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Lack of ApoA-I in ApoEKO Mice Causes Skin Xanthomas, Worsening of Inflammation, and Increased Coronary Atherosclerosis in the Absence of Hyperlipidemia

Lack of ApoA-I in ApoEKO Mice Causes Skin Xanthomas, Worsening of Inflammation, and Increased... Arteriosclerosis, Thrombosis, and Vascular Biology BASIC SCIENCES Lack of ApoA-I in ApoEKO Mice Causes Skin Xanthomas, Worsening of Inflammation, and Increased Coronary Atherosclerosis in the Absence of Hyperlipidemia Marco Busnelli ,* Stefano Manzini ,* Alice Colombo , Elsa Franchi , Fabrizia Bonacina , Matteo Chiara , Francesca Arnaboldi , Elena Donetti , Federico Ambrogi , Roberto Oleari , Antonella Lettieri , David Horner , Eugenio Scanziani , Giuseppe Danilo Norata , Giulia Chiesa BACKGROUND: HDL (high-density lipoprotein) and its major protein component, apoA-I (apolipoprotein A-I), play a unique role in cholesterol homeostasis and immunity. ApoA-I deficiency in hyperlipidemic, atheroprone mice was shown to drive cholesterol accumulation and inflammatory cell activation/proliferation. The present study was aimed at investigating the impact of apoA-I deficiency on lipid deposition and local/systemic inflammation in normolipidemic conditions. METHODS: ApoE deficient mice, apoE/apoA-I double deficient (DKO) mice, DKO mice overexpressing human apoA-I, and C57Bl/6J control mice were fed normal laboratory diet until 30 weeks of age. Plasma lipids were quantified, atherosclerosis development at the aortic sinus and coronary arteries was measured, skin ultrastructure was evaluated by electron microscopy. Blood and lymphoid organs were characterized through histological, immunocytofluorimetric, and whole transcriptome analyses. RESULTS: DKO were characterized by almost complete HDL deficiency and by plasma total cholesterol levels comparable to control mice. Only DKO showed xanthoma formation and severe inflammation in the skin-draining lymph nodes, whose transcriptome analysis revealed a dramatic impairment in energy metabolism and fatty acid oxidation pathways. An increased presence of CD4 T effector memory cells was detected in blood, spleen, and skin-draining lymph nodes of DKO. A worsening of atherosclerosis at the aortic sinus and coronary arteries was also observed in DKO versus apoE deficient. Human apoA-I overexpression in the DKO background was able to rescue the skin phenotype and halt atherosclerosis development. CONCLUSIONS: HDL deficiency, in the absence of hyperlipidemia, is associated with severe alterations of skin morphology, aortic and coronary atherosclerosis, local and systemic inflammation. GRAPHIC ABSTRACT: A graphic abstract is available for this article. Key Words: atherosclerosis ◼ hyperlipidemias ◼ inflammation ◼ lymph nodes ◼ xanthomatosis he pivotal role of HDL (high-density lipoprotein) in reg- ability has been considered as the main explanation for ulating cell cholesterol homeostasis is very well recog- the inverse correlation between HDL-C (HDL-cholesterol) Tnized. HDL and its main protein component, apoA-I (apolipoprotein A-I), are essential in mediating the removal See cover image of excess cholesterol from all the cells of our body. This Correspondence to: Marco Busnelli, Stefano Manzini, and Giulia Chiesa, Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, via Balzaretti 9, 20133 Milano, Italy. Emails marco.busnelli@unimi.it, stefano.manzini@unimi.it, giulia.chiesa@unimi.it Supplemental Material is available at https://www.ahajournals.org/doi/suppl/10.1161/ATVBAHA.122.317790. *M. Busnelli and S. Manzini contributed equally. For Sources of Funding and Disclosures, see page 854. © 2022 The Authors. Arteriosclerosis, Thrombosis, and Vascular Biology is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial-NoDerivs License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited, the use is noncommercial, and no modifications or adaptations are made. Arterioscler Thromb Vasc Biol is available at www.ahajournals.org/journal/atvb Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 July 2022 839 Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice Nonstandard Abbreviations and Acronyms HIGHLIGHTS ABCA1 ATP-binding cassette transporter A1 • ApoA-I (apolipoprotein A-I)/apoE knockout mice are normocholesterolemic and HDL (high-density ABCG1 ATP-binding cassette transporter G1 lipoprotein) deficient. apoA-I apolipoprotein A-I • Skin xanthomas and severe atherosclerosis are the DKO apoE/apoA-I double deficient phenotypic features of apoA-I/apoE knockout mice. DKO/hA-I D KO mice overexpressing human • ApoA-I/apoE deletion causes severe inflammation apoA-I in the skin-draining lymph nodes. EKO apoE deficient • RNA-seq of lymph nodes shows alterations in lipid metabolism and fatty acid oxidation pathways. FITC fluorescein isothiocyanate • Overexpression of human apoA-I is able to rescue G-CSF granulocyte-colony stimulating factor the apoA-I/apoE knockout phenotype. HDL high-density lipoprotein HDL-C HDL-cholesterol IFN interferon influence the cellular activities involved in the innate and IL interleukin adaptive immune response through the fine regulation of IP-10 interferon gamma-induced protein 10 the cellular cholesterol content. Under reduced plasma KC/CXCL1 keratinocyte-derived cytokine HDL levels, peripheral blood mononuclear cells become LDLrKO low-density lipoprotein receptor enriched in cholesterol and display signs of cholesterol knockout imbalance, resulting in monocyte adhesion to the endo- LIX/CXCL5 lipopolysaccharide-inducible CXC thelium and recruitment in peripheral tissues, including chemokine the arterial wall. Additionally, it has been demonstrated MIG/CXCL9 monokine induced by gamma that the transgenic expression of human apoA-I inhib- interferon its foam cell formation in apoE deficient (EKO) mice by PPAR peroxisome proliferator-activated promoting macrophage cholesterol efflux via the ABCA1 receptor (ATP-binding cassette transporter A1) and ABCG1 rHDL reconstituted HDL (ATP-binding cassette transporter G1). In a mouse model of hyperlipidemia combined to T central memory T lymphocytes CM apoA-I deficiency—LDLrKO (low-density lipoprotein T effector memory T lymphocytes EM receptor knockout)/apoA-IKO mice fed an atherogenic T naive T lymphocytes diet for 12 weeks—an expansion and activation of T WT wild type cells in the skin-draining lymph nodes was observed, and T and B lymphocytes showed an increased cho- 18,19 lesteryl ester content compared with LDLrKO mice. plasma levels and atherosclerotic cardiovascular disease Interestingly, LDLrKO/apoA-IKO mice also showed resulting from prospective observational studies. In addi- skin abnormalities, which included dermal cholesterol tion, severe HDL deficiency, especially when genetically accumulation. It has never been investigated if these determined, is frequently associated in the clinic with ath- alterations may occur when apoA-I deficiency is not erosclerotic cardiovascular disease. associated to a hyperlipidemic condition. While Mendelian randomization studies failed to estab- In the present study, the impact of different levels of lish a clear causal role between HDL-C and myocardial apoA-I/HDL-C on lipid accumulation in the skin, local, 4,5 infarction or coronary heart disease, they supported a and systemic immunoinflammatory activation, as well causal role for HDL in immune regulation. More recently, as atherosclerosis progression at the aortic sinus and a thorough analysis of the relationship between HDL-C coronary arteries, was evaluated in genetically modified levels and all-cause mortality highlighted a U-shaped mice fed a normal laboratory diet. Specifically, the follow- association between HDL-C levels and increased mortal- ing mouse lines were investigated: wild-type (WT) mice ity risk and also autoimmune diseases for both extremes— as control, apoE and apoA-I double knockout mice with 7,8 very low and very high levels of HDL-C —thus proposing negligible HDL-C levels (apoE/apoA-I double deficient a key role for HDL in infection and sepsis. [DKO]); DKO knockout mice expressing human apoA- A direct link between low HDL-C levels and immune I (DKO/hA-I), with elevated apoA-I and HDL-C levels; disorders has been experimentally established. Several apoE knockout mice (EKO), which display halved apoA-I animal studies have demonstrated that, beyond the levels and low HDL-C levels compared with WT mice. effect on cholesterol efflux, HDL possesses a pleth- The results indicate that HDL deficiency in DKO 11 12 ora of additional anti-inflammatory, antioxidant, and mice, in the presence of plasma total cholesterol com- antithrombotic properties. In particular, HDL is able to parable to that of WT mice, is associated with xanthoma 840 July 2022 Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 BASIC SCIENCES - AL BASIC SCIENCES - AL Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice immunoturbidimetric quantification of human apoA-I. Purified formation, accelerated atherosclerosis, both at the aortic apoA-I was used as calibration standard. sinus and coronary arteries, and increased local and sys- temic inflammation. Detection of Circulating Cytokines/Chemokines By using a customized detection MILLIPLEX MAP Mouse METHODS Cytokine/Chemokine panel (Merck Millipore, Billerica, MA), the following 13 cytokines/chemokines were evaluated in The data that support the findings of this study are available plasma-EDTA samples: eotaxin, G-CSF (granulocyte-col- from the corresponding author upon reasonable request. ony stimulating factor), IL (interleukin)-4, IL-5, IL-6, IL-10, IL-12(p70), IL-15, IL-17, IP-10 (interferon gamma-induced Mice protein 10), KC/CXCL1 (keratinocyte-derived cytokine), Procedures involving animals and their care were conducted LIX/CXCL5 (lipopolysaccharide-inducible CXC chemokine), in accordance with institutional guidelines in compliance with and MIG/CXCL9 (monokine induced by gamma interferon). national (D.L. No. 26, March 4, 2014, G.U. No. 61 March 14, These analyses were outsourced to certified laboratories 2014) and international laws and policies (E EC Council Directive (Department of Experimental Evolutionary Biology, University 2010/63, September 22, 2010: Guide for the Care and Use of of Bologna and Labospace Srl). Laboratory Animals, United States National Research Council, 2011). The experimental protocol was approved by the Italian Tissue Harvesting Ministry of Health (Protocollo 2012/4). Mice were euthanized at 30 weeks of age under general C57Bl/6J (WT) and apoE knockout (EKO) male mice (strain anesthesia with 2% isoflurane and blood was removed by 002052) were purchased from Charles River Laboratories perfusion with 1× PBS. For histological analyses, hearts (Calco, Italy). ApoE/apoA-I double knockout male mice (DKO) were harvested, fixed in 10% formalin for 24 hours, then were previously generated in our lab. DKO/hA-I were obtained transferred into 1× PBS containing 30% sucrose (w/v) for by multiple crosses between DKO mice and hemizygous mice 24 hours at 4 °C before being embedded in OCT (optimal overexpressing human apoA-I. The human apoA-I transgene cutting temperature) compound (Sakura Finetek Europe B.V., in this model is almost exclusively expressed in the liver, and—at Alphen aan den Rijn, the Netherlands) and stored at −80 °C. very low amounts—in testis. Only male mice were enrolled in From a first set of mice, half of the spleen and the right the study, to prevent the possible impact of hormonal changes axillary lymph node were harvested and immediately processed of female mice on the results. for flow cytometry analysis; the other half of the spleen and the Primers specific for murine apoE (For 5′ -GCCTAGC left axillary lymph node were snap-frozen in liquid nitrogen for CGAGGGAGAGCCG-3′; Rev1 5′-TGTGACTTGGGAGCTCTG subsequent RNA-seq and molecular analyses. From a second CAGC-3′; Rev2 5-GCCGCCCCGACTGCATCT-3′), murine set of mice, half of the spleen and the right axillary lymph node apoA-I (For 5′-CCTTCTATCGCCTTCTTGACG-3′; Rev1 5′-GTT were immersion-fixed in 10% formalin for 24 hours, dehy- CATCTTGCTGCCATACG-3′; Rev2 5′-TCTGGTCTTCCTGACAG drated in a graded scale of ethanol, and paraffin embedded. GTAGG-3′), and human apoA-I (For 5′-GATCGAGTGAAGGACC The other half of the spleen and the contralateral axillary lymph TGGC-3′; Rev 5′-CCTCCTGCCACTTCTTCTGG-3′) were used node were immersion-fixed in 10% formalin for 24 hours, then to screen genotypes. transferred into 1× PBS containing 30% sucrose (w/v) for 24 Mice were maintained under standard laboratory conditions hours at 4 °C and embedded in OCT compound. Skin biopsies, (12-hour light cycle, temperature 22 °C, humidity 55%), with free excised from the thoracic region, were dissected in 2×2 mm access to normal laboratory diet (4RF21, Mucedola, Settimo fragments and processed for transmission electron microscopy Milanese, Italy) and tap water from weaning to 30 weeks of age. analysis as previously described. Additional skin biopsies were excised from the thoracic region, dissected in 5×5 mm Plasma Lipids fragments, immersion-fixed in 10% formalin for 24 hours, then transferred into 1× PBS containing 30% sucrose (w/v) for 24 After an overnight fast, mice were anaesthetized with 2% iso- hours at 4 °C and embedded in OCT compound. flurane (Merial Animal Health, Woking, United Kingdom). Blood was collected from the retroorbital plexus into tubes contain- ing 0.1% EDTA (ethylenediamine tetraacetic acid; w/v) and Histology centrifuged in a microcentrifuge for 10 minutes at 5900×g at 4 °C. Plasma total cholesterol was measured with