Caspase-1 activation is related with HIV-associated atherosclerosis in a HIV transgenic mouse model and HIV patient cohort

Abstract

Objective:

Atherosclerotic cardiovascular disease (ASCVD) is an increasing cause of morbidity and mortality in patients with HIV (PWH) since the introduction of combination antiretroviral therapy (cART). Despite recent advances in our understanding of HIV ASCVD, controversy still exists on whether this increased risk of ASCVD is due to chronic HIV infection or other risk factors. Mounting biomarker studies indicate a role of monocyte/macrophage activation in HIV ASCVD, however little is known about the mechanisms through which HIV infection mediates monocyte/macrophage activation in such a way as to engender accelerated atherogenesis. Here, we experimentally investigated if HIV expression is sufficient to accelerate atherosclerosis, and evaluated the role of caspase-1 activation in monocytes/macrophages in HIV ASCVD.

Approach and Results:

We crossed a well-characterized HIV mouse model, Tg26 mice, which transgenically express HIV-1, with Apolipoprotein E deficient mice to promote atherogenic conditions (Tg26^+/−/ApoE^−/−). Tg26^+/−/ApoE^−/− have accelerated atherosclerosis with increased caspase-1 pathway activation in inflammatory monocytes and atherosclerotic vasculature compared to ApoE^-/−. Using a well-characterized patient cohort of PWH and tissue banked aortic plaques, we documented that serum IL-18 was higher in PWH compared to non-HIV-infected controls and in patients with plaques, IL-18 levels correlated with monocyte/macrophage activation markers and non-calcified inflammatory plaques. In autopsy derived aortic plaques, caspase-1+cells and CD163+macrophages correlated.

Conclusion:

These data demonstrate that expression of HIV is sufficient to accelerate atherogenesis. Further it highlights the importance of caspase-1 and monocyte/macrophage activation in HIV atherogenesis and the potential of Tg26^+/−/ApoE^−/− as a tool for mechanistic studies of HIV ASCVD.

Introduction:

Combination antiretroviral therapy (cART)-treated people with human immunodeficiency virus (HIV) (PWH) face an increased risk of myocardial infarction (MI), even after controlling for traditional and non-traditional risk factors¹. Persistent HIV-associated immune activation (monocyte and T-cell activation) is believed to contribute to heightened MI risk in HIV^{2, 3}. Specifically, systemic immune activation predisposes PWH to arterial inflammation (characterized by increased arterial macrophage infiltration), and accelerated atherogenesis^4–8. Strikingly, accelerated atherogenesis has even been noted among elite controllers, or PWH who maintain undetectable virus levels in the absence of cART⁹, suggesting that the process occurs independent of cART effects. However, controversy still exists as to whether chronic HIV infection and immune activation or an increased prevalence of CVD risk factors account for the accelerated atherogenesis^10–12. Despite recent advances in our understanding of HIV-associated cardiovascular disease (CVD), little is known about the mechanisms through which HIV infection mediates monocyte/macrophage activation in such a way as to engender accelerated atherogenesis.

HIV-induced caspase-1 activation is a potential molecular link between monocyte/macrophage activation and HIV-associated atherogenesis³. Productive and latent HIV infection activates monocytes/macrophages and mediates an array of molecular signaling pathways that have an established pathogenic role in traditional atherosclerosis³. One pathway of interest is inflammasome formation and caspase-1 activation¹³. Pathogens, stress and endogenous danger molecules can activate caspase-1 through cleavage and activation by inflammasome formation. Active caspase-1 then cleaves pro-inflammatory cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18) into their active forms, initiating an immune response and pathogen clearance, as well as systemic tissue inflammation. HIV infection stimulates the formation of the nod-like receptor protein 3 (NLRP3) inflammasome in human monocytes/macrophages, as well as microglia leading to caspase-1 activation and the release of active IL-1β and IL-18^{14, 15,16}. Activated levels of caspase-1, from inflammasome cleavage, increase rapidly during early HIV infection¹⁷. Caspase-1 activation also contributes to T-cell depletion and T-cell activation in HIV and simian immunodeficiency virus (SIV) infection through pyroptosis^18–22. However, the exact role of caspase-1 activation in monocytes/macrophages in HIV atherogenesis has not been explored experimentally, mainly due to the lack of a suitable small animal model.

In the last two decades, a number of rodent models have been developed to study HIV-associated disorders³. The non-infectious Tg26 mouse^23–25, which has viral transcripts but no active viral replication, develops HIV comorbidities such as HIV-associated nephropathy (HIVAN)^{24, 26–28}, B-cell lymphomas²⁹ and cardiac dysfunction³⁰. This suggests that persistence of HIV transcripts and viral proteins alone without replication is sufficient to trigger inflammation and comorbidities. Here, we have developed a novel HIV atherosclerotic mouse model (Tg26^+/−/ApoE^−/−). We demonstrate for the first time that expression of HIV transcripts and proteins in mice is sufficient to accelerate atherogenesis. We used a prospectively recruited patient cohort of PWH and non-HIV-infected controls, and autopsy-derived aortas with plaques from PWH and non-HIV-infected controls, along with Tg26^+/−/ApoE^−/− mice, to show that caspase-1 activation is associated with monocyte/macrophage activation in the development of HIV atherosclerosis.

Material and methods

The data that support the findings of this study are available from the corresponding authors on reasonable request.

Animal treatment and characterization of atherosclerotic plaques

Animal studies were approved by the Animal Care and Use Committee from Lewis Katz School of Medicine of Temple University and Tulane University School of Medicine. We used HIV Tg26^+/− transgenic mice (kindly provided to us by Dr. Paul Klotman²⁶). Tg26^+/−/ApoE^−/− mice were generated by crossing Tg26^+/− mice on B6 background with ApoE^−/− mice. To accelerate atherosclerosis, 6-week-old male and female Tg26^+/−/ApoE^−/− and littermate ApoE^−/− mice were fed with a high fat and cholesterol enriched diet (D12108C; Research Diets Inc) containing 20.1% saturated fat, 1.37% cholesterol, and 0% sodium cholate for 8 weeks. In another set of experiment, male Tg26^+/−/ApoE^−/− and ApoE^−/− mice were fed with normal chow for 8 months. Male Tg26^+/−/ApoE^−/− and ApoE^−/− mice were only used for the 8-month normal chow study because of an atherogenic effect of maternal hypercholesterolemia during pregnancies³¹. The body weight was recorded before and after experiments. At the end of experiments, mice were sacrificed by CO₂ asphyxiation and serum was prepared and stored at −80°C. After perfusion the mice with PBS, heart was harvested and embedded in OCT and frozen in liquid nitrogen for tissue sectioning. Oil red-O or Masson’s trichrome staining were performed on sections of the aortic roots (7μm thickness). The entire aorta from the heart outlet to the iliac bifurcation was also stained with Oil red-O, as previously described³². The total oil red-O stained area in the entire aorta, and stained plaque area in aortic root for each section were recorded. Measurements of the surface area of atherosclerotic plaques were acquired by manual tracing using ImageJ analysis software and analyzed by 2 individuals, who are blinded to the experimental design. The plaques are represented as percent plaque area of the entire intimal surface area as suggested in previous guidelines³³. In our study, we selected to use the percent plaque area instead of the absolute size for the comparison of the differences in the aortic root, because Tg26 has been previously documented to have significantly smaller left ventricular size than littermate control³⁰. Plaque area in aorta (%) = (plaque area/whole aortic area) × 100%. Collagen content in plaque (%) = (blue staining area/plaque area) × 100%.

