Endothelial IL17RD promotes Western diet-induced aortic myeloid cell infiltration

Abstract

The Interleukin-17 (IL17) family is a group of cytokines implicated in the etiology of several inflammatory diseases. Interleukin-17 receptor D (IL17RD), also known as Sef (similar expression to fibroblast growth factor) belonging to the family of IL17 receptors, has been shown to modulate IL17A-associated inflammatory phenotypes. The objective of this study was to test the hypothesis that IL17RD promotes endothelial cell activation and consequent leukocyte adhesion. We utilized primary human aortic endothelial cells and demonstrated that RNAi targeting of IL17RD suppressed transcript levels by 83% compared to non-targeted controls. Further, RNAi knockdown of IL17RD decreased the adhesion of THP-1 monocytic cells onto a monolayer of aortic endothelial cells in response to IL17A. Additionally, we determined that IL17A did not significantly enhance the activation of canonical MAPK and NFκB pathways in endothelial cells, and further did not significantly affect the expression of VCAM-1 and ICAM-1 in aortic endothelial cells, which is contrary to previous findings. We also determined the functional relevance of our findings in vivo by comparing the expression of endothelial VCAM-1 and ICAM-1 and leukocyte infiltration in the aorta in Western diet-fed Il17rd null versus wild-type mice. Our results showed that although Il17rd null mice do not have significant alteration in aortic expression of VCAM-1 and ICAM-1 in endothelial cells, they exhibit decreased accumulation of proinflammatory monocytes and neutrophils, suggesting that endothelial IL17RD induced in vivo myeloid cell accumulation is not dependent on upregulation of VCAM-1 and ICAM-1 expression. Collectively, our results underscore a critical role for Il17rd in the regulation of aortic myeloid cell infiltration in the context of Western diet feeding.

INTRODUCTION

Endothelial cell activation is one of the key phenomena underlying the pathophysiology of inflammatory diseases including Crohn’s disease [1], systemic lupus erythematosus [2], sepsis [3], rheumatoid arthritis [4] and atherosclerotic cardiovascular disease[5]. It consists of dynamic alterations in endothelial cell phenotype and function in response to stimulation with biological agents such as cytokines, pathogen products, or turbulent hemodynamic forces [5, 6]. This process initiates a series of events involving leukocyte arrest, rolling, and transendothelial migration (TEM), designated as the leukocyte adhesion cascade [7]. Therefore, cellular remodeling processes associated with endothelial cell activation contribute to a significant proportion of disease burden [8].

The phenomenon of endothelial cell activation involves an upregulation in the expression of adhesion molecules on the endothelial cell membrane in response to environmental cues such as inflammatory cytokines. These molecules facilitate the adhesion and TEM of leukocytes across the vessel wall, which eventually initiates the pathophysiological manifestation of the disease [9, 10]. Principal EC cellular markers associated with leukocyte TEM include immunoglobulin-like adhesion molecules comprising cellular adhesion molecules (CAM) such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) [9]. Recent evidence has underscored an important role for the pleiotropic cytokine IL17A secreted by Th17 cells and its cognate receptor IL17RA in promoting atherosclerotic plaque formation by enhancing myeloid cell accumulation in Apoe−/− mice upon WD feeding [11]. Further, IL17A was shown to enhance leukocyte adhesion on endothelial cells in a manner dependent on ICAM-1 [12, 13].

IL17RD is a transmembrane protein that was initially characterized as an inhibitor of FGFR1, and subsequently reclassified as a member of the IL17 signaling family based on the presence of a cytosolic SEFIR domain commonly shared between all IL17 receptors [14–16]. Recent evidence shows that IL17RD functions as an integral component of the IL17RA-IL17RC receptor complex and enhances IL17A-induced proinflammatory phenotypes in mouse models of inflammatory disease [17–20] Additionally, dysregulation in IL17RD expression has been implicated in endothelial dysfunction during disorders of vascular remodeling [21]. Collectively, these results led us to postulate an important role for IL17RD in regulating endothelial cell response to IL17A. We hypothesized that endothelial IL17RD modulates IL17A-induced downstream pathways activation to enhance expression of VCAM-1 and ICAM-1 and consequent leukocyte adhesion. In the present study, this hypothesis was investigated using the RNAi knockdown of gene expression and in vivo knockout mouse models.