an enzy- Heart matic method (CPA11 A01634, ABX Diagnostics, Montpellier, To determine the presence and length of atherosclerotic France). Cholesterol distribution among lipoproteins was ana- plaques in coronary arteries, serial transverse cryosections (7 lyzed by fast protein liquid chromatography as described. μm thick) of the entire heart from the apex to the ostia of HDL-C was measured after precipitation of apoB-con- the coronary arteries at the aortic root were cut. Lipid deposi- taining lipoproteins with polyethylene glycol (20%, w/v) in 0.2 tion in the coronary arteries was determined through Oil Red mol/L glycine (pH 10). Plasma non-HDL-C was measured as O (ORO; Sigma-Aldrich, MO) staining of all slides. To obtain the difference between total cholesterol and HDL-C. Plasma reliable data, the number of coronary lesions has been deter- human apoA-I concentration was determined by immunoturbi- mined only in coronary arteries with an orthogonal course with dimetric assays, using an antiserum specific for human apoA-I respect to the orientation of the sections. Plaque length was (LTA, Milan, Italy), as previously described. Coomassie staining determined by summing the sequential sections in which the was used to quantify plasma murine apoA-I and to confirm the presence of the ORO signal within arteries was observed, Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 July 2022 841 Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice taking into account the thickness of each section. Hematoxylin Data were acquired in FCS (flow cytometry standard) format, and eosin stained sections were used to detect atheroscle- processed and analyzed using the FCS Express V3 Research rotic plaque area at the aortic sinus, as previously described. edition (De Novo Software, Inc). CD45 was used for the identi- + − + Both coronary arteries and aortic sinus histology were per- fication of total leukocytes. CD4 CD44 CD62L cells are indi- + + − formed in accordance with American Heart Association cated as T naive (T ), CD4 CD44 CD62L cells are indicated as 29 + + + recommendations. Macrophages and T lymphocytes were T effector memory (T ), and CD4 CD44 CD62L cells are indi- EM + + lo detected using an anti-Mac2 antibody (CL8942, Cedarlane, cated as T central memory (T ). CD11b CD115 Ly6C cells are CM + + hi Ontario, Canada; RRID:AB_10060258) and an anti-CD3 indicated as patrolling monocytes and CD11b CD115 Ly6C as hi + lo antibody (MAB4841, R&D Systems, MN; RRID:AB_358426), inflammatory monocytes. CD11c CD11b B220 cells are indi- lo − hi respectively. Detection was performed using an ImmPRESS cated as myeloid and CD11c CD11b B220 as plasmacytoid + + reagent kit (Vector Laboratories, Peterborough, United dendritic cells. CD19 CD5 cells are indicated as B cells and 1a + − Kingdom). Diaminobenzidine was used as the chromogen CD19 CD5 as B . 1b (ImmPACT DAB Substrate, Peroxidase HRP SK-4105, Vector In Vitro T-Cell Proliferation and Cytokine Production Laboratories), and the sections were counterstained with Discoidal reconstituted HDL (rHDL) containing human apoA-I Gill’s hematoxylin (Bio-Optica, Milan, Italy). and 1-palmitoyl-2-oleoyl phosphatidylcholine were prepared by Axillary Lymph Node, Spleen, and Skin cholate dialysis method, as described. Lymphocyte suspen- Serial sections (5 μm thick) of the axillary lymph node were sion from EKO lymph nodes was incubated with 5 μmol/L obtained and stained with hematoxylin and eosin. In addition, of carboxyfluorescein succinimidyl ester (Merck, Catalog no. in the axillary lymph node, macrophages, B and T lympho- 21888) for 10 minutes at room temperature in the dark, diluted cytes were detected using an anti-Iba-1 (019-19741, WAKO, 10 times in PBS/FBS 2%/2 mmol/L EDTA, washed 3 times United States; RRID AB_839504), an anti-CD45R/B220 and resuspended in TexMACS medium. A total of 0.2×10 (BD Pharmingen, United States; RRID AB_396673) and an cells were plated in 96-well plate (U-bottom wells), coated with anti-CD3 epsilon (Sc-1127, Santa Cruz Biotechnology, United 0.5 μg/mL anti-CD3 (Biolegend, Catalog no. 152302; RRID States; RRID AB_631128), respectively. Cryosections (7 μm AB_2650621) and 2.5 μg/mL anti-CD28 (Biolegend, Catalog thick) from OCT embedded organs were stained with ORO no. 102102; RRID AB_312867), in 200 μL of medium con- to detect neutral lipid accumulation. Lipid deposition was taining 20 U/mL IL-2 (Preprotec, Catalog no. 200-02), with or measured as the percent of ORO positive area over the total without rHDL (10, 25, 50, and 100 μg/mL). Cells were incu- area. bated for 4 days at 37 °C with 5% CO . The Aperio ScanScope GL Slide Scanner (Aperio For cytokine production, at the end of the incubation, cells Technologies, CA) was used to acquire digital images that were were pulsed with 0.1 μg/mL PMA (phorbol-12-myristate- subsequently processed with the ImageScope software. Two 13-acetate; Merk, Catalog no. P1585) and 1 μg/mL ionomycin operators blinded to mouse genotypes quantified all histologi- (Invitrogen, Catalog no. I24222) for 4 hours at 37 °C with 5% cal parameters. CO in the presence of Brefeldin A (1:1000, BD Bioscience, Skin semithin sections, 2 μm thick, were stained with tolu- Catalog no. 555028). Cytokine analysis was performed by flow idine blue and observed with a Nikon Eclipse E600 microscope cytometry following the instructions from the fixation/permea- equipped with a Nikon digital camera DXM1200 (Nikon, Tokyo, bilization kit (BD Bioscience, Catalog no. 555028). Japan). Ultrathin sections were obtained with an Ultracut ultra- T cells were analyzed for the expression of extracellular microtome (Reichert Ultracut R-Ultramicrotome, Leika, Wien, markers, intracellular cytokines, proliferation, cellular viability, Austria) and stained with uranyl acetate/lead citrate before and lipid abundance by flow cytometry. observation by a Jeol CX100 transmission electron microscope Immunophenotyping was performed on resting and (Jeol, Tokyo, Japan). activated lymphocytes. For live and dead cells discrimina- tion, cell suspension was stained with 100 μL of a 1:1000 dilution of the LIVE/DEAD Fixable Aqua Dead Cell Stain Flow Cytometry Analysis on Blood and Kit (Invitrogen, Catalog no. L34965) in PBS at 4 °C for 30 Lymphoid Organs minutes. Single cell suspensions were then incubated with Analyses were performed with a Calibur Flow Cytometer (BD antibody mixtures at 4 °C for 30 minutes and then washed Biosciences, Milan, Italy) with 2 lasers was used. The panels with PBS/FBS 2%/EDTA 2 mmol/L. For intracellular stain- consisted of the subsequent cellular surface markers: CD4 ing, first cells were fixed and permeabilized according to (PE YTS 191.1.2, Immunotools, Friesoythe, Germany), CD44 manufacturer instructions and then incubated with specific (fluorescein isothiocyanate [FITC], IM7, BD Pharmingen; RRID antibodies at 4 °C for 30 minutes, washed and analyzed as AB _2076224), CD45 (FITC, 30-F11, BD Pharmingen; RRID described. Antibodies are listed in the Major Resources AB_394610), CD62L (antigen-presenting cell, mMEL-14, BD Table in the Supplemental Material. Pharmingen; RRID AB_10895379), Ly-6C (FITC AL-21, BD For detection of lipid rafts, cells were incubated with 2 μg/ Pharmingen; RRID AB_394628), CD115 (antigen-presenting mL of Cholera Toxin-FITC (Merck, Catalog no. C1655) con- cell AF598, eBioscience; RRID AB_2314130), CD11b (PE jugated for 30 minutes at 4 °C or with 25 μg/mL 100 filipin M1/70.15, Immunotools), CD11c (antigen-presenting cell HL3, (Merck, Catalog no. F9765) for 30 minutes at 30 °C, washed BD Pharmingen RRID AB_398460), B220 (PerCP Cy5,5 and analyzed. RA3-6B2, BD Pharmingen; RRID AB_2034009), CD19 (FITC Samples were acquired with BD LSRII Fortessa and ana- 1D3, BD Pharmingen RRID AB_395049), and CD5 (antigen- lyzed with Novoexpress 1.3.3 software (Agilent Technologies presenting cell 53-7.3, BD Pharmingen; RRID: AB_394562). Inc). Gating strategy was reported in Figure S16. 842 July 2022 Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 BASIC SCIENCES - AL BASIC SCIENCES - AL Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice by Dunn post hoc test, according to the check of normality RNA Extraction of residuals (Shapiro-Wilk test) and homoscedasticity of data Total RNA was isolated from mouse tissues and extracted as (Levene test). Statistical analyses are detailed in each figure or previously described. RNA was quantified and checked, and table caption, specifying for which parameters the nonparamet- 1 μg RNA was retrotranscribed to cDNA. Possible gDNA con- ric procedure was applied. tamination was ruled out as described. Numerical data is shown as box plots, where the upper and lower ends of the boxes indicate the 25th and 75th percen- RNA-Seq Analysis tiles, respectively. The length of the box shows the interquartile range within which 50% of the values are located. The solid For RNA-seq analysis, the quality of the mRNA was tested using gray lines denote the median. Precise adjusted P values are the Agilent 2100 Bioanalyzer (Agilent Technologies, CA), and shown in Table S1. In most figures, approximate P values are only libraries with RNA integrity number ≥7 were included. RNA provided to improve readability (ex. P<0.05, P<0.01). samples were processed using the RNA-seq Sample Prep kit from Illumina (Illumina, Inc, CA). Eight to 9 tagged libraries were loaded on one lane of an Illumina flowcell, and clusters were created using the Illumina Cluster Station (Illumina, Inc, CA). RESULTS Clusters were sequenced on a Genome Analyzer IIx (Illumina, ApoA-I Deficiency in EKO Mice Leads to Inc, CA) to produce 50nt single-reads. Data sets can be accessed at NCBI (Gene Expression Omnibus) GSE202237. Normocholesterolemia Associated to Negligible HDL Levels Bioinformatics Analysis At the end of the experimental period, body weight was comparable in WT, DKO, and DKO/hA-I mice and signifi- Preprocessing of Reads cantly higher in EKO mice (Figure S1A). Raw sequence reads were trimmed using Trimmomatic soft- 35 Plasma total cholesterol concentration in DKO and ware and applying the leading, trailing, and slidingwindows WT mice was comparable, and ≈3-fold lower than the operations, with the following quality score cutoffs, respectively: concentration observed in EKO mice and DKO/hA-I Qs ≥15, Qs ≥10, and average Qs ≥15. mice (Figure 1A). Differential Gene Expression As expected, apoA-I was absent in plasma of DKO Transcript abundance estimation and differential expression mice. ApoA-I concentration in EKO mice was approxi- analyses were performed using the standard Bowtie-Tophat- mately half of that measured in WT. The overexpression of Cuffdiff pipeline on the Refseq annotation of the mm10 mouse the human APOA1 transgene strongly increased apoA-I reference genome assembly, as obtained from the following: plasma levels of DKO/hA-I (Figure 1B). HDL-C concen- https://ftp.ncbi.nlm.nih.gov/refseq/ M_musculus/annota- tration was about 10-fold lower in DKO and 3-fold lower tion_releases/current/. Transcriptome assembly functions of in EKO than in WT mice, whereas DKO/hA-I mice showed cufflinks were deactivated and only established transcripts annotations were used. higher HDL-C levels than those measured in EKO and Differential expression analyses were executed by per- DKO (Figure 1C). Non-HDL-C levels were significantly forming direct pairwise comparisons between the conditions different among all genotypes (Table S1). Specifically, under study. A false discovery rate cutoff value of 0.05 was plasma non-HDL-C concentration was 12.2±3.7 mg/dL applied for the identification of differentially expressed genes. in WT, 316.3±23.0 mg/dL in EKO, 247.3±31.2 mg/dL in Functional enrichment analyses, Kyoto Encyclopedia of Genes DKO/hA-I, and 106.2±16.4 mg/dL in DKO. and Genome (KEGG) and reactome pathways, were performed Fast protein liquid chromatography analysis confirmed with reString (version 0.1.18). a dramatic cholesterol accumulation in the VLDL (very- low-density lipoprotein) and LDL fractions of EKO mice Statistical Analyses and a greatly reduced HDL-C peak compared with that Sample size for histological and imunohistochemical analyses was of WT mice (Figure 1D). In DKO mice, the HDL-C peak calculated based on previously published data on atherosclerosis was almost absent and cholesterol in the VLDL and LDL 38,39 in EKO mice and EKO mice overexpressing human apoA-I. fractions was much lower than in EKO mice. DKO/hA-I Plaque size in EKO mice was expected to be about 5 times larger mice displayed an HDL-C peak close to that of WT and a than in DKO/hA-I mice. A lack of atherosclerosis development was relevant cholesterol accumulation in the VLDL and LDL expected in WT mice, and the development of plaques in DKO mice fractions. was hypothesized 2 times larger than in EKO mice. Based on these premises, a sample size of 6 per experimental group achieves 92% power to detect the hypothesized differences among the means HDL Deficiency Worsens Atherosclerotic using an F test at 5% significance level. A higher number of sam- ples was cautiously used for plasma lipid measurements and for Plaque Development at the Aortic Sinus and in most of the analyses on lymphocyte class distribution. Coronary Arteries Analyses were performed with R and GraphPad Prism ver- The apoA-I deficiency in the apoEKO background (DKO) sion 9.3.0. Significant differences were determined by ANOVA caused an increase of plaque extent at the aortic sinus followed by Tukey post hoc test or by Kruskal-Wallis followed Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 July 2022 843 Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice