Renal end-stage organ injury induction by R848 treatment

We next investigated renal injury in female and male B6.Nba2 mice. First, we found that R848-treated female mice exhibited a profound increase in renal NGAL expression compared to acetone controls. However, neither acetone nor R848 treatment had any effect on NGAL expression (Supplementary Figure 1A). Next, we assessed plasma levels of BUN, a clinically relevant marker of renal injury, in our mice, and found that R848-treated female mice had a profound increase in plasma BUN compared with acetone controls. Again, there was no significant difference between male acetone or R848 treatment (Supplementary Figure 1B). Lastly, we investigated renal fibrosis and injury in female and male B6.Nba2 mice histologically; we found that both R848-treated female and male mice exhibited profound renal injury compared with their respective vehicle controls at the 16-week time point, with the highest injury scores and damage presented in the R848-treated female mice group (Supplementary Figure 1C).

DISCUSSION

In the current study, we demonstrated that treatment with R848 in SLE-prone B6.Nba2 mice promote classical symptomology of SLE, including IgG complex deposition and elevated plasma anti-Sm autoantibodies in female mice, whereas males only exhibited IgG complex deposition. Moreover, we found a profound increase in splenomegaly and cardiomegaly in female mice but only enlargement of the spleen in males. A key finding was the R848 treatment–induced significant cardiac dysfunction by the 16-week time point through a reduction in EF and elevated E/e′, ESV, and EDV, which was not found in treated male mice. We found that vascular activation markers sICAM-1 and sVCAM-1, along with cardiac perivascular fibrosis, cardiac inflammation, and renal glomerular injury, were significantly increased in female mice treated with R848. Lastly, we found that female R848-treated mice had profound elevation in renal injury markers including NGAL and BUN, which was absent in male mice independent of treatment. Thus, R848 treatment induces prominent SLE-like cardiac dysfunction and systemic endothelial activation in female B6.Nba2 mice, with mild systemic endothelial activation in males. Importantly, this novel preclinical murine model provides investigators a unique model to investigate signaling mechanisms associated with the development of CVD in SLE on the most common genetic background for cardiovascular studies.

SLE pathogenesis and symptomology present with high levels of plasma autoantibodies, IgG complex deposition, and an elevated interferon gene signature (interferon-α [IFN-α] and IFN-β).¹² An interesting finding was that plasma levels of autoantibodies (anti-dsDNA and anti-Sm) were not different in males at the 16-week time point, but treated males had exacerbated IgG complex deposition in target organs. This dichotomy could be a temporary development in our four-week treatment model or a systemic versus specific end-stage organ deposition. In this accord, we found that only female treated mice had elevated renal Isg15 and anti-Sm autoantibodies, highlighting possible key pathogenetic differences in the development of SLE-like symptomology between female and male B6.Nba2 mice.

The highly inflammatory pathogenesis of SLE contributes to the development vascular dysfunction, including aortic aneurysm, hypertension, and cardiac and renal dysfunction including myocardial infarction, heart failure, and lupus nephritis.^4,13 Studies have identified the development of CVD, specifically hypertension, in genetic models of SLE-prone mice. Clinically, it has been demonstrated that patients with SLE had a higher incidence of heart failure compared with age-matched controls.³ Moreover, the number of patients with SLE and concomitant heart failure was three times higher than the general population and was equal to patients with diabetes mellitus.³ These studies demonstrate a critical link between cardiovascular complications and patients with SLE. In accordance with these studies, our data demonstrate that TLR7 treatment induces profound cardiac dysfunction, including cardiomegaly, reduced EF, increased ESV and EDV, and increased cardiac macrophages in female mice. Conversely, hydroxychloroquine, which interferes with TLR7 protein maturation and subsequent signaling, prevents CVD complications.¹⁴ Of critical importance, the development of cardiac dysfunction was not seen in male SLE-prone B6.Nba2 mice, highlighting possible key sex-dependent pathogenesis of SLE-like symptomology.

Not only does SLE pathogenesis effect the cardiac tissues, but it has been demonstrated to promote vascular dysfunction and end-stage organ tissue fibrosis.^15,16 Studies have indicated that impaired endothelial function and/or activation is present in both preclinical and clinical settings of SLE.⁷ Endothelial activation and up-regulation of ICAM-1 and VCAM-1 has been indicated to be a contributing mechanism for immune cell adhesion and diapedesis promoting inflammation and end-stage organ damage often present in patients with SLE and CVD.¹⁷ Our data from the present study demonstrate that, in our preclinical model, systemic vascular endothelial activation is up-regulated independent of sex. Female mice, but not male mice, exhibited tissue-specific endothelial cell activation, demonstrating a tissue-specific vascular endothelial activation regulation that is dependent on sex. Importantly, our model being on the C57Bl/6 background allows for direct comparisons to numerous studies on endothelial activation and vascular dysfunction in female and male mice.

Although our study provides important data on a novel preclinical murine model of SLE-like CVD, future studies are required to determine the specific pathophysiologic organ-specific mechanisms contributing to SLE-like CVD end-stage organ disease. A limitation to our study is that we did not directly measure blood pressure in our mice. However, our data demonstrate that female B6.Nba2 mice develop cardiomegaly, reduced EF, renal injury, and endothelial dysfunction, indicative of a hypertensive pathophysiology. Future studies are needed to investigate the role of blood pressure status in the development of the cardiovascular phenotype in our lupus-prone mice. Another limitation to this study is that we measured systemic vascular endothelial cell activation and did not perform in vivo physiologic vascular measurements in the heart, kidney, and brain of our mice. Recently, Mihailovic et al demonstrated that men with SLE had a significant increase for both renal and cardiovascular complications, specifically coronary artery disease and myocardial infarctions, compared with women,¹⁸ highlighting the critical importance of studying vascular dysfunction. Lastly, we know that immune cell activation and infiltration can contribute to the development of not only SLE, but also CVD and end-stage organ injury. Although we did not specifically study inflammatory mediators in the development of our model, our future studies will investigate the role of the immune system in the development of SLE-like CVD complications.

The present study provides a new preclinical murine model of sex-dependent SLE-like CVD with cardiac dysfunction, systemic endothelial activation, and renal injury on the common murine C57Bl/6 background to provide future investigators with a model to study signaling mechanisms contribution in this deleterious pathology. This model can provide insights into potential therapeutic targets and novel biomarkers for patients with SLE-associated CVD.

AUTHOR CONTRIBUTIONS

All authors contributed to at least one of the following manuscript preparation roles: conceptualization AND/OR methodology, software, investigation, formal analysis, data curation, visualization, and validation AND drafting or reviewing/editing the final draft. As corresponding author, Dr Van Beusecum confirms that all authors have provided the final approval of the version to be published, and takes responsibility for the affirmations regarding article submission (eg, not under consideration by another journal), the integrity of the data presented, and the statements regarding compliance with institutional review board/Helsinki Declaration requirements.

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