MATERIALS AND METHODS

Cell culture, siRNA transfection, and in vitro stimulation

Human Aortic Endothelial Cells (HAEC; catalog no: CC-2535, Lot no: 20TL231227) and Human Umbilical Vein Endothelial Cells (HUVEC; catalog no: CC-2519, Lot no: not available) were obtained from Lonza (Portsmouth, NH) and used up to passage 8 in all experiments. HAECs and HUVECs were cultured on plates coated with 0.2% gelatin (Sigma;St. Louis, MO; catalog no: G9391), in Endothelial Cell Growth Medium-2 (EGM-2; Lonza, catalog no: CC-3162) for the majority of the experiments. The immortalized THP-1 monocytic cells were obtained from American Type Culture Collection (ATCC; Manassas, Virginia; catalog no: TIB-202) and maintained in Roswell Park Memorial Institute – 1640 media (RPMI-1640; HyClone; Logan, UT; catalog no: SH30027) containing 10% FBS, 20mM 4–2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES; Sigma; catalog no: H4034), 1mM sodium pyruvate (Thermo Fisher; Waltham, MA; catalog no:11360070) and 50 μM β-mercaptoethanol (Sigma; catalog no: M6250). THP-1 cells were maintained at a concentration of 0.3 – 0.9 × 10⁶ cells/ml and split every three days. For qPCR analysis, HAECs and HUVECs were grown to 100% confluence and used for qPCR analysis (see RNA extraction and RT-qPCR procedure in subsequent sections). For siRNA transfection, HAECs were grown to 50–60% confluence and transfected with 25 nM IL17RD targeting (Horizon Discovery; Lafayette, CO, catalog no: L-007946–02) or 25 nM non-targeting scrambled control (Horizon Discovery; catalog no: D-001810–10) ON-TARGETplus SMARTpool siRNA (see Table 1) using 2μl/ml of RNAiMax (Thermo Fisher; catalog no: 13778075) in Opti-MEM Reduced Serum Medium (ThermoFisher; catalog no: 31985070) for 6 hours. The cells were then washed with Dulbecco’s Phosphate Buffered Saline (without calcium and magnesium) (dPBS; HyClone; catalog no: SH30028), and allowed to recover in fresh EGM-2 for 48 hours before being used for the indicated experiments. Efficiency of RNAi suppression was evaluated by qPCR.

Table 1:

siRNA sequences

siRNA Target	Sequence
NT SMARTpool	UGGUUUACAUGUGACUAA, UGGUUUACAUGUUGUGUGA, UGGUUUACAUGUUUUCUGA, UGGUUUACAUGUUUUCCUA
IL17RD SMARTpool	GUGUGAUGAUAAAUGGUAA, GAGAGUGACUUCUGCCUAA, ACUACAAGCUCAAGCACGA, UCGACGGACCAGACAGAAA

For experiments involving analysis of endothelial activation markers expression, HAECs transfected with NT or IL17RD siRNA were stimulated with 100 ng/ml recombinant human IL17A (Advent Bio; Elk Grove, IL; catalog no: 11694) or vehicle control (0.2% Bovine Serum Albumin, BSA; Sigma; catalog no: 10735086001) for 24 hours. For short- term signaling studies, HAECs were cultured in Endothelial Basal Media-2 (EBM-2; Lonza; catalog no: CC-3156) for 8 hours to deplete growth factor activity, before being stimulated with IL17A for the indicated time points.

Immunoblotting

Cells were washed with dPBS and lysed in an appropriate volume of 2X SDS sample buffer containing 0.5M Tris-HCl pH 6.8 (Thermo Fisher; catalog no: A144), 10% β-mercaptoethanol, 4% SDS (Sigma; catalog no: S7920), 20% glycerol (Sigma; catalog no: G22020), 0.02% bromophenol blue (Sigma; catalog no: B8026) on ice. The samples were homogenized by sonication and further denatured by boiling at 95°C for 5 minutes. For immunoblotting, proteins were loaded into 8% or 10% SDS-polyacrylamide gels made using Trizma (Sigma; T6066), 30% acrylamide (BioRad; Hercules, CA; catalog no: 1610156), SDS, APS (Sigma; catalog no: A3778), TEMED (Sigma; catalog no: T9281). The samples were separated by electrophoresis and transferred onto nitrocellulose membranes (BioRad; catalog no: 162–0115) at 16 V for 120 minutes in transfer buffer containing 25 mM Tris, 192 mM glycine (Sigma; catalog no: G8898), 0.0002% SDS and 20% methanol (Millipore Sigma; catalog no: MX0485). The membranes were then blocked in a solution of 5% nonfat milk in Tris buffer saline –Tween-20 (TBS-T), containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl (Sigma; catalog no: S3014); 0.05% Tween 20 (Sigma; catalog no: P1379) for 2 hours at room temperature. The membrane was then probed with the indicated primary antibodies (See Table 2) in 5% BSA in TBS at indicated dilutions at 4 °C overnight. The primary antibody was then washed off with TBS-T, and the membranes were further incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Table 3) for 2 hours at room temperature. The membranes were washed for 30 minutes each with TBS-T and detected by autoradiography using chemiluminescent substrate (West Pico, Millipore Sigma; catalog no: WBKLS0500) obtained by mixing Substrate Peroxide solution and Substrate Luminol Reagent (1:1). For analysis, the indicated protein(s) was determined based upon migration of prestained molecular weight standards (Bio-Rad; catalog no: 1610394).