Figure 1. Plasma cholesterol and apoA-I (apolipoprotein A-I) levels. Plasma total cholesterol (A), apoA-I (B), HDL-C (high-density lipoprotein cholesterol (C) concentrations, and cholesterol distribution among lipoproteins by fast protein liquid chromatography in the 4 genotypes (n=8). Statistically significant differences were determined by ANOVA followed by Tukey post hoc. ***P<0.001. Precise adjusted P values are shown in Table S1. compared with that measured in EKO mice. Conversely, DKO/hA-I mice (Figure S3E). However, no differences apoA-I overexpression in DKO/hA-I mice dramatically were observed when CD3 cell counts were normalized reduced atherosclerotic plaque size at the aortic sinus to plaque area (Figure S3F). In addition, the presence of compared with both DKO and EKO mice. As expected, CD3 cells was evaluated in the myocardial tissue imme- no plaques developed at the aortic sinus of WT mice diately surrounding the aortic sinus: the count of CD3 (Figure 2A through 2D and 2K). cells was significantly increased in EKO and DKO mice The area occupied by macrophages in the plaques of compared with that in DKO/hA-I mice (Figure S3G). DKO/hA-I mice was about ten times lower than in EKO A feature observed only in DKO and EKO mice was and DKO mice, since DKO/hA-I developed smaller plaques the presence of lesions at the left atrioventricular valves (Figure S2C and S2E). Macrophage area expressed as (Figure 2E and 2F). percentage of total area was instead comparable among The presence of atherosclerotic plaques within the DKO/hA-I, DKO, and EKO mice (Figure S2F). left and right coronary arteries arising from the aortic An increased count of CD3 T lymphocytes was sinus was detected in 100% of DKO and 30% of EKO observed in the atherosclerotic plaques of DKO versus mice (Figure 2G through 2J). The average number of 844 July 2022 Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 BASIC SCIENCES - AL BASIC SCIENCES - AL Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice Figure 2. Atherosclerosis development at the aortic sinus and coronary arteries. Representative hematoxylin and eosin (H&E) stained pictures of atherosclerosis development at the aortic sinus of wild type (WT; A), apoE/ apoA-I double deficient (DKO) mice overexpressing human apoA-I (DKO/hA-I; B), apoE deficient (EKO; C) and DKO mice (D). Representative oil red O (ORO) stained pictures of plaque development at the left atrioventricular valves of EKO (E) and DKO mice (F), and in the right coronary artery of EKO (G) and DKO mice (H). Magnifications of the same coronary plaques are shown (I and J). Atherosclerotic plaque quantification at the aortic sinus (K), number of plaques in coronary arteries and coronary branches (L), and average plaque length in coronary arteries (M) are also shown (n=6). Bar length: 250 μm. Statistically significant differences were determined by ANOVA followed by Tukey post hoc. *P<0.05; **P<0.01; and ***P<0.001. Precise adjusted P values are shown in Table S1. Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 July 2022 845 Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice atherosclerotic plaques in left and right ventricular coro- WT, EKO, and DKO/hA-I mice, whereas in DKO mice a nary branches was more than doubled in DKO mice com- dramatic lipid accumulation was detected (Figure 4F). pared with EKO mice (Figure 2L and Figure S4). The average length of coronary plaques was comparable in HDL Deficiency Increases the Amount of CD4 DKO and EKO mice (Figure 2M). No plaques developed T in Blood, Skin-Draining Lymph Nodes and in the coronary arteries of DKO/hA-I and WT mice (Fig- EM Spleen ure 2L and 2M). Total white blood cells (CD45 ) were significantly higher in DKO versus WT mice. Comparable CD45 counts The Pathological Skin Phenotype Determined were observed in the spleen of the 4 genotypes. CD45 by HDL Deficiency Is Rescued by hA-I cells were increased in the axillary lymph node of DKO Overexpression mice versus all the other groups. Total CD4 counts were In accordance with our previous findings, DKO mice not affected by genotype in blood, spleen, and axillary displayed hair loss and a pale color of the skin. These lymph node (Figure S8). features were not observed in any other genotype. The analysis of CD4 T-cell subsets (T , T , and T ) N EM CM Structural and ultrastructural features of the skin were revealed that, in peripheral blood and spleen, DKO mice investigated in semithin and in ultrathin sections stained had an increased percentage of T and a concomitant EM with toluidine blue, respectively. By light microscopy, reduced percentage of T lymphocytes compared with all the dermal and the epidermal compartment exhibited a the other genotypes (Figure 5A and 5B). In the axillary normal organization in WT, in EKO, and DKO/hA-I mice lymph node, DKO mice showed a significantly increased (Figure 3A through 3C). In DKO mice, the subpapillary percentage of T compared with the other genotypes EM dermis appeared disarranged and filled with cholesterol and a tendency towards a reduced percentage of T , sig- clefts (Figure 3D and 3E) and foam cells were inter- nificant only versus WT mice (Figure 5C). The percent- spersed in the reticular dermis (Figure 3D and 3E). Infil- age of T cells resulted always unaffected by genotype. CM trated lymphocytes were present in the dermis of DKO No differences were found among the 4 genotypes hi mice (Figure 3F). In addition, neutral lipid deposition was in monocyte subsets distribution (inflammatory Ly6C lo increased in the thickened dermis of DKO mice, com- and patrolling Ly6C ) in peripheral blood. B lympho- 1a pared with the other genotypes (Figure S5). cytes were increase in EKO mice compared with DKO/ hA-I and DKO mice, both in spleen and in axillary lymph node (Figure S9). HDL Deficiency Results in Deep Alterations in The concentration of plasma cytokines did not show Skin-Draining Lymph Nodes differences among genotypes (Table). The size and weight of the spleen in WT, DKO/hA-I, and DKO were comparable; on the contrary, spleens from + + rHDL Influence CD4 and CD8 Cells EKO mice had an increased weight compared with DKO/ Proliferation In Vitro hA-I and DKO mice (Figure 4A). Histological analysis did not reveal differences among groups. No lipid deposition To investigate the potential role of apoA-I/HDL in was observed in any genotype (Figure S6). When spleen modulating T lymphocyte proliferation/activation, lym- weight was normalized to body weight, no significant dif- phocytes were isolated from lymph nodes of EKO mice ferences were observed (Figure S1B). to test cell proliferation and cytokine release by poly- DKO mice were characterized by increased size and clonal stimulation (anti-CD3/28 antibodies plus IL-2) weight of the skin-draining axillary lymph node, compared in the presence of increasing concentration of rHDL with the other genotypes (Figure 4B), a finding further (10, 25, 50, and 100 μg/mL) for 4 days. rHDL reduced + + confirmed by the normalization of lymph node weight to both CD4 and CD8 T-cell proliferation in a concen- body weight (Figure S1C). At the histological level, 3 main tration dependent manner (Figure S13A and S13B). features were observed only in DKO mice: (1) the accu- Changes in cytokine production were evaluated in acti- mulation of large macrophages, often characterized by a vated T cells following stimulation with PMA/ionomycin. foamy cytoplasm, as single cell or in small groups in the IFN (Interferon)-γ and IL-17 production (marking T-cell cortex, surrounded by lymphoid cells (Figure 4C and Fig- polarization toward Th1 and Th17, respectively ) was + + ure S7); (2) the presence of granulomatous reactions in decreased in CD4 and CD8 T cells following incuba- the inner cortex and the medulla, localized around a large tion with rHDL (Figure S13C through S13F). A similar number of multinucleated macrophages and cholesterol trend was observed for IL-15R and IL-4, despite a sta- crystals (Figure 4D and Figure S7); and (3) a dilation tistical significance was appreciated only for CD8 T of subcapsular, corticomedullary, and medullary sinuses cells (Figure S14). (Figure 4E). Moreover, a moderate deposition of neutral To further investigate the ability of HDL to affect cell lipids within the lymph node parenchyma was found in membrane lipids, lipid rafts abundance and cholesterol 846 July 2022 Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 BASIC SCIENCES - AL BASIC SCIENCES - AL Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice Figure 3. Light and transmission electron microscopy of mouse skin. Araldite semithin sections after toluidine blue staining of skin from wild type (WT; A), apoE/apoA-I double deficient (DKO) mice overexpressing human apoA-I (DKO/hA-I; B), apoE deficient (EKO; C), and DKO mice (D). Araldite ultrathin sections of DKO skin (E and F): cholesterol clefts (arrows) and foam cells (asterisks) accumulation in the dermis are shown in D and E; lymphocytes in the dermis of DKO are shown in F. Bars: A–D: 60 μm; E: 2 μm; F: 1 μm. + + content in CD4 and CD8 T-cell membrane were evalu- in the spleen of DKO and DKO/hA-I mice (Figure 6A ated by Cholera Toxin-subunit B staining and filipin through 6D). staining. HDL significantly reduced, in a concentration A total of 784 genes were identified as differentially dependent manner, both lipid raft abundance and cho- expressed genes in the axillary lymph node: 513 were + + lesterol content in CD4 and CD8 T cells (Figure S15). upregulated, whereas 271 were downregulated in DKO compared with DKO/hA-I mice (Figure 6A). In DKO mice, functional enrichment analysis of dif- The Transcriptional Expression Profile of Skin- ferentially expressed genes indicated an increased acti- vation of the immune system, with particular reference Draining Lymph Nodes of HDL-Deficient Mice to the phagocytic activity (mmu04610 Complement Reveals Increased Activation of the Immune and coagulation cascades, mmu04145 Phagosome, System and an Unbalanced Expression of mmu04142 Lysosome, mmu04060 cytokine-cytokine Genes Involved in Energy Metabolism receptor interaction; Figure 7A). The reactome associ- A comparison of the transcriptional expression pro- ated to the genes with an increased expression in DKO files was performed in the axillary lymph nodes and mice refined this observation: the activation of both the Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 July 2022 847 Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice Figure 4. Weight of spleen and axillary lymph node in the 4 genotypes and histology of apoE/apoA-I double deficient (DKO) axillary lymph node. Gross anatomic appearance and weights (n=7–13) of the spleen (A) and the axillary lymph node (B). Representative photomicrographs of axillary lymph node from DKO mice. Hematoxylin and eosin (H&E) images show large, foamy macrophages in the cortex, surrounded by lymphoid cells (C), presence of granulomatous reactions around cholesterol crystals in the inner cortex and the medulla (D), dilation of subcapsular, corticomedullary and medullary sinuses (a detail of medullary sinuses is shown in E). Oil Red O (O.R.O.) staining (n=6) of axillary lymph node cryosections (F). Statistically significant differences were determined in A and B by Kruskal-Wallis followed by Dunn post hoc test and in F by ANOVA followed by Tukey post hoc. CC indicates cholesterol crystals; DS, dilated sinuses; EKO, apoE deficient; FM, foamy macrophages; GR, granulomatous reactions; hA-I, human apoA-I; LC, lymphoid cells; and WT, wild type. *P<0.05; **P<0.01; and ***P<0.001. Bar length =100 μm. Precise adjusted P values are shown in Table S1. 848 July 2022 Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 BASIC SCIENCES - AL BASIC SCIENCES - AL Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice Figure 5. CD4 T-cell subsets in blood, spleen, and axillary lymph node. Flow cytofluorimetric evaluation of the percentage composition of CD4 T-cell subsets is shown in blood (A–C), spleen (D–F), and axillary lymph node (G–I; n=7–13). Statistically significant differences were determined by ANOVA followed by Tukey post hoc test, except for (C) and (I) which were analyzed by Kruskal-Wallis followed by Dunn post hoc test. *P<0.05; **P<0.01; and ***P<0.001. Precise adjusted P values are shown in Table S1. DKO indicates apoE/apoA-I double deficient; DKO/hA-I, DKO mice overexpressing human apoA-I; EKO, apoE deficient; and WT, wild type. innate (MMU-168249) and adaptive immune system nonlymphoid cells (MMU-198933; Figure 7B). In addi- (MMU-1280218) was accompanied by an increased tion, extracellular matrix organization (MMU-1474244) immunoregulatory interaction between lymphoid and was modulated as well (Figure 7B). Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 July 2022 849 Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice Table. Plasma Cytokine Concentrations in the 4 Genotypes (pg/mL) WT EKO DKO/hA-I DKO Eotaxin 326.4±65.2 639.2±164 591.9±313.2 911.4±535.5 G-CSF 825±697.2 1891.1±1280.8 1663.6±1161.7 898.4±422.3 IL-4 109.8±114.3 44.5±25.3 57.1±26.3 33±29.5 IL-5 73.9±47.7 74.8±68.2 33.8±22.8 42.5±36.5 IL-6 36.6±37.1 48.5±34.6 56.9±81 124.4±168.8 IL-10 269.1±268.6 79.2±21.7 154.8±84.2 92.2±70.1 IL-12 (p70) 285.4±227.9 511.2±468.7 197.4±177.4 238.5±129.2 IL-15 91.2±5.7 96.3±6.6 88.4±4.7 92.5±4.2 IL-17 63.1±65.4 29.4±20.2 102.7±133.9 86.8±131.9 IP-10 96.5±12.2 153.7±91.5 192.6±79.8 147.2±45.7 CXCL1/KC 90.5±120.1 108.7±117.9 124.5±88 159.1±152.9 CXCL5/LIX 6376.7±1575.7 5416±4646.5 9177.2±7529.3 9411.7±3366.3 CXCL9/MIG 121.6±61.7 102.9±25.2 123.2±11.7 137.0±43.4 n=5–6 mice per group. Data are expressed as mean±SD. Statistically significant differences were determined by ANOVA followed by Tukey post hoc or Kruskal-Wallis followed by Dunn post hoc (IL-17). Precise adjusted P values are shown in Table S1. DKO indicates apoE/apoA-I double deficient; EKO, apoE deficient; G-CSF, granulocyte-colony stimulating factor; hA-I, human apoA-I; IL, interleukin; IP-10, interferon gamma-induced protein 10; KC/CXCL1, keratinocyte-derived cytokine; LIX/CXCL5, lipopolysaccharide-inducible CXC chemokine; MIG/CXCL9, monokine induced by gamma interferon; and WT, wild type. Interestingly, macrophage-specific markers (Fig- with a massive dermal accumulation of cholesterol ure 6E and Figure S10), particularly those associated clefts, foam cells, and T lymphocytes. The skin features with the differentiation of macrophages to lipid-laden of this mouse model, partially described previously foam cells, displayed and increased expression in DKO were further investigated with the inclusion of DKO/ mice, the group characterized by exacerbated athero- hA-I. The presence of xanthomas in the skin of DKO sclerosis (Figure 6F and Figure S11). mice was paralleled by partial hair loss, a thickened der- In the axillary lymph node of DKO mice a reduced mal layer filled with inflammatory cells and increased expression of several genes involved in cell metabolism neutral lipid deposition. Interestingly, apoA-I expression, was observed (Figure 6G and Figure S12). Those genes both at low (EKO mice) and high (DKO/hA-I mice) lev- were largely involved in pathways related to energy els, was able to reverse these skin abnormalities. The metabolism (mmu03320 PPAR [peroxisome prolifera- skin and plasma lipid phenotype observed in DKO mice tor-activated receptor] signaling pathway, mmu00640 strongly resembles the condition of human subjects propanoate metabolism, mmu01212 fatty acid metabo- affected by genetic apoA-I deficiency, who are similarly lism, mmu00620 pyruvate metabolism, and mmu00020 characterized by xanthoma formation in the absence of citrate cycle; Figure 7C). Similarly, the reactome indicated elevated plasma lipid levels. in DKO mice a reduction in metabolism of lipids (MMU- DKO/hA-I and EKO mice were both characterized 556833), fatty acid metabolism (MMU-8978868), by plasma total and non-HDL-C levels significantly pyruvate metabolism and TCA (tricarboxylic acid) cycle higher than DKO mice. Despite that, atherosclerotic (MMU-71406), TCA cycle and respiratory electron trans- plaque development at the aortic sinus of DKO mice port (MMU-1428517), and mitochondrial fatty acid beta- was about twice as high as in EKO. Of note, individuals oxidation (MMU-77289; Figure 7D). with apoA-I/HDL deficiency in the absence of other In the spleen, the comparison between the gene plasma lipid alterations, generally develop premature expression profiles of DKO and DKO/hA-I mice did not coronary heart disease. highlight relevant differences except for a handful of In DKO/hA-I, plaque area was dramatically lower genes. Of those, 7 were upregulated (Apol11b, Cyr61, compared with DKO and EKO mice. A reduced ath- Dnaja1, Dnajb1, Hspa1a, Hspa1b, and Hsph1) and one erosclerosis development by apoA-I overexpression was downregulated in DKO mice (Rpl31-ps12; Fig- has been previously described in hyperlipidemic EKO 21,38,44 ure 6B and 6C). and LDLrKO mice and atherosclerosis worsen- ing by apoA-I deletion has been shown in LDLrKO mice fed a high-fat/high-cholesterol diet, where DISCUSSION cholesterolemia was not affected by apoA-I abla- DKO mice, almost completely devoid of HDL and with tion. Interestingly, in our study, DKO mice showed plasma total cholesterol levels comparable to those of a strong exacerbation of atherosclerosis develop- WT mice, showed deep alterations in the skin structure, ment versus EKO, in spite of much lower non-HDL-C 850 July 2022 Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 BASIC SCIENCES - AL BASIC SCIENCES - AL Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice Figure 6. Transcriptional expression profiles of axillary lymph node and spleen of apoE/apoA-I double deficient (DKO) and DKO mice overexpressing human apoA-I (DKO/hA-I) mice. The average of normalized counts per gene between DKO/hA-I (x axis) and DKO (y axis) were compared in axillary lymph node (A) and spleen (B). The values spanned several orders of magnitude and were log -transformed. The log fold change (DKO vs DKO/ 2 2 hA-I) is shown for all transcripts in lymph nodes (x axis) and in the spleen (y axis; C). Values greater than 0 denote genes with higher expression in DKO; values lower than 0 denote genes with higher expression in DKO/hA-I. Heatmap with the Z-scored average expression for all genes in the secondary lymphoid organs (D). Heatmaps with the gene expression signature for macrophages (E) and foam cells (F). Heatmap of metabolic genes with significantly increased expression in the axillary lymph node of DKO/hA-I mice (G). n=3 for DKO and DKO/hA-I. DE indicates differentially expressed genes. Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 July 2022 851 Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice Figure 7. Functional enrichment of differentially expressed genes in the axillary lymph nodes of apoE/apoA-I double deficient (DKO) and DKO mice overexpressing human apoA-I (DKO/hA-I) mice. The most relevant KEGG and reactome pathways enriched in DKO (A and B, respectively) and in DKO/hA-I (C and D, respectively) are reported. PPAR indicates peroxisome proliferator-activated receptor. levels. Altogether, apoA-I, in our experimental setting, it was inversely related to apoA-I plasma concentra- appeared to be a major determinant of atherosclero- tions or to HDL-C levels. sis development, regardless of the lipidemic status. Observational studies in humans have established an In fact, atherosclerosis extent in the 3 atheroprone inverse correlation between HDL-C levels and cardio- genotypes was independent of non-H DL- C levels and vascular disease. However, a strong elevation of HDL-C 852 July 2022 Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 BASIC SCIENCES - AL BASIC SCIENCES - AL Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice levels caused by genetic variants or pharmacological experimental models with dysfunctional lymphatic drain- treatments have been associated to a higher cardiovas- age. In this regard, it has been demonstrated that athero- 8,45,46 cular mortality. A similar condition has been found sclerosis regression secondary to a reduction of plasma in mice double knockout for SR-B1 and apoE, charac- cholesterol levels, in ezetimibe-treated EKO mice, is terized by a considerable elevation of HDL-C levels and obtained only in the presence of an efficient aortic lym- by a premature and severe atherosclerosis. These evi- phatic drainage that prevents cholesterol and immune dences clearly indicate that plasma HDL-C concentra- cell accumulation in the aortic adventitia. tion may not always reflect HDL functionality, especially There is considerable evidence from studies in mice at very high levels. To reconcile these observations with demonstrating that apoA-I possesses anti-inflammatory 54 15 our results, we may hypothesize that HDL in both EKO properties and immunoregulatory functions possibly and DKO/hA-I mice are functional and associate to ath- affecting atherosclerosis development. For this reason, eroprotection in a dose-dependent manner. we have characterized the presence of T , T , or T EM CM N In addition to the aortic sinus, EKO mice develop ath- CD4 T lymphocytes in the axillary skin-draining lymph erosclerosis in the aorta and in brachiocephalic arter- node, spleen and blood. The percentage of T was sig- EM 48,49 ies, whereas plaques in coronary arteries are less nificantly higher in DKO mice in the 3 districts analyzed. consistently observed. Genetically modified mouse This increase was counterbalanced by a reduced per- models of coronary atherosclerosis have been devel- centage of T . The percentage of T resulted always N CM oped over the years, but they require high-fat/high- unaffected by genotype. Of note, alterations in the dis- 51,52 cholesterol diets to exhibit coronary lesions. The only tribution of T lymphocytes were not observed in the model developing coronary atherosclerosis without any other genotypes, including EKO mice. That suggests that dietary challenge is the EKO/SR-B1KO mouse, charac- even low levels of apoA-I are sufficient to switch off the terized by severe hypercholesterolemia and a high rate inflammation and T lymphocyte activation consequent to of mortality by 6 weeks of age. In the present study, apoA-I deficiency. normal laboratory diet-fed DKO mice showed an exag- The present study was not designed to evaluate the gerated plaque development also in the coronary arter- minimal effective level of apoA-I required to correct the ies, with several lesions observed in coronary branches. phenotype driven by apoA-I/HDL deficiency. However, 18,56 This condition did not associate to an increased mortality based on previous evidences, it can be hypothesized rate within the time frame considered (up to 30 weeks that even extremely low levels of apoA-I can preserve of age). DKO mice might, therefore, represent a valuable leukocyte homeostasis. model of coronary atherosclerosis. In vitro experiments with rHDL, showed that apoA-I + + As the alteration of the HDL system was shown directly affects CD4 and CD8 T-cell activation. HDL to affect local and systemic immunoinflammatory also modulated cholesterol and lipid rafts abundance responses, we deeply characterized tissue and sys- in T cells plasma membrane and this possibly influ- temic immune profile. Interestingly, DKO mice showed enced their polarization and activation status. The enlarged skin-draining axillary lymph nodes, character- modulation of lipid raft composition and cholesterol ized by the presence of foamy macrophages, granu- abundance in plasma membrane of T cells has been lomatous reactions, cholesterol crystals, dilation of postulated as a key mechanism affecting T-cell biol- sinuses, and increased lipid deposition. In contrast, WT ogy ; of note cholesterol/lipid rafts abundance at the and EKO mice did not develop any of these alterations immunologic synapse contributes to cluster the sig- and human apoA-I overexpression in DKO/hA-I mice naling receptors involved in the activation of adaptive 58–60 reversed the DKO alterations. immune response. Similarly to our observation, Wilhelm et al thoroughly Although several studies have already investigated described an enlargement of skin-draining lymph nodes the transcriptional profile of skin-draining lymph nodes 61,62 caused by increased lipid accumulation and expansion under skin inflammatory conditions, to our knowl- of macrophages, dendritic cells, T and B lymphocytes edge, this is the first report in which the gene expression in cholesterol-fed LDLrKO/A-IKO mice compared with profile of skin-draining lymph nodes specifically obtained LDLrKO mice. from dyslipidemic mouse models is described. In accor- Of note, cholesterol accumulation in skin-draining dance with the histological findings, it was not surpris- lymph nodes of LDLrKO/A-IKO mice appeared to be ing to detect a greater expression of genes attributable mainly triggered by hypercholesterolemia, whereas in our to an increased immune response in the lymph node of model an overtly altered lymph node histology arose in DKO versus DKO/hA-I mice. It was also interesting to normocholesterolemic conditions and was possibly trig- note the enrichment of pathways indicative of increased gered by HDL deficiency itself. phagocytic and lysosomal activity, which was reported to This condition, possibly deriving from the almost com- be critical in the immune response. Consistent with the plete HDL deficiency, somehow mimics the impaired unchanged accumulation of lipids in the splenic paren- cholesterol flow through the lymphatics observed in chyma, the expression of genes involved in phagocytosis, Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 July 2022 853 Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice (G.D. Norata); Telethon Foundation grant. No. GGP19146 (G.D. Norata); PRIN lysosomal degradation, as well as foam cell formation 2017K55HLC (G.D. Norata). markers, was also comparable between genotypes. Beyond the increased immune/inflammatory Disclosures None. response, the transcriptome analysis also revealed a reduced enrichment of several lipid-related pathways Supplemental Material in the lymph node of DKO versus DKO/hA-I mice. 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Curr Opin Lipidol. 2019;30:462–469. doi: Sources of Funding 10.1097/MOL.0000000000000642 This work has received funding from the European Union’s Horizon 2020 re- 15. Yvan-Charvet L, Bonacina F, Guinamard RR, Norata GD. Immunometabolic search and innovation programme under the ERA-Net Cofund action no. 727565 function of cholesterol in cardiovascular disease and beyond. Cardiovasc (OCTOPUS project) and the MIUR (G. Chiesa). This work was also supported by Res. 2019;115:1393–1407. doi: 10.1093/cvr/cvz127 the European Community’s Seventh Framework Programme (FP7/2007–2013) 16. Sorci-Thomas MG, Thomas MJ. Microdomains, inflammation, and athero- AtheroRemo, grant no. 201668 (G. Chiesa), by the European Community’s Sev- sclerosis. Circ Res. 2016;118:679–691. doi: 10.1161/CIRCRESAHA. enth Framework Programme (FP7/2012–2017) RiskyCAD, grant no. 305739 115.306246 (G. Chiesa), by Fondazione CARIPLO (2011-0645; G. Chiesa), by grants from 17. 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J Mol Cell Cardiol. 2019;127:270–276. 58. Westerterp M, Gautier EL, Ganda A, Molusky MM, Wang W, Fotakis P, doi: 10.1016/j.yjmcc.2019.01.003 Wang N, Randolph GJ, D’Agati VD, Yvan-Charvet L, Tall AR. Cholesterol 66. Diskin C, Pålsson-McDermott EM. Metabolic modulation in macrophage accumulation in dendritic cells links the inflammasome to acquired immu- effector function. Front Immunol. 2018;9:270. doi: 10.3389/fimmu. nity. Cell Metab. 2017;25:1294–1304.e6. doi: 10.1016/j.cmet.2017.04.005 2018.00270 856 July 2022 Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 BASIC SCIENCES - AL http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Arteriosclerosis Thrombosis and Vascular Biology Wolters Kluwer Health