Immunofluorescence and histology

To determine the macrophage content in mice plaque, frozen sections of aortic root (7 μm) were stained with rat anti-mouse CD68, (clone: FA-1, AbD Serotec MCA1957), followed by fluorescein Alexa488-conjugated anti-rat antibody or rabbit anti-mouse CD163 (1:50 abcam ab182422) with HRP-conjugated secondary antibody (Dako). Images were detected by fluorescence microscope (BZ-X700, Keyence) and quantified using Image ProPlus 6.0 software. Macrophage content in plaque (%) = (staining positive area/ plaque area) x100% or total cell count.

Measurements of caspase-1 activity

Caspase-1 activity in monocytes was determined by APO LOGIX kit (Cell Tech. Mountain View, CA). The kit contains a carboxyfluorescein (FAM)-labeled caspase-1 inhibitor fluoromethyl ketone (FMK) (FAM-YVAD-FMK), which irreversibly binds to active caspase-1. All procedures were performed following the manufacturer’s instructions. Briefly, blood from Tg26^+/−/ApoE^−/− and littermate ApoE^−/− were collected by tail bleeding. After red blood cells were lysed, PBMCs were first stained with monocytes markers including PE-Cy7 anti-mouse CD11b, APC anti-mouse Ly6G and eFlour450 anti-mouse Ly6C (eBioscience, San Diego, CA) for 30min at room temperature. After washing with PBS, cells were then incubated with caspase-1 inhibitor FAM-YVAD-FMK at 37°C for 1 h, followed by washing with 1x washing buffer provided in the kit. LSRII flow cytometer was used to determine caspase-1 activity in CD11b+Ly6G-Ly6c+ monocytes. Data were analyzed with the FlowJo software (Tree Star, Ashland, OR).

Protein extraction and Western blot analysis

Total protein from cultured cells or mice aorta were extracted by M-PER Protein Extraction Reagent (Pierce, Rockford, IL) containing protease and phosphatase inhibitors according to the manufacturer’s instructions. Protein concentration was determined with a bicinchoninic acid assay (Pierce, Rockford, IL). 30–50ug protein was loaded and separated on 10% SDS-PAGE gels, transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Wattford, UK), and blocked with 5% non-fat milk in Tris-buffered saline solution containing 0.1% Tween-20(TBST) for 1 h. Membranes were then incubated with the primary antibodies against caspase-1 (1:500 Santa Cruz sc1218) and β-actin overnight at 4 °C, followed by corresponding fluorescent-conjugated secondary antibody for 1 h at room temperature. After washing with TBST, the signals were visualized using ODYSSEY® CLx Imaging system (LI-COR, Inc.). Band densities were quantified using Image Studio Software.

Macrophage foam cell formation

Mouse peritoneal macrophages were isolated and cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) 3 days after i.p. injection of 3% thioglycollate broth medium. After being washed with PBS, cells were seeded at 1×10⁶/ml in 6-well plate and allowed to adhere to the substrate by culturing them for 2–4h at 37°C. Non-adherent cells were removed by gently washing with warm PBS. To induce foam cell formation, adherent macrophages were treated with 100 μg/ml oxidized low density lipoprotein (oxLDL, Alfa Aesar, Haverhill, MA) for 24h. Cells were stained with 0.4 % Oil Red O and quantified for percent of foam cell formation³⁴.

ELISA and qRT-PCR

Mouse IL-1β, IL-18 ELISA kits were purchased from R&D Systems. The levels of cytokines were determined according to the manufacturer’s instructions. IL-1β procedure was modified with orbital shaking to decrease the lower detection limit of the kit to 2.35 pg/mL as previously described³⁵. Total RNA was isolated from mice tissue using TRIZOL Reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. 1ug RNA was reversely transcribed to complementary DNA(cDNA) using Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, San Jose, CA). For Real-time PCR(RT-PCR), equal amounts of cDNA were added to SYBR Green master mix (Qiagen, Valencia,CA) to detect target transcripts using Roche light cycler 480. Data for relative expression were presented using the 2ΔΔCt method.

Bone marrow (BM) transplantation

Eight-week old ApoE^−/− mice were lethally irradiated (9.5Gy) and reconstituted with 10⁶ donor BM cells of Tg26^+/−/ApoE^−/− or ApoE^−/− by i.v. injection using RS2000 irradiator (Rad Source, Buford, GA) as detailed in³⁶. The chimeric mice were fed a normal chow for 5 weeks to allow fully reconstitution, then switched to the high fat and cholesterol enriched diet for another 8 weeks. At the end of experiment, the aorta were isolated and the plaque was identified in the aortic root by oil red-O staining. The plaque area in aortic root for each section was quantified as previously described in³³. The full reconstitution in the chimeric mice was determined by measuring HIV transcripts of the spleen of the mice with real-time PCR.

Serum lipid measurement

Blood was collected by heart puncture. Serum was separated by 2000xg centrifugation for 10 min at 4°C. Serum cholesterol and triglyceride profiles were measured at NIH/NIAAA as described³⁷.