Lack of ApoA-I in ApoEKO Mice Causes Skin Xanthomas, Worsening of Inflammation, and Increased Coronary Atherosclerosis in the Absence of Hyperlipidemia

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Abstract

Arteriosclerosis, Thrombosis, and Vascular Biology BASIC SCIENCES Lack of ApoA-I in ApoEKO Mice Causes Skin Xanthomas, Worsening of Inflammation, and Increased Coronary Atherosclerosis in the Absence of Hyperlipidemia Marco Busnelli ,* Stefano Manzini ,* Alice Colombo , Elsa Franchi , Fabrizia Bonacina , Matteo Chiara , Francesca Arnaboldi , Elena Donetti , Federico Ambrogi , Roberto Oleari , Antonella Lettieri , David Horner , Eugenio Scanziani , Giuseppe Danilo Norata , Giulia Chiesa BACKGROUND: HDL (high-density lipoprotein) and its major protein component, apoA-I (apolipoprotein A-I), play a unique role in cholesterol homeostasis and immunity. ApoA-I deficiency in hyperlipidemic, atheroprone mice was shown to drive cholesterol accumulation and inflammatory cell activation/proliferation. The present study was aimed at investigating the impact of apoA-I deficiency on lipid deposition and local/systemic inflammation in normolipidemic conditions. METHODS: ApoE deficient mice, apoE/apoA-I double deficient (DKO) mice, DKO mice overexpressing human apoA-I, and C57Bl/6J control mice were fed normal laboratory diet until 30 weeks of age. Plasma lipids were quantified, atherosclerosis development at the aortic sinus and coronary arteries was measured, skin ultrastructure was evaluated by electron microscopy. Blood and lymphoid organs were characterized through histological, immunocytofluorimetric, and whole transcriptome analyses. RESULTS: DKO were characterized by almost complete HDL deficiency and by plasma total cholesterol levels comparable to control mice. Only DKO showed xanthoma formation and severe inflammation in the skin-draining lymph nodes, whose transcriptome analysis revealed a dramatic impairment in energy metabolism and fatty acid oxidation pathways. An increased presence of CD4 T effector memory cells was detected in blood, spleen, and skin-draining lymph nodes of DKO. A worsening of atherosclerosis at the aortic sinus and coronary arteries was also observed in DKO versus apoE deficient. Human apoA-I overexpression in the DKO background was able to rescue the skin phenotype and halt atherosclerosis development. CONCLUSIONS: HDL deficiency, in the absence of hyperlipidemia, is associated with severe alterations of skin morphology, aortic and coronary atherosclerosis, local and systemic inflammation. GRAPHIC ABSTRACT: A graphic abstract is available for this article. Key Words: atherosclerosis ◼ hyperlipidemias ◼ inflammation ◼ lymph nodes ◼ xanthomatosis he pivotal role of HDL (high-density lipoprotein) in reg- ability has been considered as the main explanation for ulating cell cholesterol homeostasis is very well recog- the inverse correlation between HDL-C (HDL-cholesterol) Tnized. HDL and its main protein component, apoA-I (apolipoprotein A-I), are essential in mediating the removal See cover image of excess cholesterol from all the cells of our body. This Correspondence to: Marco Busnelli, Stefano Manzini, and Giulia Chiesa, Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, via Balzaretti 9, 20133 Milano, Italy. Emails marco.busnelli@unimi.it, stefano.manzini@unimi.it, giulia.chiesa@unimi.it Supplemental Material is available at https://www.ahajournals.org/doi/suppl/10.1161/ATVBAHA.122.317790. *M. Busnelli and S. Manzini contributed equally. For Sources of Funding and Disclosures, see page 854. © 2022 The Authors. Arteriosclerosis, Thrombosis, and Vascular Biology is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial-NoDerivs License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited, the use is noncommercial, and no modifications or adaptations are made. Arterioscler Thromb Vasc Biol is available at www.ahajournals.org/journal/atvb Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 July 2022 839 Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice Nonstandard Abbreviations and Acronyms HIGHLIGHTS ABCA1 ATP-binding cassette transporter A1 • ApoA-I (apolipoprotein A-I)/apoE knockout mice are normocholesterolemic and HDL (high-density ABCG1 ATP-binding cassette transporter G1 lipoprotein) deficient. apoA-I apolipoprotein A-I • Skin xanthomas and severe atherosclerosis are the DKO apoE/apoA-I double deficient phenotypic features of apoA-I/apoE knockout mice. DKO/hA-I D KO mice overexpressing human • ApoA-I/apoE deletion causes severe inflammation apoA-I in the skin-draining lymph nodes. EKO apoE deficient • RNA-seq of lymph nodes shows alterations in lipid metabolism and fatty acid oxidation pathways. FITC fluorescein isothiocyanate • Overexpression of human apoA-I is able to rescue G-CSF granulocyte-colony stimulating factor the apoA-I/apoE knockout phenotype. HDL high-density lipoprotein HDL-C HDL-cholesterol IFN interferon influence the cellular activities involved in the innate and IL interleukin adaptive immune response through the fine regulation of IP-10 interferon gamma-induced protein 10 the cellular cholesterol content. Under reduced plasma KC/CXCL1 keratinocyte-derived cytokine HDL levels, peripheral blood mononuclear cells become LDLrKO low-density lipoprotein receptor enriched in cholesterol and display signs of cholesterol knockout imbalance, resulting in monocyte adhesion to the endo- LIX/CXCL5 lipopolysaccharide-inducible CXC thelium and recruitment in peripheral tissues, including chemokine the arterial wall. Additionally, it has been demonstrated MIG/CXCL9 monokine induced by gamma that the transgenic expression of human apoA-I inhib- interferon its foam cell formation in apoE deficient (EKO) mice by PPAR peroxisome proliferator-activated promoting macrophage cholesterol efflux via the ABCA1 receptor (ATP-binding cassette transporter A1) and ABCG1 rHDL reconstituted HDL (ATP-binding cassette transporter G1). In a mouse model of hyperlipidemia combined to T central memory T lymphocytes CM apoA-I deficiency—LDLrKO (low-density lipoprotein T effector memory T lymphocytes EM receptor knockout)/apoA-IKO mice fed an atherogenic T naive T lymphocytes diet for 12 weeks—an expansion and activation of T WT wild type cells in the skin-draining lymph nodes was observed, and T and B lymphocytes showed an increased cho- 18,19 lesteryl ester content compared with LDLrKO mice. plasma levels and atherosclerotic cardiovascular disease Interestingly, LDLrKO/apoA-IKO mice also showed resulting from prospective observational studies. In addi- skin abnormalities, which included dermal cholesterol tion, severe HDL deficiency, especially when genetically accumulation. It has never been investigated if these determined, is frequently associated in the clinic with ath- alterations may occur when apoA-I deficiency is not erosclerotic cardiovascular disease. associated to a hyperlipidemic condition. While Mendelian randomization studies failed to estab- In the present study, the impact of different levels of lish a clear causal role between HDL-C and myocardial apoA-I/HDL-C on lipid accumulation in the skin, local, 4,5 infarction or coronary heart disease, they supported a and systemic immunoinflammatory activation, as well causal role for HDL in immune regulation. More recently, as atherosclerosis progression at the aortic sinus and a thorough analysis of the relationship between HDL-C coronary arteries, was evaluated in genetically modified levels and all-cause mortality highlighted a U-shaped mice fed a normal laboratory diet. Specifically, the follow- association between HDL-C levels and increased mortal- ing mouse lines were investigated: wild-type (WT) mice ity risk and also autoimmune diseases for both extremes— as control, apoE and apoA-I double knockout mice with 7,8 very low and very high levels of HDL-C —thus proposing negligible HDL-C levels (apoE/apoA-I double deficient a key role for HDL in infection and sepsis. [DKO]); DKO knockout mice expressing human apoA- A direct link between low HDL-C levels and immune I (DKO/hA-I), with elevated apoA-I and HDL-C levels; disorders has been experimentally established. Several apoE knockout mice (EKO), which display halved apoA-I animal studies have demonstrated that, beyond the levels and low HDL-C levels compared with WT mice. effect on cholesterol efflux, HDL possesses a pleth- The results indicate that HDL deficiency in DKO 11 12 ora of additional anti-inflammatory, antioxidant, and mice, in the presence of plasma total cholesterol com- antithrombotic properties. In particular, HDL is able to parable to that of WT mice, is associated with xanthoma 840 July 2022 Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 BASIC SCIENCES - AL BASIC SCIENCES - AL Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice immunoturbidimetric quantification of human apoA-I. Purified formation, accelerated atherosclerosis, both at the aortic apoA-I was used as calibration standard. sinus and coronary arteries, and increased local and sys- temic inflammation. Detection of Circulating Cytokines/Chemokines By using a customized detection MILLIPLEX MAP Mouse METHODS Cytokine/Chemokine panel (Merck Millipore, Billerica, MA), the following 13 cytokines/chemokines were evaluated in The data that support the findings of this study are available plasma-EDTA samples: eotaxin, G-CSF (granulocyte-col- from the corresponding author upon reasonable request. ony stimulating factor), IL (interleukin)-4, IL-5, IL-6, IL-10, IL-12(p70), IL-15, IL-17, IP-10 (interferon gamma-induced Mice protein 10), KC/CXCL1 (keratinocyte-derived cytokine), Procedures involving animals and their care were conducted LIX/CXCL5 (lipopolysaccharide-inducible CXC chemokine), in accordance with institutional guidelines in compliance with and MIG/CXCL9 (monokine induced by gamma interferon). national (D.L. No. 26, March 4, 2014, G.U. No. 61 March 14, These analyses were outsourced to certified laboratories 2014) and international laws and policies (E EC Council Directive (Department of Experimental Evolutionary Biology, University 2010/63, September 22, 2010: Guide for the Care and Use of of Bologna and Labospace Srl). Laboratory Animals, United States National Research Council, 2011). The experimental protocol was approved by the Italian Tissue Harvesting Ministry of Health (Protocollo 2012/4). Mice were euthanized at 30 weeks of age under general C57Bl/6J (WT) and apoE knockout (EKO) male mice (strain anesthesia with 2% isoflurane and blood was removed by 002052) were purchased from Charles River Laboratories perfusion with 1× PBS. For histological analyses, hearts (Calco, Italy). ApoE/apoA-I double knockout male mice (DKO) were harvested, fixed in 10% formalin for 24 hours, then were previously generated in our lab. DKO/hA-I were obtained transferred into 1× PBS containing 30% sucrose (w/v) for by multiple crosses between DKO mice and hemizygous mice 24 hours at 4 °C before being embedded in OCT (optimal overexpressing human apoA-I. The human apoA-I transgene cutting temperature) compound (Sakura Finetek Europe B.V., in this model is almost exclusively expressed in the liver, and—at Alphen aan den Rijn, the Netherlands) and stored at −80 °C. very low amounts—in testis. Only male mice were enrolled in From a first set of mice, half of the spleen and the right the study, to prevent the possible impact of hormonal changes axillary lymph node were harvested and immediately processed of female mice on the results. for flow cytometry analysis; the other half of the spleen and the Primers specific for murine apoE (For 5′ -GCCTAGC left axillary lymph node were snap-frozen in liquid nitrogen for CGAGGGAGAGCCG-3′; Rev1 5′-TGTGACTTGGGAGCTCTG subsequent RNA-seq and molecular analyses. From a second CAGC-3′; Rev2 5-GCCGCCCCGACTGCATCT-3′), murine set of mice, half of the spleen and the right axillary lymph node apoA-I (For 5′-CCTTCTATCGCCTTCTTGACG-3′; Rev1 5′-GTT were immersion-fixed in 10% formalin for 24 hours, dehy- CATCTTGCTGCCATACG-3′; Rev2 5′-TCTGGTCTTCCTGACAG drated in a graded scale of ethanol, and paraffin embedded. GTAGG-3′), and human apoA-I (For 5′-GATCGAGTGAAGGACC The other half of the spleen and the contralateral axillary lymph TGGC-3′; Rev 5′-CCTCCTGCCACTTCTTCTGG-3′) were used node were immersion-fixed in 10% formalin for 24 hours, then to screen genotypes. transferred into 1× PBS containing 30% sucrose (w/v) for 24 Mice were maintained under standard laboratory conditions hours at 4 °C and embedded in OCT compound. Skin biopsies, (12-hour light cycle, temperature 22 °C, humidity 55%), with free excised from the thoracic region, were dissected in 2×2 mm access to normal laboratory diet (4RF21, Mucedola, Settimo fragments and processed for transmission electron