Study Participants

Human studies were approved by the institutional review boards of Massachusetts General Hospital. Written informed consent was received from participants prior to inclusion in the study. IL-18 was measured in sera from participants from an existing cohort of people with HIV and seronegative controls. One hundred fifty-three men and women with HIV infection were previously recruited from HIV clinics and community health care centers in the greater Boston area. Sixty-seven HIV-seronegative control men and women were simultaneously recruited from the same communities. Neither group was recruited on the basis of presence of heart disease, and in fact, history or symptoms of heart disease were exclusionary for both groups. Other than HIV disease, inclusion and exclusion criteria were identical for both groups. Participants aged 18–60 years without prior history of cardiac disease or symptoms suggestive of cardiac disease were recruited. HIV-infected and non-HIV control participants with known renal disease or estimated creatinine clearance <60 mL/min were excluded to minimize risk of contrast nephropathy. HIV-infected participants receiving combination antiretroviral therapy (ART) at the time of the study were required to have been receiving stable therapy for >3 months. We have continued to expand this cohort and now report novel data not previously published on caspase-1 pathway activation^4–6.

Study Procedures and Assessment of Cardiovascular Risk Factors

Data on sociodemographic factors, cardiovascular risk factors, medical history, family history, behavior (including smoking), use of medications were obtained. Diagnosis of diabetes mellitus was based on clinical history or laboratory assessments meeting American Diabetes Association criteria for diabetes mellitus. Cardiac multidetector row CT and CT angiography imaging were performed using a 64-slice CT scanner (Siemens Medical Solutions) as previously described^4–6. Assessment of coronary atherosclerotic plaque burden and stenosis were determined by a consensus reading between 2 cardiac imaging specialists with significant experience in the interpretation of cardiac CTs. The presence of any coronary atherosclerotic plaque, whether calcified or noncalcified, was determined as previously described^4–6.

Inflammatory, Metabolic, Biochemical, and Immunologic Parameters

Plasma sCD163, MCP-1, IL-6, sCD14, CXCL10 and CRP were quantified by enzyme-linked immunosorbent assay (ELISA) as previously described^4–6. Human IL-1β, IL-18, and a type II soluble IL-1R (IL-1 RII or sIL-1R) ELISA kits were purchased from R&D Systems. The levels of IL-18 and IL-1β were determined according to the manufacturer’s instructions. The levels of IL-1β were below the limit of detection (0.125 pg/ml). All participants fasted at least 12 hours before blood draws. Total cholesterol, high- and low-density lipoprotein, triglycerides, glucose, and creatinine levels were determined using standard techniques. CD4⁺ T-cell counts were assessed by flow cytometry. HIV RNA levels were determined by ultrasensitive reverse-transcription polymerase chain reaction (RT-PCR; Roche Amplicor Monitor; lower limit of detection, 50 copies/mL). HIV testing was performed by ELISA (Abbott), and results were confirmed by western blot.

Autopsy aortas from HIV+ and HIV- subjects

Aortas from HIV- (n=8) and HIV+ (n=9) subjects were obtained from the MHBB, member of the NNTC. Both HIV+ and HIV- subjects were selected on the basis of having severe systemic atherosclerotic disease, as assessed by gross examination of the aorta and its major branches at the time of autopsy. In the majority of cases, sections of ascending aorta from the thoracic cavity were obtained. Tissues were fixed in formalin, and routinely processed for paraffin embedding and sectioning at 4 microns. Sections were stained with hematoxylin and eosin, Verhoeff elastin stain, and Masson’s trichrome for light microscopic analysis. Immunohistochemistry was performed using primary antibodies to recognize CD163 (1:250 Serotech MCA1835), CD68 (1:400 Dako M0814) and Caspase-1 (1:50 Abcam ab1872). Slides were washed and incubated with isotype-specific, HRP-conjugated secondary antibody (Dako). Cell type and expression was visualized using the 3,3’-Diaminobenzidine (DAB) substrate system, counterstained with hematoxylin. Slides were dehydrated, cleared, and mounted for microscopy. Slides were imaged using a BZ-X700 Keyence Microscope at 200X magnification. Images were quantified for plaque area, media area, plaque cell count, media cell count, and intimal-media thickness.

Statistics

All statistical analyses were performed using Prism Software and LMP (SAS institute). If the data passed normality and equal variance test, one-way ANOVA or t-test was used for the comparison analysis. To compare values obtained from three or more groups, one-factor analysis of variance (ANOVA) was used followed by post-hoc analysis. Non-parametric Spearman correlation was used for all correlations except for data in human studies describing relationships with logIL-18, where Pearson correlation coefficient was used. The difference between the two groups was examined with an unpaired parametric two-tailed t-test unless noted as unpaired one-tailed t-test. Experimental results are shown as the mean ± SEM except for human serum measurements which are reported as mean ±SD. A p-value of <0.05 was considered significant for all tests performed. In the patient cohort, IL-18 was not normally distributed so IL-18 was log transformed and t-test was performed.

Results:

Expression of HIV transcripts accelerate atherogenesis in mice:

In order to determine if persistent and chronic HIV infection is sufficient to accelerate atherogenesis and to explore the mechanisms underlying HIV atherosclerosis experimentally, we generated a novel mouse model. The Tg26 mouse model is one of three HIV transgenic lines originally generated on an FVB genetic background by using a noninfectious HIV-1 DNA construct with a deletion of a 3kb region of the gag/pol genes²⁴. It has been widely used to study the pathogenesis of HIV-associated comorbidities, such as HIVAN^{24, 26–28}, B-cell lymphomas²⁹ and cardiac dysfunction³⁰. Although these mice do not exhibit any active HIV replication, the expression of viral transcripts and viral proteins, such as HIV gp120 and matrix protein p17, have been detected in various tissues^{23–25, 29, 38}. The HIV Tg26 mice were backcrossed onto the C57BL/6 (B6) genetic background seven times^{33, 39} to correct for the development of HIVAN and subsequent early lethality^{24, 26–28}. In addition, mice are resistant to the development of atherosclerosis without introduction into an atherogenic background^{32–34, 39}, so the Tg26 (B6) (Tg26^+/−) were introduced to the apolipoprotein E deficient (ApoE^−/−) background to generate Tg26^+/−/ApoE^−/− mice. Use of the ApoE^−/− background with an atherogenic diet is a widely accepted and established protocol^{32–34, 39} for atherogenesis. We found that both Tg26^+/−/ApoE^−/− and Tg26^+/− mice express high levels of HIV transcripts in multiple immune tissues normally infected with HIV (Figure 1A). There was no significant difference at the HIV transcript level in the tissues the Tg26^+/− or Tg26^+/−ApoE^−/− mice, (Figure 1A) suggesting that the atherogenic background does not alter HIV transcription.

Figure 1: Tg26^+/−/ApoE^−/− mice develop accelerated atherosclerosis on 8 week high fat diet.

(A) No difference in HIV transcript levels in multiple immune tissues from male and female Tg26^+/−/ApoE^−/− and Tg26^+/− mice. To determine if HIV background effects HIV transcript levels qRT-PCR analysis of HIV mRNA expression represented as mean ± SEM of multiple tissues in 8 week old mice (n=8). Fold changes were normalized to heart HIV transcript levels. BM: bone marrow. P>0.05. Levels between all tissues were compared using one-way ANOVA. Specific immune tissues were compared by unpaired two-tailed t-test of Tg26^+/−/ApoE^−/− levels vs. Tg26^+/− transcript levels. (B) Representative images of atherosclerosis of en face aorta with oil red O staining in male and female ApoE^−/− and Tg26^+/−/ApoE^−/− mice on an atherogenic diet for 8 weeks. (C) Plaque of 8 week atherogenic diet en face analysis reported as a plaque area per total aortic area (Tg26^+/−/ApoE^−/− 13.01 ± 1.6% vs. ApoE^−/− 8.959 ± 1.1%, P<0.05). (D) Representative images of aortic root plaque stained with oil red O staining in ApoE^−/− and Tg26^+/−/ApoE^−/− mice on an atherogenic diet for 8 weeks. (E) Quantification of aortic root plaques as a percentage of the aortic sinus area (Tg26^+/−/ApoE^−/− 51.56 ± 1.8% vs. ApoE^−/− 42.92 ± 3.3%, P<0.05). (F) Representative images of trichrome staining in ApoE^−/− and Tg26^+/−/ApoE^−/− mice on an atherogenic diet for 8 weeks. (G) Significant increase in collagen content analyzed by trichrome staining reported as a percentage of the total plaque area in Tg26^+/−/ApoE^−/− 15.32 ± 3.0% vs. ApoE^−/− 8.15 ± 1.73%, P<0.05. (H) BUN measured from serum of 8 week atherogenic mice reported in (mg/dL) no significant difference between Tg26^+/−/ApoE^−/− (26.33 ±1.04mg/dL; n=12) and ApoE^−/− mice (29.82 ±1.39mg/dL; n=11). (I) No difference in serum cholesterol reported in mg/dL between Tg26^+/−/ApoE^−/− (1101 ±45.29 mg/dL) and ApoE^−/− mice (1233 ±118.8 mg/dL) on 8 week atherogenic diet. (J) Triglycerides measured in serum from Tg26^+/−/ApoE^−/− (138.7 ±8.80 mg/dL) and ApoE^−/− mice (139.3 ±18.29 mg/dL) on 8 week atherogenic diet. (K) Weight (g) of Tg26^+/−/ApoE^−/− (24.24 ±0.73g) and ApoE^−/− mice (25.11 ±1.10g) after 8 weeks atherogenic diet. All graphs reported as mean ± SEM. *P < 0.05, vs ApoE^-/−. Analyzed by two-tailed unpaired t-test.

To investigate whether Tg26^+/−/ApoE^−/− mice have accelerated atherogenesis, we fed Tg26^+/−/ApoE^−/− and littermate ApoE^−/− mice an atherogenic diet for 8 weeks. We document that Tg26^+/−/ApoE^−/− mice on an atherogenic diet developed significantly more atherosclerotic plaques by en face (Figure 1B and 1C, 13.01 ± 1.6% vs. 8.959 ± 1.1%, P<0.05) and aortic root analysis (Figure 1D and 1E, 51.56 ± 1.8% vs. 42.92 ± 3.3%, P<0.05) compared to ApoE^−/− mice. Both Tg26^+/−/ApoE^−/− and Tg26^+/− mice fed with the atherogenic diet had normal renal function measured by the levels of blood urea nitrogen (BUN) (Figure 1H, and Figure IA in the online-only data supplement) without any early lethality. This result confirms that backcrossing to the B6 background corrected the renal failure and early lethality seen in FVB Tg26 mice, and there is no alteration in renal function with the atherogenic diet^{24, 26–28}. There were no significant differences in serum cholesterol (Figure 1I), triglycerides (Figure 1J), and body weight (Figure 1K), between Tg26^+/−/ApoE^−/− and ApoE^−/− mice on the atherogenic diet. Consistently, we did not detect any significant differences at the levels of cholesterol (Figure IB in the online-only data supplement) and triglycerides (Figure IC in the online-only data supplement) between Tg26^+/− and littermate B6 control on the atherogenic diet. However, there was a significant difference in cholesterol and triglyceride levels between those mice on the atherogenic background and those not (Fig IB–C in the online-only data supplement). To study any gender effect on plaque development, two cohorts of experimental mice fed with an atherogenic diet for 8 weeks were examined. We found Tg26^+/−/ApoE^−/− but not Tg26^+/− mice develop atherosclerosis, and Tg26^+/− mice have significantly lower cholesterol and triglyceride levels compared to Tg26^+/−/ApoE^−/− mice demonstrating the necessity of an atherosclerotic background³³ (Figure IB–D in the online-only data supplement). Both male and female Tg26^+/−/ApoE^−/− mice had more severe atherosclerosis by en face aorta analysis compared to littermate controls (Figure IE in the online-only data supplement). Interestingly, we also found that atherosclerotic plaques in Tg26^+/−/ApoE^−/− have increased collagen deposition (Figure 1F and 1G, 15.32 ± 3.0% vs. 8.15 ± 1.73%, P<0.05) consistent with our recent study that showed potential proteins implicated HIV ASCVD were involved in myocardial fibrosis/collagen formation and monocyte chemoattraction and changed with statin treatment⁴⁰. However, no difference in macrophage content was found (Figure IIA and IIB in the online-only data supplement). Although not significant, Tg26^+/−/ApoE^−/− mice had a trend towards increased CD163+ macrophages in the plaque, that were specific to Tg26^+/−/ApoE^−/− females, but not to males (Figure IID and IIE in the online-only data supplement).

Consistently, using mice fed a normal chow for 8 months, we observed that Tg26^+/−/ApoE^−/− mice have greater atherogenesis compared to ApoE^−/− mice (Figure 2A and 2B, 7.12 ± 3.5% vs. 4.26 ±0.96%, P<0.05). Tg26^+/−/ApoE^−/− mice fed normal chow for 8-months had normal renal function measured by the levels of BUN (Figure 2C) without any early lethality. This result confirms that backcrossing to the B6 background corrected the renal failure and early lethality seen in FVB Tg26 mice, and there is no alteration in renal function even at 8 months of age^{24, 26–28}. There were no significant differences in serum cholesterol (Figure 2D), triglycerides (Figure 2E), and body weight (Figure 2F), between Tg26^+/−/ApoE^−/− and ApoE^−/− mice on 8-month normal chow. No difference in macrophage content was found in animals in the normal chow diet (Figure IIC in the online-only data supplement).