microscopy Milanese, Italy) and tap water from weaning to 30 weeks of age. analysis as previously described. Additional skin biopsies were excised from the thoracic region, dissected in 5×5 mm Plasma Lipids fragments, immersion-fixed in 10% formalin for 24 hours, then transferred into 1× PBS containing 30% sucrose (w/v) for 24 After an overnight fast, mice were anaesthetized with 2% iso- hours at 4 °C and embedded in OCT compound. flurane (Merial Animal Health, Woking, United Kingdom). Blood was collected from the retroorbital plexus into tubes contain- ing 0.1% EDTA (ethylenediamine tetraacetic acid; w/v) and Histology centrifuged in a microcentrifuge for 10 minutes at 5900×g at 4 °C. Plasma total cholesterol was measured with an enzy- Heart matic method (CPA11 A01634, ABX Diagnostics, Montpellier, To determine the presence and length of atherosclerotic France). Cholesterol distribution among lipoproteins was ana- plaques in coronary arteries, serial transverse cryosections (7 lyzed by fast protein liquid chromatography as described. μm thick) of the entire heart from the apex to the ostia of HDL-C was measured after precipitation of apoB-con- the coronary arteries at the aortic root were cut. Lipid deposi- taining lipoproteins with polyethylene glycol (20%, w/v) in 0.2 tion in the coronary arteries was determined through Oil Red mol/L glycine (pH 10). Plasma non-HDL-C was measured as O (ORO; Sigma-Aldrich, MO) staining of all slides. To obtain the difference between total cholesterol and HDL-C. Plasma reliable data, the number of coronary lesions has been deter- human apoA-I concentration was determined by immunoturbi- mined only in coronary arteries with an orthogonal course with dimetric assays, using an antiserum specific for human apoA-I respect to the orientation of the sections. Plaque length was (LTA, Milan, Italy), as previously described. Coomassie staining determined by summing the sequential sections in which the was used to quantify plasma murine apoA-I and to confirm the presence of the ORO signal within arteries was observed, Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 July 2022 841 Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice taking into account the thickness of each section. Hematoxylin Data were acquired in FCS (flow cytometry standard) format, and eosin stained sections were used to detect atheroscle- processed and analyzed using the FCS Express V3 Research rotic plaque area at the aortic sinus, as previously described. edition (De Novo Software, Inc). CD45 was used for the identi- + − + Both coronary arteries and aortic sinus histology were per- fication of total leukocytes. CD4 CD44 CD62L cells are indi- + + − formed in accordance with American Heart Association cated as T naive (T ), CD4 CD44 CD62L cells are indicated as 29 + + + recommendations. Macrophages and T lymphocytes were T effector memory (T ), and CD4 CD44 CD62L cells are indi- EM + + lo detected using an anti-Mac2 antibody (CL8942, Cedarlane, cated as T central memory (T ). CD11b CD115 Ly6C cells are CM + + hi Ontario, Canada; RRID:AB_10060258) and an anti-CD3 indicated as patrolling monocytes and CD11b CD115 Ly6C as hi + lo antibody (MAB4841, R&D Systems, MN; RRID:AB_358426), inflammatory monocytes. CD11c CD11b B220 cells are indi- lo − hi respectively. Detection was performed using an ImmPRESS cated as myeloid and CD11c CD11b B220 as plasmacytoid + + reagent kit (Vector Laboratories, Peterborough, United dendritic cells. CD19 CD5 cells are indicated as B cells and 1a + − Kingdom). Diaminobenzidine was used as the chromogen CD19 CD5 as B . 1b (ImmPACT DAB Substrate, Peroxidase HRP SK-4105, Vector In Vitro T-Cell Proliferation and Cytokine Production Laboratories), and the sections were counterstained with Discoidal reconstituted HDL (rHDL) containing human apoA-I Gill’s hematoxylin (Bio-Optica, Milan, Italy). and 1-palmitoyl-2-oleoyl phosphatidylcholine were prepared by Axillary Lymph Node, Spleen, and Skin cholate dialysis method, as described. Lymphocyte suspen- Serial sections (5 μm thick) of the axillary lymph node were sion from EKO lymph nodes was incubated with 5 μmol/L obtained and stained with hematoxylin and eosin. In addition, of carboxyfluorescein succinimidyl ester (Merck, Catalog no. in the axillary lymph node, macrophages, B and T lympho- 21888) for 10 minutes at room temperature in the dark, diluted cytes were detected using an anti-Iba-1 (019-19741, WAKO, 10 times in PBS/FBS 2%/2 mmol/L EDTA, washed 3 times United States; RRID AB_839504), an anti-CD45R/B220 and resuspended in TexMACS medium. A total of 0.2×10 (BD Pharmingen, United States; RRID AB_396673) and an cells were plated in 96-well plate (U-bottom wells), coated with anti-CD3 epsilon (Sc-1127, Santa Cruz Biotechnology, United 0.5 μg/mL anti-CD3 (Biolegend, Catalog no. 152302; RRID States; RRID AB_631128), respectively. Cryosections (7 μm AB_2650621) and 2.5 μg/mL anti-CD28 (Biolegend, Catalog thick) from OCT embedded organs were stained with ORO no. 102102; RRID AB_312867), in 200 μL of medium con- to detect neutral lipid accumulation. Lipid deposition was taining 20 U/mL IL-2 (Preprotec, Catalog no. 200-02), with or measured as the percent of ORO positive area over the total without rHDL (10, 25, 50, and 100 μg/mL). Cells were incu- area. bated for 4 days at 37 °C with 5% CO . The Aperio ScanScope GL Slide Scanner (Aperio For cytokine production, at the end of the incubation, cells Technologies, CA) was used to acquire digital images that were were pulsed with 0.1 μg/mL PMA (phorbol-12-myristate- subsequently processed with the ImageScope software. Two 13-acetate; Merk, Catalog no. P1585) and 1 μg/mL ionomycin operators blinded to mouse genotypes quantified all histologi- (Invitrogen, Catalog no. I24222) for 4 hours at 37 °C with 5% cal parameters. CO in the presence of Brefeldin A (1:1000, BD Bioscience, Skin semithin sections, 2 μm thick, were stained with tolu- Catalog no. 555028). Cytokine analysis was performed by flow idine blue and observed with a Nikon Eclipse E600 microscope cytometry following the instructions from the fixation/permea- equipped with a Nikon digital camera DXM1200 (Nikon, Tokyo, bilization kit (BD Bioscience, Catalog no. 555028). Japan). Ultrathin sections were obtained with an Ultracut ultra- T cells were analyzed for the expression of extracellular microtome (Reichert Ultracut R-Ultramicrotome, Leika, Wien, markers, intracellular cytokines, proliferation, cellular viability, Austria) and stained with uranyl acetate/lead citrate before and lipid abundance by flow cytometry. observation by a Jeol CX100 transmission electron microscope Immunophenotyping was performed on resting and (Jeol, Tokyo, Japan). activated lymphocytes. For live and dead cells discrimina- tion, cell suspension was stained with 100 μL of a 1:1000 dilution of the LIVE/DEAD Fixable Aqua Dead Cell Stain Flow Cytometry Analysis on Blood and Kit (Invitrogen, Catalog no. L34965) in PBS at 4 °C for 30 Lymphoid Organs minutes. Single cell suspensions were then incubated with Analyses were performed with a Calibur Flow Cytometer (BD antibody mixtures at 4 °C for 30 minutes and then washed Biosciences, Milan, Italy) with 2 lasers was used. The panels with PBS/FBS 2%/EDTA 2 mmol/L. For intracellular stain- consisted of the subsequent cellular surface markers: CD4 ing, first cells were fixed and permeabilized according to (PE YTS 191.1.2, Immunotools, Friesoythe, Germany), CD44 manufacturer instructions and then incubated with specific (fluorescein isothiocyanate [FITC], IM7, BD Pharmingen; RRID antibodies at 4 °C for 30 minutes, washed and analyzed as AB _2076224), CD45 (FITC, 30-F11, BD Pharmingen; RRID described. Antibodies are listed in the Major Resources AB_394610), CD62L (antigen-presenting cell, mMEL-14, BD Table in the Supplemental Material. Pharmingen; RRID AB_10895379), Ly-6C (FITC AL-21, BD For detection of lipid rafts, cells were incubated with 2 μg/ Pharmingen; RRID AB_394628), CD115 (antigen-presenting mL of Cholera Toxin-FITC (Merck, Catalog no. C1655) con- cell AF598, eBioscience; RRID AB_2314130), CD11b (PE jugated for 30 minutes at 4 °C or with 25 μg/mL 100 filipin M1/70.15, Immunotools), CD11c (antigen-presenting cell HL3, (Merck, Catalog no. F9765) for 30 minutes at 30 °C, washed BD Pharmingen RRID AB_398460), B220 (PerCP Cy5,5 and analyzed. RA3-6B2, BD Pharmingen; RRID AB_2034009), CD19 (FITC Samples were acquired with BD LSRII Fortessa and ana- 1D3, BD Pharmingen RRID AB_395049), and CD5 (antigen- lyzed with Novoexpress 1.3.3 software (Agilent Technologies presenting cell 53-7.3, BD Pharmingen; RRID: AB_394562). Inc). Gating strategy was reported in Figure S16. 842 July 2022 Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 BASIC SCIENCES - AL BASIC SCIENCES - AL Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice by Dunn post hoc test, according to the check of normality RNA Extraction of residuals (Shapiro-Wilk test) and homoscedasticity of data Total RNA was isolated from mouse tissues and extracted as (Levene test). Statistical analyses are detailed in each figure or previously described. RNA was quantified and checked, and table caption, specifying for which parameters the nonparamet- 1 μg RNA was retrotranscribed to cDNA. Possible gDNA con- ric procedure was applied. tamination was ruled out as described. Numerical data is shown as box plots, where the upper and lower ends of the boxes indicate the 25th and 75th percen- RNA-Seq Analysis tiles, respectively. The length of the box shows the interquartile range within which 50% of the values are located. The solid For RNA-seq analysis, the quality of the mRNA was tested using gray lines denote the median. Precise adjusted P values are the Agilent 2100 Bioanalyzer (Agilent Technologies, CA), and shown in Table S1. In most figures, approximate P values are only libraries with RNA integrity number ≥7 were included. RNA provided to improve readability (ex. P<0.05, P<0.01). samples were processed using the RNA-seq Sample Prep kit from Illumina (Illumina, Inc, CA). Eight to 9 tagged libraries were loaded on one lane of an Illumina flowcell, and clusters were created using the Illumina Cluster Station (Illumina, Inc, CA). RESULTS Clusters were sequenced on a Genome Analyzer IIx (Illumina, ApoA-I Deficiency in EKO Mice Leads to Inc, CA) to produce 50nt single-reads. Data sets can be accessed at NCBI (Gene Expression Omnibus) GSE202237. Normocholesterolemia Associated to Negligible HDL Levels Bioinformatics Analysis At the end of the experimental period, body weight was comparable in WT, DKO, and DKO/hA-I mice and signifi- Preprocessing of Reads cantly higher in EKO mice (Figure S1A). Raw sequence reads were trimmed using Trimmomatic soft- 35 Plasma total cholesterol concentration in DKO and ware and applying the leading, trailing, and slidingwindows WT mice was comparable, and ≈3-fold lower than the operations, with the following quality score cutoffs, respectively: concentration observed in EKO mice and DKO/hA-I Qs ≥15, Qs ≥10, and average Qs ≥15. mice (Figure 1A). Differential Gene Expression As expected, apoA-I was absent in plasma of DKO Transcript abundance estimation and differential expression mice. ApoA-I concentration in EKO mice was approxi- analyses were performed using the standard Bowtie-Tophat- mately half of that measured in WT. The overexpression of Cuffdiff pipeline on the Refseq annotation of the mm10 mouse the human APOA1 transgene strongly increased apoA-I reference genome assembly, as obtained from the following: plasma levels of DKO/hA-I (Figure 1B). HDL-C concen- https://ftp.ncbi.nlm.nih.gov/refseq/ M_musculus/annota- tration was about 10-fold lower in DKO and 3-fold lower tion_releases/current/. Transcriptome assembly functions of in EKO than in WT mice, whereas DKO/hA-I mice showed cufflinks were deactivated and only established transcripts annotations were used. higher HDL-C levels than those measured in EKO and Differential expression analyses were executed by per- DKO (Figure 1C). Non-HDL-C levels were significantly forming direct pairwise comparisons between the conditions different among all genotypes (Table S1). Specifically, under study. A false discovery rate cutoff value of 0.05 was plasma non-HDL-C concentration was 12.2±3.7 mg/dL applied for the identification of differentially expressed genes. in WT, 316.3±23.0 mg/dL in EKO, 247.3±31.2 mg/dL in Functional enrichment analyses, Kyoto Encyclopedia of Genes DKO/hA-I, and 106.2±16.4 mg/dL in DKO. and Genome (KEGG) and reactome pathways, were performed Fast protein liquid chromatography analysis confirmed with reString (version 0.1.18). a dramatic cholesterol accumulation in the VLDL (very- low-density lipoprotein) and LDL fractions of EKO mice Statistical Analyses and a greatly reduced HDL-C peak compared with that Sample size for histological and imunohistochemical analyses was of WT mice (Figure 1D). In DKO mice, the HDL-C peak calculated based on previously published data on atherosclerosis was almost absent and cholesterol in the VLDL and LDL 38,39 in EKO mice and EKO mice overexpressing human apoA-I. fractions was much lower than in EKO mice. DKO/hA-I Plaque size in EKO mice was expected to be about 5 times larger mice displayed an HDL-C peak close to that of WT and a than in DKO/hA-I mice. A lack of atherosclerosis development was relevant cholesterol accumulation in the VLDL and LDL expected in WT mice, and the development of plaques in DKO mice fractions. was hypothesized 2 times larger than in EKO mice. Based on these premises, a sample size of 6 per experimental group achieves 