Figure. 2: Tg26^+/−/ApoE^−/− mice develop accelerated atherosclerosis on 8 month normal chow (A-F) and transgenic expression of HIV only in hematopoietic cells promotes the atherogenesis (G and H).

(A) Representative en face aorta analysis from 8 months of normal chow reported as a percentage of plaque per total aortic area. (B) Significant increase in en face aortic plaque in male Tg26^+/−/ApoE^−/− 7.12 ± 3.5% vs. male ApoE^−/− 4.26 ±0.96% (1 tailed t-test P<0.05). (C) BUN measured from serum of 8 month normal chow mice reported in mg/dL. No significant difference between Tg26^+/−/ApoE^−/− (33.71±2.78mg/dL) and ApoE^−/− mice (31.33 ±1.60mg/dL). (D) No difference in serum cholesterol reported in mg/dL between Tg26^+/−/ApoE^−/− (488.3 ±47.57mg/dL) and ApoE^−/− mice (492.2±38.03mg/dL) on 8 month normal chow (E) Triglycerides measured in serum from Tg26^+/−/ApoE^−/− (191.7±27.51mg/dL) and ApoE^−/− mice (198.7 ±24.40mg/dL) on 8 month normal chow showing no significant difference. (F) Weight (g) of Tg26^+/−/ApoE^−/− (27.46 ±0.95g) and ApoE^−/− mice (29.07±0.86g) after 8 months’ normal chow showing no significant difference. (G) The plaque area in arch of the chimeric mice: the ApoE^−/− mice reconstituted with the bone marrow of Tg26^+/−/ApoE^−/− mice is greater than that of ApoE^−/− transplanted into ApoE^−/− mice (14.95 ± 1.70 vs. 9.38 ± 1.27). (H) Real-time RT PCR analysis of HIV transcripts in Tg26^+/−/ApoE^−/− and chimeric mice. All graphs reported as mean ± SEM. Analyzed by two-tailed unpaired t-test. *P<0.05.

HIV infects T lymphocytes and cells of the myeloid lineage, including monocytes and tissue macrophage. We further defined the role of transgenic expression of HIV in hematopoietic versus stromal cells on atherogenesis using BM transplantation. The irradiated ApoE^−/− mice were reconstituted with BM obtained from either Tg26^+/−/ApoE^−/− or ApoE^−/− mice, then fed on a atherogenic diet for two months. As shown in Figure 2G and 2H, we documented that the ApoE^−/− mice reconstituted withTg26^+/−/ApoE^−/− BM developed significantly more plaque in aortic arch than the mice reconstituted with ApoE^−/− BM. This result indicates that the expression of HIV transcripts in hematopoietic cells is responsible for the accelerated atherosclerosis in the Tg26^+/−/ApoE^−/− mouse model. In summary, we have created a novel model, Tg26^+/−/ApoE^−/− mice, that for the first time shows that HIV expression in hematopoietic cells accelerates ASCVD in vivo. This model can be used to further explore the potential atherogenic mechanisms in HIV infection.

Increased activation of caspase-1 pathway including its upstream and downstream events in Tg26^+/−/ApoE^−/− mice:

To explore HIV-mediated immune activation in atherogenesis, we investigated caspase-1 pathway activation throughout the course of the atherogenic diet in inflammatory monocytes and the vasculatures of Tg26^+/−/ApoE^−/− and ApoE^−/− mice. Considering that Tg26^+/− did not develop any plaques and had a significantly different lipid profile, they were excluded as a control in the following mechanistic studies. We found through a time course study that Tg26^+/−/ApoE^−/− mice by 6 weeks of the atherogenic diet had higher caspase-1 activation in Ly6+ monocytes (inflammatory monocytes) than ApoE^−/− mice (Figure 3). Further, these Tg26^+/−/ApoE^−/− mice by 8 weeks on the atherogenic diet had a significantly increased caspase-1 activation as determined by the ratio of caspase-1 p20 vs pro-caspase-1 in the atherosclerotic aorta as measured by western blot (Figure 4A–D, 0.93 ± 0.06 vs. 0.35 ± 0.24, P<0.05). Ex vivo macrophages from Tg26^+/−/ApoE^−/− had increased foam cell formation compared to ApoE^−/−, and are a possible source of the elevated caspase-1 activation in Tg26^+/−/ApoE^−/− aortas (Figure III in the online-only data supplement).

Figure 3. Expression of HIV increases caspase-1 activity in inflammatory monocytes in Tg26^+/−/ApoE^−/− mice.

PBMCs from male and female Tg26^+/−/ApoE^−/− and littermate ApoE^−/− were collected and stained with anti-CD11b, -Ly6G, -Ly6C mouse Abs and FAM-FLICA-Caspase-1 Ab, and analyzed by flow cytometry. A-E. Representative flow cytometry depicting CD11b⁺ monocyte (MC) caspase-1 activity. (A) Single cells gated according to the forward scatter(FSC)-A and FSC-H. (B) White Blood Cells (WBCs) were then selected according to the FSC-A and side scatter (SSC)-A. (C) Neutrophils were gated out by selecting SSC-H low and CD11b+ cells and Ly6G low and CD11b+ cells. (D) Inflammatory monocytes were identified as CD11b⁺Ly6C⁺ high monocytes. (E) Representative histograms depicting caspase-1 activity as a percentage of total cells in CD11b⁺Ly6C⁺ monocytes from ApoE^−/− and Tg26^+/−/ApoE^−/− mice respectively. (F) Two experiments with significant increased monocyte caspase-1 activity in Tg26^+/−/ApoE^−/− compared to ApoE^−/− after 6 weeks on an atherogenic diet (38.40 + 10.05 vs. 20.70 +2.40%caspase-1+ cells, P<0.05 and 19.5 +0.98 vs. 16.18 +0.71% caspase-1+ cells, p<0.05 respectively). Analyzed by two-tailed unpaired t-test,

*P<0.05.

Figure 4. HIV increases caspase-1 activity in the vasculature of Tg26^+/−/ApoE^−/− mice.