92% power to detect the hypothesized differences among the means HDL Deficiency Worsens Atherosclerotic using an F test at 5% significance level. A higher number of sam- ples was cautiously used for plasma lipid measurements and for Plaque Development at the Aortic Sinus and in most of the analyses on lymphocyte class distribution. Coronary Arteries Analyses were performed with R and GraphPad Prism ver- The apoA-I deficiency in the apoEKO background (DKO) sion 9.3.0. Significant differences were determined by ANOVA caused an increase of plaque extent at the aortic sinus followed by Tukey post hoc test or by Kruskal-Wallis followed Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 July 2022 843 Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice Figure 1. Plasma cholesterol and apoA-I (apolipoprotein A-I) levels. Plasma total cholesterol (A), apoA-I (B), HDL-C (high-density lipoprotein cholesterol (C) concentrations, and cholesterol distribution among lipoproteins by fast protein liquid chromatography in the 4 genotypes (n=8). Statistically significant differences were determined by ANOVA followed by Tukey post hoc. ***P<0.001. Precise adjusted P values are shown in Table S1. compared with that measured in EKO mice. Conversely, DKO/hA-I mice (Figure S3E). However, no differences apoA-I overexpression in DKO/hA-I mice dramatically were observed when CD3 cell counts were normalized reduced atherosclerotic plaque size at the aortic sinus to plaque area (Figure S3F). In addition, the presence of compared with both DKO and EKO mice. As expected, CD3 cells was evaluated in the myocardial tissue imme- no plaques developed at the aortic sinus of WT mice diately surrounding the aortic sinus: the count of CD3 (Figure 2A through 2D and 2K). cells was significantly increased in EKO and DKO mice The area occupied by macrophages in the plaques of compared with that in DKO/hA-I mice (Figure S3G). DKO/hA-I mice was about ten times lower than in EKO A feature observed only in DKO and EKO mice was and DKO mice, since DKO/hA-I developed smaller plaques the presence of lesions at the left atrioventricular valves (Figure S2C and S2E). Macrophage area expressed as (Figure 2E and 2F). percentage of total area was instead comparable among The presence of atherosclerotic plaques within the DKO/hA-I, DKO, and EKO mice (Figure S2F). left and right coronary arteries arising from the aortic An increased count of CD3 T lymphocytes was sinus was detected in 100% of DKO and 30% of EKO observed in the atherosclerotic plaques of DKO versus mice (Figure 2G through 2J). The average number of 844 July 2022 Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 BASIC SCIENCES - AL BASIC SCIENCES - AL Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice Figure 2. Atherosclerosis development at the aortic sinus and coronary arteries. Representative hematoxylin and eosin (H&E) stained pictures of atherosclerosis development at the aortic sinus of wild type (WT; A), apoE/ apoA-I double deficient (DKO) mice overexpressing human apoA-I (DKO/hA-I; B), apoE deficient (EKO; C) and DKO mice (D). Representative oil red O (ORO) stained pictures of plaque development at the left atrioventricular valves of EKO (E) and DKO mice (F), and in the right coronary artery of EKO (G) and DKO mice (H). Magnifications of the same coronary plaques are shown (I and J). Atherosclerotic plaque quantification at the aortic sinus (K), number of plaques in coronary arteries and coronary branches (L), and average plaque length in coronary arteries (M) are also shown (n=6). Bar length: 250 μm. Statistically significant differences were determined by ANOVA followed by Tukey post hoc. *P<0.05; **P<0.01; and ***P<0.001. Precise adjusted P values are shown in Table S1. Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 July 2022 845 Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice atherosclerotic plaques in left and right ventricular coro- WT, EKO, and DKO/hA-I mice, whereas in DKO mice a nary branches was more than doubled in DKO mice com- dramatic lipid accumulation was detected (Figure 4F). pared with EKO mice (Figure 2L and Figure S4). The average length of coronary plaques was comparable in HDL Deficiency Increases the Amount of CD4 DKO and EKO mice (Figure 2M). No plaques developed T in Blood, Skin-Draining Lymph Nodes and in the coronary arteries of DKO/hA-I and WT mice (Fig- EM Spleen ure 2L and 2M). Total white blood cells (CD45 ) were significantly higher in DKO versus WT mice. Comparable CD45 counts The Pathological Skin Phenotype Determined were observed in the spleen of the 4 genotypes. CD45 by HDL Deficiency Is Rescued by hA-I cells were increased in the axillary lymph node of DKO Overexpression mice versus all the other groups. Total CD4 counts were In accordance with our previous findings, DKO mice not affected by genotype in blood, spleen, and axillary displayed hair loss and a pale color of the skin. These lymph node (Figure S8). features were not observed in any other genotype. The analysis of CD4 T-cell subsets (T , T , and T ) N EM CM Structural and ultrastructural features of the skin were revealed that, in peripheral blood and spleen, DKO mice investigated in semithin and in ultrathin sections stained had an increased percentage of T and a concomitant EM with toluidine blue, respectively. By light microscopy, reduced percentage of T lymphocytes compared with all the dermal and the epidermal compartment exhibited a the other genotypes (Figure 5A and 5B). In the axillary normal organization in WT, in EKO, and DKO/hA-I mice lymph node, DKO mice showed a significantly increased (Figure 3A through 3C). In DKO mice, the subpapillary percentage of T compared with the other genotypes EM dermis appeared disarranged and filled with cholesterol and a tendency towards a reduced percentage of T , sig- clefts (Figure 3D and 3E) and foam cells were inter- nificant only versus WT mice (Figure 5C). The percent- spersed in the reticular dermis (Figure 3D and 3E). Infil- age of T cells resulted always unaffected by genotype. CM trated lymphocytes were present in the dermis of DKO No differences were found among the 4 genotypes hi mice (Figure 3F). In addition, neutral lipid deposition was in monocyte subsets distribution (inflammatory Ly6C lo increased in the thickened dermis of DKO mice, com- and patrolling Ly6C ) in peripheral blood. B lympho- 1a pared with the other genotypes (Figure S5). cytes were increase in EKO mice compared with DKO/ hA-I and DKO mice, both in spleen and in axillary lymph node (Figure S9). HDL Deficiency Results in Deep Alterations in The concentration of plasma cytokines did not show Skin-Draining Lymph Nodes differences among genotypes (Table). The size and weight of the spleen in WT, DKO/hA-I, and DKO were comparable; on the contrary, spleens from + + rHDL Influence CD4 and CD8 Cells EKO mice had an increased weight compared with DKO/ Proliferation In Vitro hA-I and DKO mice (Figure 4A). Histological analysis did not reveal differences among groups. No lipid deposition To investigate the potential role of apoA-I/HDL in was observed in any genotype (Figure S6). When spleen modulating T lymphocyte proliferation/activation, lym- weight was normalized to body weight, no significant dif- phocytes were isolated from lymph nodes of EKO mice ferences were observed (Figure S1B). to test cell proliferation and cytokine release by poly- DKO mice were characterized by increased size and clonal stimulation (anti-CD3/28 antibodies plus IL-2) weight of the skin-draining axillary lymph node, compared in the presence of increasing concentration of rHDL with the other genotypes (Figure 4B), a finding further (10, 25, 50, and 100 μg/mL) for 4 days. rHDL reduced + + confirmed by the normalization of lymph node weight to both CD4 and CD8 T-cell proliferation in a concen- body weight (Figure S1C). At the histological level, 3 main tration dependent manner (Figure S13A and S13B). features were observed only in DKO mice: (1) the accu- Changes in cytokine production were evaluated in acti- mulation of large macrophages, often characterized by a vated T cells following stimulation with PMA/ionomycin. foamy cytoplasm, as single cell or in small groups in the IFN (Interferon)-γ and IL-17 production (marking T-cell cortex, surrounded by lymphoid cells (Figure 4C and Fig- polarization toward Th1 and Th17, respectively ) was + + ure S7); (2) the presence of granulomatous reactions in decreased in CD4 and CD8 T cells following incuba- the inner cortex and the medulla, localized around a large tion with rHDL (Figure S13C through S13F). A similar number of multinucleated macrophages and cholesterol trend was observed for IL-15R and IL-4, despite a sta- crystals (Figure 4D and Figure S7); and (3) a dilation tistical significance was appreciated only for CD8 T of subcapsular, corticomedullary, and medullary sinuses cells (Figure S14). (Figure 4E). Moreover, a moderate deposition of neutral To further investigate the ability of HDL to affect cell lipids within the lymph node parenchyma was found in membrane lipids, lipid rafts abundance and cholesterol 846 July 2022 Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 BASIC SCIENCES - AL BASIC SCIENCES - AL Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice Figure 3. Light and transmission electron microscopy of mouse skin. Araldite semithin sections after toluidine blue staining of skin from wild type (WT; A), apoE/apoA-I double deficient (DKO) mice overexpressing human apoA-I (DKO/hA-I; B), apoE deficient (EKO; C), and DKO mice (D). Araldite ultrathin sections of DKO skin (E and F): cholesterol clefts (arrows) and foam cells (asterisks) accumulation in the dermis are shown in D and E; lymphocytes in the dermis of DKO are shown in F. Bars: A–D: 60 μm; E: 2 μm; F: 1 μm. + + content in CD4 and CD8 T-cell membrane were evalu- in the spleen of DKO and DKO/hA-I mice (Figure 6A ated by Cholera Toxin-subunit B staining and filipin through 6D). staining. HDL significantly reduced, in a concentration A total of 784 genes were identified as differentially dependent manner, both lipid raft abundance and cho- expressed genes in the axillary lymph node: 513 were + + lesterol content in CD4 and CD8 T cells (Figure S15). upregulated, whereas 271 were downregulated in DKO compared with DKO/hA-I mice (Figure 6A). In DKO mice, functional enrichment analysis of dif- The Transcriptional Expression Profile of Skin- ferentially expressed genes indicated an increased acti- vation of the immune system, with particular reference Draining Lymph Nodes of HDL-Deficient Mice to the phagocytic activity (mmu04610 Complement Reveals Increased Activation of the Immune and coagulation cascades, mmu04145 Phagosome, System and an Unbalanced Expression of mmu04142 Lysosome, mmu04060 cytokine-cytokine Genes Involved in Energy Metabolism receptor interaction; Figure 7A). The reactome associ- A comparison of the transcriptional expression pro- ated to the genes with an increased expression in DKO files was performed in the axillary lymph nodes and mice refined this observation: the activation of both the Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 July 2022 847 Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice Figure 4. Weight of spleen and axillary lymph node in the 4 genotypes and histology of apoE/apoA-I double deficient (DKO) axillary lymph node. Gross anatomic appearance and weights (n=7–13) of the spleen (A) and the axillary lymph node (B). Representative photomicrographs of axillary lymph node from DKO mice. Hematoxylin and eosin (H&E) images show large, foamy macrophages in the cortex, surrounded by lymphoid cells (C), presence of granulomatous reactions around cholesterol crystals in the inner cortex and the medulla (D), dilation of subcapsular, corticomedullary and medullary sinuses (a detail of medullary sinuses is shown in E). Oil Red O (O.R.O.) staining (n=6) of axillary lymph node cryosections (F). Statistically significant differences were determined in A and B by Kruskal-Wallis followed by Dunn post hoc test and in F by ANOVA followed by Tukey post hoc. CC indicates cholesterol crystals; DS, dilated sinuses; EKO, apoE deficient; FM, foamy macrophages; GR, granulomatous reactions; hA-I, human apoA-I; LC, lymphoid cells; and WT, wild type. *P<0.05; **P<0.01; and ***P<0.001. Bar length =100 μm. Precise adjusted P values are shown in Table S1. 848 July 2022 Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 BASIC SCIENCES - AL BASIC SCIENCES - AL Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice Figure 5. CD4 T-cell subsets in blood, spleen, and axillary lymph node. Flow cytofluorimetric evaluation of the percentage composition of CD4 T-cell subsets is shown in blood (A–C), spleen (D–F), and axillary lymph node (G–I; n=7–13). Statistically significant differences were determined by ANOVA followed by Tukey post hoc test, except for (C) and (I) which were analyzed by Kruskal-Wallis followed by Dunn post hoc test. *P<0.05; **P<0.01; and ***P<0.001. Precise adjusted P values are shown in Table S1. DKO indicates apoE/apoA-I double deficient; DKO/hA-I, DKO mice overexpressing human apoA-I; EKO, apoE deficient; and WT, wild type. innate (MMU-168249) and adaptive immune system nonlymphoid cells (MMU-198933; Figure 7B). In addi- (MMU-1280218) was accompanied by an increased tion, extracellular matrix organization (MMU-1474244) immunoregulatory interaction between lymphoid and was modulated as well (Figure 7B). Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 July 2022 849 Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice Table. Plasma Cytokine Concentrations in the 4 Genotypes (pg/mL) WT EKO DKO/hA-I DKO Eotaxin 326.4±65.2 639.2±164 591.9±313.2 911.4±535.5 G-CSF 825±697.2 1891.1±1280.8 1663.6±1161.7 898.4±422.3 IL-4 109.8±114.3 44.5±25.3 57.1±26.3 33±29.5 IL-5 73.9±47.7 74.8±68.2 33.8±22.8 42.5±36.5 IL-6 36.6±37.1 48.5±34.6 56.9±81 124.4±168.8 IL-10 269.1±268.6 79.2±21.7 154.8±84.2 92.2±70.1 IL-12 (p70) 285.4±227.9 511.2±468.7 197.4±177.4 238.5±129.2 IL-15 91.2±5.7 96.3±6.6 88.4±4.7 92.5±4.2 IL-17 63.1±65.4 29.4±20.2 102.7±133.9 86.8±131.9 IP-10 96.5±12.2 153.7±91.5 192.6±79.8 147.2±45.7 CXCL1/KC 90.5±120.1 108.7±117.9 124.5±88 159.1±152.9 CXCL5/LIX 6376.7±1575.7 5416±4646.5 9177.2±7529.3 9411.7±3366.3 CXCL9/MIG 121.6±61.7 102.9±25.2 123.2±11.7 137.0±43.4 n=5–6 mice per group. Data are expressed as mean±SD. Statistically significant differences were determined by ANOVA followed by Tukey post hoc or Kruskal-Wallis followed by Dunn post hoc (IL-17). Precise adjusted P values are shown in Table S1. DKO indicates apoE/apoA-I double deficient; EKO, apoE deficient; G-CSF, granulocyte-colony stimulating factor; hA-I, human apoA-I; IL, interleukin; IP-10, interferon gamma-induced protein 10; KC/CXCL1, keratinocyte-derived cytokine; LIX/CXCL5, lipopolysaccharide-inducible CXC chemokine; MIG/CXCL9, monokine induced by gamma interferon; and WT, wild type. Interestingly, macrophage-specific markers (Fig- with a massive dermal accumulation of cholesterol ure 6E and Figure S10), particularly those associated clefts, foam cells, and T lymphocytes. The skin features with the differentiation of macrophages to lipid-laden of this mouse model, partially described previously foam cells, displayed and increased expression in DKO were further investigated with the inclusion of DKO/ mice, the group characterized by exacerbated athero- hA-I. The presence of xanthomas in the skin of DKO sclerosis (Figure 6F and Figure S11). mice was paralleled by partial hair loss, a thickened der- In the axillary lymph node of DKO mice a reduced mal layer filled with inflammatory cells and increased expression of several genes involved in cell metabolism neutral lipid deposition. Interestingly, apoA-I expression, was observed (Figure 6G and Figure S12). Those genes both at low (EKO mice) and high (DKO/hA-I mice) lev- were largely involved in pathways related to energy els, was able to reverse these skin abnormalities. The metabolism (mmu03320 PPAR [peroxisome prolifera- skin and plasma lipid phenotype observed in DKO mice tor-activated receptor] signaling pathway, mmu00640 strongly resembles the condition of human subjects propanoate metabolism, mmu01212 fatty acid metabo- affected by genetic apoA-I deficiency, who are similarly lism, mmu00620 pyruvate metabolism, and mmu00020 characterized by xanthoma formation in the absence of citrate cycle; Figure 7C). Similarly, the reactome indicated elevated plasma lipid levels. in DKO mice a reduction in metabolism of lipids (MMU- DKO/hA-I and EKO mice were both characterized 556833), fatty acid metabolism (MMU-8978868), by plasma total and non-HDL-C levels significantly pyruvate metabolism and TCA (tricarboxylic acid) cycle higher than DKO mice. Despite that, atherosclerotic (MMU-71406), TCA cycle and respiratory electron trans- plaque development at the aortic sinus of DKO mice port (MMU-1428517), and mitochondrial fatty acid beta- was about twice as high as in EKO. Of note, individuals oxidation (MMU-77289; Figure 7D). with apoA-I/HDL deficiency in the absence of other In the spleen, the comparison between the gene plasma lipid alterations, generally develop premature expression profiles of DKO and DKO/hA-I mice did not coronary heart disease. highlight relevant differences except for a handful of In DKO/hA-I, plaque area was dramatically lower genes. Of those, 7 were upregulated (Apol11b, Cyr61, compared with DKO and EKO mice. A reduced ath- Dnaja1, Dnajb1, Hspa1a, Hspa1b, and Hsph1) and one erosclerosis development by apoA-I overexpression was downregulated in DKO mice (Rpl31-ps12; Fig- has been previously described in hyperlipidemic EKO 21,38,44 ure 6B and 6C). and LDLrKO mice and atherosclerosis worsen- ing by apoA-I deletion has been shown in LDLrKO mice fed a high-fat/high-cholesterol diet, where DISCUSSION cholesterolemia was not affected by apoA-I abla- DKO mice, almost completely devoid of HDL and with tion. Interestingly, in our study, DKO mice showed plasma total cholesterol levels comparable to those of a strong exacerbation of atherosclerosis develop- WT mice, showed deep alterations in the skin structure, ment versus EKO, in spite of much lower non-HDL-C 850 July 2022 Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 BASIC SCIENCES - AL BASIC SCIENCES - AL Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice Figure 6. Transcriptional expression profiles of axillary lymph node and spleen of apoE/apoA-I double deficient (DKO) and DKO mice overexpressing human apoA-I (DKO/hA-I) mice. The average of normalized counts per gene between DKO/hA-I (x axis) and DKO (y axis) were compared in axillary lymph node (A) and spleen (B). The values spanned several orders of magnitude and were log -transformed. The log fold change (DKO vs DKO/ 2 2 hA-I) is shown for all transcripts in lymph nodes (x axis) and in the spleen (y axis; C). Values greater than 0 denote genes with higher expression in DKO; values lower than 0 denote genes with higher expression in DKO/hA-I. Heatmap with the Z-scored average expression for all genes in the secondary lymphoid organs (D). Heatmaps with the gene expression signature for macrophages (E) and foam cells (F). Heatmap of metabolic genes with significantly increased expression in the axillary lymph node of DKO/hA-I mice (G). n=3 for DKO and DKO/hA-I. DE indicates differentially expressed genes. Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 July 2022 851 Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice Figure 7. Functional enrichment of differentially expressed genes in the axillary lymph nodes of apoE/apoA-I double deficient (DKO) and DKO mice overexpressing human apoA-I (DKO/hA-I) mice. The most relevant KEGG and reactome pathways enriched in DKO (A and B, respectively) and in DKO/hA-I (C and D, respectively) are reported. PPAR indicates peroxisome proliferator-activated receptor. levels. Altogether, apoA-I, in our experimental setting, it was inversely related to apoA-I plasma concentra- appeared to be a major determinant of atherosclero- tions or to HDL-C levels. sis development, regardless of the lipidemic status. Observational studies in humans have established an In fact, atherosclerosis extent in the 3 atheroprone inverse correlation between HDL-C levels and cardio- genotypes was independent of non-H DL- C levels and vascular disease. However, a strong elevation of HDL-C 852 July 2022 Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 BASIC SCIENCES - AL BASIC SCIENCES - AL Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice levels caused by genetic variants or pharmacological experimental models with dysfunctional lymphatic drain- treatments have been associated to a higher cardiovas- age. In this regard, it has been demonstrated that athero- 8,45,46 cular mortality. A similar condition has been found sclerosis regression secondary to a reduction of plasma in mice double knockout for SR-B1 and apoE, charac- cholesterol levels, in ezetimibe-treated EKO mice, is terized by a considerable elevation of HDL-C levels and obtained only in the presence of an efficient aortic lym- by a premature and severe atherosclerosis. These evi- phatic drainage that prevents cholesterol and immune dences clearly indicate that plasma HDL-C concentra- cell accumulation in the aortic adventitia. tion may not always reflect HDL functionality, especially There is considerable evidence from studies in mice at very high levels. To reconcile these observations with demonstrating that apoA-I possesses anti-inflammatory 54 15 our results, we may hypothesize that HDL in both EKO properties and immunoregulatory functions possibly and DKO/hA-I mice are functional and associate to ath- affecting atherosclerosis development. For this reason, eroprotection in a dose-dependent manner. we have characterized the presence of T , T , or T EM CM N In addition to the aortic sinus, EKO mice develop ath- CD4 T lymphocytes in the axillary skin-draining lymph erosclerosis in the aorta and in brachiocephalic arter- node, spleen and blood. The percentage of T was sig- EM 48,49 ies, whereas plaques in coronary arteries are less nificantly higher in DKO mice in the 3 districts analyzed. consistently observed. Genetically modified mouse This increase was counterbalanced by a reduced per- models of coronary atherosclerosis have been devel- centage of T . The percentage of T resulted always N CM oped over the years, but they require high-fat/high- unaffected by genotype. Of note, alterations in the dis- 51,52 cholesterol diets to exhibit coronary lesions. The only tribution of T lymphocytes were not observed in the model developing coronary atherosclerosis without any other genotypes, including EKO mice. That suggests that dietary challenge is the EKO/SR-B1KO mouse, charac- even low levels of apoA-I are sufficient to switch off the terized by severe hypercholesterolemia and a high rate inflammation and T lymphocyte activation consequent to of mortality by 6 weeks of age. In the present study, apoA-I deficiency. normal laboratory diet-fed DKO mice showed an exag- The present study was not designed to evaluate the gerated plaque development also in the coronary arter- minimal effective level of apoA-I required to correct the ies, with several lesions observed in coronary branches. phenotype driven by apoA-I/HDL deficiency. However, 18,56 This condition did not associate to an increased mortality based on previous evidences, it can be hypothesized rate within the time frame considered (up to 30 weeks that even extremely low levels of apoA-I can preserve of age). DKO mice might, therefore, represent a valuable leukocyte homeostasis. model of coronary atherosclerosis. In vitro experiments with rHDL, showed that apoA-I + + As the alteration of the HDL system was shown directly affects CD4 and CD8 T-cell activation. HDL to affect local and systemic immunoinflammatory also modulated cholesterol and lipid rafts abundance responses, we deeply characterized tissue and sys- in T cells plasma membrane and this possibly influ- temic immune profile. Interestingly, DKO mice showed enced their polarization and activation status. The enlarged skin-draining axillary lymph nodes, character- modulation of lipid raft composition and cholesterol ized by the presence of foamy macrophages, granu- abundance in plasma membrane of T cells has been lomatous reactions, cholesterol crystals, dilation of postulated as a key mechanism affecting T-cell biol- sinuses, and increased lipid deposition. In contrast, WT ogy ; of note cholesterol/lipid rafts abundance at the and EKO mice did not develop any of these alterations immunologic synapse contributes to cluster the sig- and human apoA-I overexpression in DKO/hA-I mice naling receptors involved in the activation of adaptive 58–60 reversed the DKO alterations. immune response. Similarly to our observation, Wilhelm et al thoroughly Although several studies have already investigated described an enlargement of skin-draining lymph nodes the transcriptional profile of skin-draining lymph nodes 61,62 caused by increased lipid accumulation and expansion under skin inflammatory conditions, to our knowl- of macrophages, dendritic cells, T and B lymphocytes edge, this is the first report in which the gene expression in cholesterol-fed LDLrKO/A-IKO mice compared with profile of skin-draining lymph nodes specifically obtained LDLrKO mice. from dyslipidemic mouse models is described. In accor- Of note, cholesterol accumulation in skin-draining dance with the histological findings, it was not surpris- lymph nodes of LDLrKO/A-IKO mice appeared to be ing to detect a greater expression of genes attributable mainly triggered by hypercholesterolemia, whereas in our to an increased immune response in the lymph node of model an overtly altered lymph node histology arose in DKO versus DKO/hA-I mice. It was also interesting to normocholesterolemic conditions and was possibly trig- note the enrichment of pathways indicative of increased gered by HDL deficiency itself. phagocytic and lysosomal activity, which was reported to This condition, possibly deriving from the almost com- be critical in the immune response. Consistent with the plete HDL deficiency, somehow mimics the impaired unchanged accumulation of lipids in the splenic paren- cholesterol flow through the lymphatics observed in chyma, the expression of genes involved in phagocytosis, Arterioscler Thromb Vasc Biol. 2022;42:839–856. DOI: 10.1161/ATVBAHA.122.317790 July 2022 853 Busnelli et al Severe Systemic Inflammation in HDL-Deficient Mice (G.D. Norata); Telethon Foundation grant. No. GGP19146 (G.D. Norata); PRIN lysosomal degradation, as well as foam cell formation 2017K55HLC (G.D. Norata). markers, was also comparable between genotypes. Beyond the increased immune/inflammatory Disclosures None. response, the transcriptome analysis also revealed a reduced enrichment of several lipid-related pathways Supplemental Material in the lymph node of DKO versus DKO/hA-I mice. 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Arteriosclerosis Thrombosis and Vascular BiologyWolters Kluwer Health

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