(A) Western blot of pro-caspase-1, active caspase-1 p20, and pro-IL-1β in atherosclerotic aortic arch from male and female mice on an atherogenic diet for 8 weeks. (B) Significant increase in the activation of caspase-1 in the vasculature as measured by the ratio of caspase-1 p20 to pro-caspase-1 in Tg26^+/−/ApoE^−/− mice (0.93 ±0.06) compared to in ApoE^−/− mice (0.35 ±0.24). (C) A trend towards significantly higher pro-caspase-1 protein expression in the vasculature of Tg26^+/−/ApoE^−/− mice (1.21 ±0.30) compared to in ApoE^−/− mice (0.46 ±0.20). (D) A trend towards significantly higher active caspase-1 p20 protein expression in Tg26^+/−/ApoE^−/− mice (1.17 ±0.36) compared to in ApoE^−/− mice (0.26 ±0.23). Data depicted as mean ± SEM of values in the detectable range. Analyzed by two-tailed unpaired T-Test,

*P<0.05, ** P<0.01.

We further explore the molecular mechanism underlying caspase-1 pathway activation. HIV infection activates danger signaling cascades in infected immune and endothelial cells, which triggers Nod-like receptor protein 3 (NLRP3) inflammasome formation^13–16. Previous studies also documented HIV infection increased NLRP3 production, contributing to increased caspase-1 activation in HIV-associated comorbidities, such as kidney disease and HIV-associated neurological disorder (HAND)^{16, 41, 42}. These findings prompted us to evaluate the upstream event of caspase-1 activation; NLRP3 in Tg26^+/−/ApoE^−/− mice. We demonstrated that Tg26^+/−ApoE^−/− mice had significantly higher NLRP3 mRNA level in the spleen compared to ApoE^−/− mice (Figure 5A). Further demonstrating caspase-1 pathway activation we measured downstream cleavage products of activation, cytokines IL-1β and IL-18, in serum from 8-week atherogenic diet mice. IL-1β (Figure 5B; 20.74 ± 4.19 pg/mL vs. 5.86 ± 1.19 pg/mL, P<0.01) and IL-18 (Figure 5C; 584.4 ± 74.91 pg/mL vs. 421.& ± 46.07 pg/mL, P=0.07) were significantly elevated in Tg26^+/−/ApoE^−/− mice compared to ApoE^−/− mice. Considering that macrophages make up a majority of the cells in the plaque, caspase-1 activation in monocytes/macrophages can contribute to the accelerated atherogenesis seen in Tg26^+/−/ApoE^−/− mice. Together these results indicate that chronic, persistent HIV expression in mice, activates the caspase-1 pathway in the periphery and vasculature leading to monocyte/macrophage activation and HIV atherogenesis.

Figure 5: Upstream and downstream caspase-1 pathway activation in Tg26+/−ApoE−/− mice.

(A) NLRP3 mRNA level in the spleen of male and female ApoE^−/− and Tg26^+/−ApoE^−/− mice on atherogenic diet for 8 weeks. The NLRP3 mRNA transcript was first normalized by GAPDH and compared between two group. The expression level of NLRP3 in ApoE−/− group was set as 1. (Mean± s.e.m, *p<0.05 using two-tailed unpaired t-test. (B&C) Serum IL-1β and IL-18 levels measured in mice on atherogenic diet for 8 weeks using R&D systems ELISA. (B) Significant increase in serum IL-1β in Tg26^+/−/ApoE^−/− mice (20.74 ±4.19pg/mL) compared to in ApoE^−/− mice (5.86 ±1.19pg/mL) measured by ELISA with orbital shaking to decrease the lower detection limit of the kit to 2.35 pg/mL. (C) A trend towards a significant increase in serum IL-18 in in Tg26^+/−/ApoE^−/− mice (584.4 ±74.91pg/mL) compared to ApoE^−/− mice (421.7 ±46.07pg/mL). Data depicted as mean ± SEM of values in the detectable range. Analyzed by two-tailed unpaired T-Test,

*P<0.05, ** P<0.01.

Baseline characteristics of PWH and non-HIV-infected controls:

We investigated whether the downstream products of caspase-1 activation, IL-18 and IL-1β, were increased in PWH compared to non-HIV-infected controls and if the increased cytokines, indicative of enhanced caspase-1 activity were associated with monocyte activation and plaque phenotypes. This cross-sectional study used a cohort of well-characterized PWH (n=153) and non-HIV-infected controls (n=67) controls with no past history of CVD and low Framingham risk scores. Demographic and clinical characteristics of the subjects are displayed in Table I in the online-only data supplement . Age, sex, race, body mass index (BMI), diabetes, and smoking did not differ significantly between groups. Compared with non-HIV-infected subjects, PWH exhibited elevated triglycerides (139 ± 113 mg/dL vs. 104 ± 62 mg/dL, P=0.004) but still within the normal range, were more often on statin therapy (13% vs. 3%, P=0.02) and had significantly larger numbers of noncalcified coronary atherosclerotic plaque segments (0.97 ± 1.5 vs. 0.45 ± 1.2, P=0.009), in line with our previous published findings(Table I in the online-only data supplement )^4–6.

Circulating IL-18 levels by HIV status and plaque status and relationship to inflammatory biomarkers:

To investigate the atherogenic role of the caspase-1 pathway in HIV infection, serum from the PWH and non-HIV-infected controls was examined for IL-18 and IL-1β as well as IL-1 RII, which modulates the action of IL-1⁴³ Log serum IL-18 was significantly elevated in the PWH compared to non-HIV-infected (Figure 6A; 5.59 ± 0.48 log pg/mL vs. 5.29 ± 0.51 log pg/mL, P<0.0001). Log serum IL-18 was significantly elevated in the all subjects with plaque compared to those without plaque (Figure 6B; 5.58 ± 0.50 log pg/mL vs. 5.42 ± 0.49 log pg/mL, P=0.02). IL-1β levels were below the limit of detection (0.125 pg/mL). Of significance, elevated log IL-18 levels correlated with total number of segments with plaque (r= 0.15, P=0.03) and number of segments with non-calcified vulnerable plaques (r=0.14, P=0.04), but not number of segments with calcified plaque (r=0.002, P=0.98) (Table II in the online-only data supplement). Serum IL-18 levels were analyzed for potential correlations with inflammatory markers previously shown to be important in HIV ASCVD, including soluble CD163 (sCD163), monocyte chemoattractant protein 1 (MCP-1), lippopolysaccride (LPS), soluble CD14 (sCD14), C-X-C motif chemokine 10 (CXCL10) and traditional risk markers IL-6 and C-reactive protein (CRP). Log IL-18 positively correlated with plasma sCD163 (r=0.51, P<.0001), MCP-1 (r=0.22, P=0.001), CXCL10 (r=0.64, P<0.0001), LPS (r=0.20, P=0.005), but not with sCD14, IL-6 or CRP (Table II in the online-only data supplement). IL-1 RII was measured in both HIV and non-HIV patients and there was no significant difference between the groups. There was a significant correlation between IL-18 and IL-1 RII (r=0.16, p=0.02). IL-1 RII did not correlate with plaque parameters including total plaque segments, non-calcified plaque, calcified plaque, and calcium score. Additionally, there was no correlation between IL-1 RII and inflammatory markers (MCP-1, LPS, sCD14, IL-6, and CRP) with the exception of sCD163 (r=0.20, p=0.002) and CXCL10 (r=0.30, p=0.008). These data document the relationship between IL-18 and non-calcified inflammatory aortic plaques and peripheral macrophage inflammation whose elevation are a finding of HIV atherogenesis.

Figure 6: Circulating IL-18 levels increased in PWH and in patients with plaque.

Serum from PWH and non-HIV-infected controls was examined for IL-18 and IL-1β. (A) Log serum IL-18 was significantly elevated in the PWH compared to non-HIV-infected controls (5.59 ± 0.48 log pg/mL vs. 5.29 ± 0.51log pg/mL, P<0.0001). (B) Log serum IL-18 was significantly elevated in all subjects with plaque compared to those without plaque (5.58 ± 0.50 log pg/mL vs. 5.42 ± 0.49 log pg/mL, P=0.02). IL-1β levels were below the limit of detection (data not shown). Data reported as mean ± SD and were analyzed by two-tailed unpaired t-test.

Analysis of monocyte/macrophage and caspase-1 in human plaques:

There are currently no detailed pathological studies comparing systemic atherosclerotic plaques of PWH and non-HIV-infected controls. HIV- (n=8) and HIV+ (n=9) aortas with severe atherosclerosis as determined by gross inspection at the time of autopsy were obtained from the Manhattan HIV Brain Bank (MHBB), member of the National NeuroAIDS Tissue Consortium (NNTC). The sections were characterized for the extent of atherosclerotic plaque, and immunohistochemical stains were completed to examine macrophage populations CD163 (M2-macrophages), CD68 (resident pan macrophages) and caspase-1+ cells (Table III in the online-only data supplement). There were no significant differences between the two groups with regard to plaque morphology or the extent of plaque area within the sections; 6 samples (4 HIV+ and 2 HIV-) had microscopic evidence of mineralization, and 6 (4 HIV- and 2 HIV+) had large lipid cores (in contrast to small lipid pools or intermediate histologies). We found no significant difference in plaque or media levels of CD163 or CD68 macrophages (Table III in the online-only data supplement), and similarly, no difference between caspase-1+ cells in the plaques. Of importance caspase-1 levels positively correlated to plaque CD163 macrophages, but not media CD163 macrophages or CD68 macrophages in the plaques or media (Figure 7; r=0.6, P= 0.02). The results obtained from these two human cohort studies link caspase-1 pathway activation to monocyte/macrophage activation for the first time and indicate its role in HIV atherogenesis, warranting further investigation.

Figure 7: The number of caspase-1+ cells and CD163+ macrophages correlate in tissue banked aortic plaques.

Immunohistochemical analyses of tissue banked aortas from HIV- and HIV+ specimens were performed for CD163 (M2- macrophages) and caspase-1. (A) Representative images of CD163+ macrophages and caspase-1+ cells in aortic plaques. (B) The numbers of caspase-1+ and CD163+ macrophages positively correlated in plaque (r=0.6, P= 0.02). Correlation was determined using Spearman non-parametric analysis.

Discussion:

Experiments in Tg26^+/−/ApoE^−/− mice demonstrate for the first time that non-replicating HIV expression is sufficient to accelerate atherosclerosis. Extensive clinical evidence suggests that well controlled HIV infection with or without cART (eg. PWH or HIV+ elite controllers) accelerates atherogenesis^{1–7, 9, 44}. In these patients, viral loads are low or undetectable in the peripheral blood and the virus remains in a latent state in immune cells⁴⁵. These results suggest that even without viral replication, the persistence of HIV transcripts or viral proteins can continue to cause HIV comorbidities, including ASCVD. However, this notion is still challenged by few clinical studies showing that other risk factors such as smoking, but not chronic HIV infection in PWH, contribute to the development of atherosclerosis^10–12. Until now, no studies have demonstrated that chronic HIV infection alone without confounding factors (HIVAN, diabetes, etc.) is sufficient to accelerate atherosclerosis. Here, we show that Tg26^+/−/ApoE^−/− mice on B6 genetic background develop an accelerated atherosclerosis under both the atherogenic diet and normal chow conditions. There was no significant difference in the levels of cholesterol and triglycerides, and the body weight between Tg26^+/−/ApoE^−/− and ApoE^−/− mice on the atherogenic diet or normal chow, thus not influencing the difference in ASCVD observed. Additionally, we demonstrated that Tg26^+/−/ApoE^−/− mice had higher levels of HIV transcripts in hematopoietic tissues such as thymus, and spleen than that in the vascular wall (Figure IV in the online-only data supplement). Further, we document that the expression of HIV transcripts in hematopoietic cells promotes atherosclerosis in the Tg26^+/−/ApoE^−/− mouse model. This result indicates that the expression of HIV transcripts in hematopoietic cells containing T cells and monocytes are the main contributors to the accelerated atherosclerosis in Tg26^+/−/ApoE^−/− mice. Together, our experimental results further highlight the importance of chronic HIV in atherogenesis and also prompt the utilization of our model to further explore an inflammatory mechanism.

To explore the potential of caspase-1 activation in HIV ASCVD, we characterized our newly developed HIV atherogenic mouse model. We showed that the accelerated aortic atherogenesis in these mice occurs with concomitant increased activation of caspase-1 in circulating inflammatory monocytes and atherosclerotic vasculature as well as increased expression level of NLRP3, an upstream event of caspase-1 activation in immune cells. Tg26^+/−/ApoE^−/− mice also significantly higher levels of serum IL-1β and IL-18, a downstream event of caspase-1 activation compared to ApoE^−/− mice. In this paper, we explore the translational significance of this caspase-1 activation using a well-characterized patient cohort of PWH and tissue banked aortic plaques. Demonstrating the importance of caspase-1 in HIV ASCVD, in our clinical cohort, serum IL-18, a downstream product of caspase-1 activation, was elevated in PWH (n=153) compared to non-HIV-infected controls (n=67). Serum IL-18 correlated with total coronary plaque segments and the number of non-calcified inflammatory coronary plaque segments in all patients with plaque. PWH with low Framingham risk scores and subclinical ASCVD have an unique phenotype of increased inflammatory noncalcified plaques as seen by coronary CT angiogram (CCTA)^{4, 5}. Clinical biomarker studies by our group and others indicate that macrophage activation contributes to HIV ASCVD^{3, 5, 44, 46–48}. Our clinical data goes further with regard to macrophage activation by documenting for the first time that IL-18 levels in this cohort also significantly correlated to macrophage inflammatory markers including sCD163, which we have previously shown to correlate to aortic and coronary inflammation^{5, 7}.

We expanded on the relationship between caspase-1 activation and macrophage activation by analyzing autopsy aortic plaques. We document for the first time that the caspase-1+ cells in tissue banked aortic plaques positively correlate with plaque CD163+ macrophages, but not CD68 macrophages. However, the association of caspase-1+ cells and atherosclerotic plaque may not be unique to HIV; in our tissue study, we focused on samples (both HIV+ and HIV-) with extensive systemic atherosclerosis, and unsurprisingly, found no quantitative differences in the extent of plaque-associated inflammatory indices (Table II in the online-only data supplement). In prior vascular imaging studies, while cART-treated PWH had increased vascular wall inflammation in contrast to demographically and Framingham risk matched non-HIV-infected controls, they appeared similar to non-HIV-infected individuals with known atherosclerotic disease¹⁶. Although our prior study had reported elevated CD163 levels in HIV+ tissue banked aortic samples when contrasted to HIV- tissues, we did not examine the if there was a difference in the extent of atherosclerotic plaque in the samples analyzed⁴⁹. Here, we controlled for the extent of plaque analyzed within samples with high disease burden. Our findings are also similar to histologic analyses of arteries from the Circle of Willis, where intimal CD68+ populations correlated with the extent of intracranial atherosclerosis, but not HIV status⁵⁰. In Tg26^+/−ApoE^−/− mice there was a trend towards an increase in CD163+ macrophages in aortic root plaques, with no significant difference in CD68+ macrophages. This suggests that CD163+ macrophages may play a role in inflammatory aortic plaques in Tg26^+/−ApoE^−/− mice. Future studies will explore comfounding factors in HIV infection such as LPS and gut barrier breakdown that could further amplify this inflammatory plaque phenotype as seen in PWH. Future studies should examine the atherogenic role of caspase-1 in the monocyte activation in Tg26^+/−/ApoE^−/− mice with BM transplantation coupled with caspase-1 deficient mice. The relationship between caspase-1 activation and foam cell formation and cholesterol sequestration warrants further investigation. In a parallel study, using Tg26^+/−ApoE^−/− mice on an atherogenic diet, circulating TNF-α and IL-6, as well as the inflammatory indoleamine- 2,3-dioxygenase (IDO) pathway were elevated (Kearns et al. submitted). These data aid in the hypothesis that HIV ASCVD is an immune-driven inflammatory process, even though we did not see difference in macrophage content in the vasculature of Tg26^+/−ApoE^−/− mice. However, it is important to note that the source of the elevated serum cytokines in these studies not extensively examined, although the increased caspase-1 activity in monocytes is contributory, the contribution of caspase-1 activation in T-cells was not explored.

A recent study illustrated that among cART-treated PWH, caspase-1 expression in PBMCs was higher in immunologic non-responders (CD4 count <350 cells/ul) than in immune responders⁵¹. In our recent study, we demonstrated significantly greater active caspase-1 in lymph node and spleen of SIV-infected macaques compared to non-SIV-infected controls; however, cART intervention was not able to return to pre-infection levels. Although, cART was successful in acutely decreasing caspase-1 inflammation in the circulation, caspase-1 activation persisted in tissue compartments⁵². These data highlight that caspase-1 activation is highly relevant to immune dysfunction and activation even among cART-treated individuals, and cART is not sufficient to prevent ASCVD, and other therapeutics are needed.

The Canakinumab Anti-inflammatory Thrombosis Outcome Study (CANTOS) was a randomized double blinded study of non-HIV patients that demonstrated a lowered incidence of MI after treatment with a monoclonal antibody against IL-1β (Canakinumab)⁵³. CANTOS supported the inflammatory hypothesis of ASCVD. The inflammatory hypothesis may be stronger in PWH as there is a greater association with ASCVD and inflammatory markers, an abundance of inflammatory non-calcified plaques and arterial inflammation in HIV. A recent clinical pilot study in PWH treated with Canakinumab showed a substantial reduction in vascular inflammation, bone marrow metabolic activity, and peripheral inflammatory markers (sCD163, hsCRP and IL-6)⁵⁴. This study shows that the validity of a potent anti-inflammatory strategy to reduce atherosclerotic inflammation in HIV and the potential importance of targeting other components of the caspase-1 pathway. This animal model can now be used to determine the atherogenic role of caspase-1 pathway activation in HIV infection, and the exact upstream and downstream mechanisms of HIV ASCVD. Further studies can examine combination therapeutic strategies targeting macrophage activation and caspase-1 activity in HIV ASCVD.

Highlights:

1. Expression of HIV is sufficient to accelerate atherogenesis

2. Tg26^+/−/ApoE^−/− is a new mouse model for mechanistic studies of HIV atherogenesis

3. Activation of caspase-1 pathway may contribute to HIV-associated atherogenesis.

Non standard Abbreviations and Acronyms:

ASCVD

Atherosclerotic cardiovascular disease

ApoE^−/−

ApolipoproteinE deficient mice

BM

Bone marrow

CANTOS

CanakinumabAntiinflammatoryThrombosis Outcome Study

CRP

C-reactive protein

CVD

Cardiovascular disease

CXCL10

C-X-C motif chemokine 10

CCTA

Coronary CT angiogram

HIV

Human immunodeficiency virus type 1

HIV+

HIV-1-infected

HIV−

Non HIV-1-infected

HIVAN

HIV-associated nephropathy

IDO

indoleamine-2,3-dioxygenase

MCP-1

Monocyte chemoattractant protein 1

MHBB

Manhattan HIV Brain Bank

MI

Myocardial infarction

NLRP3

Nod-like receptor protein 3

NNTC

National NeuroAIDSTissue Consortium

PBMCs

Peripheral blood mononuclear cells

PWH

People with HIV

Tg26

HIV Tg26 transgenic mice

sCD163

Soluble CD163

sCD14

Soluble CD14

SIV

simian immunodeficiency virus