Table 2:

List of primary antibodies used for immunoblotting assays

Target antigen	Vendor	Catalog number	Working dilution
dpERK	Cell Signaling Technology	4370	1:1000
Total ERK	Cell Signaling Technology	9102	1:1000
pAkt	Cell Signaling Technology	9271	1:1000
Total Akt	Cell Signaling Technology	4691	1:1000
pp38	Cell Signaling Technology	9211	1:1000
Total p38	Cell Signaling Technology	8690	1:1000
pJNK	Cell Signaling Technology	9251	1:500
Total JNK	Cell Signaling Technology	9252	1:1000
pp65	Cell Signaling Technology	3033	1:1000
Total p65	Cell Signaling Technology	8242	1:1000
β-Tubulin	Cell Signaling Technology	86298	1:5000
GAPDH	Cell Signaling Technology	2118	1:5000
VCAM-1	Cell Signaling Technology	13662	1:5000
ICAM-1	Cell Signaling Technology	4915	1:500
β-actin	Sigma	8226	1:5000

Table 3:

List of secondary antibodies used for immunoblotting assays

Target antigen	Vendor	Catalog number	IB working dilution
Goat anti-mouse IgG, HRP-linked	Bio-Rad	1706516	1:5000
Goat anti-rabbit IgG, HRP-linked	Bio-Rad	1706515	1:5000

The densitometric intensity of each protein was calculated using the measure feature in ImageJ. The raw values were obtained by subtracting the densitometric intensity of each experimental condition from the background intensity on the same blot. The raw values were then normalized to loading control (Total ERK, Total p38, Total JNK, Total p65, Total Akt or β-actin as indicated) for each experimental condition, and further normalized to unstimulated siNT control to obtain relative densitometric intensity quantification.

Monocyte adhesion assay

HAECs were transfected with NT or IL17RD siRNA according to the procedure described in the preceding sections. The cells were allowed to recover in fresh EGM-2 for 24 hours and were then seeded at a density of 10⁵ cells per well of a 96-well plate. The cells were allowed to attain confluence for 24 hours before being stimulated with vehicle control (0.2% BSA) or recombinant IL17A (100 ng/ml) for 24 hours. THP-1 cells, at a concentration of 3 × 10⁵ cells/well, were labeled with 20μM Calcein Red-Orange (ThermoFisher; catalog no: 34851) for 30 minutes in EBM-2. The labeled THP-1 cells were washed twice with EBM-2 and subsequently added to endothelial cells cultured in EBM-2 for 1 hour. The adherent cells were then washed twice with dPBS (with calcium and magnesium) (HyClone; catalog no: SH30264), and the number of adherent monocytes was quantitatively determined by spectrophotometric measurement at 580 nm using Glomax^® Microplate Reader (Promega; Fitchburg,WI) using a standard curve with a known number of THP-1 cells. The absorbance of labeled THP-1 cells was subtracted from the absorbance of the endothelial monolayer. The wells were then imaged using EVOS M5000 (Thermo Fisher) Inverted fluorescent microscope.

RNA extraction, cDNA preparation and RT-qPCR analysis

Total RNA was extracted from cells using the RNEasy mini kit (Qiagen; Germantown, MD; catalog no: 74106) using the Nanodrop 1000 UV-Vis Spectrophotometer (Thermo Scientific). 1μg of the extracted RNA was reverse transcribed into cDNA using qScript cDNA SuperMix (BioRad; catalog no: 1708890), following which the reaction mixture was diluted in a ratio of 2ng cDNA:1 μl of nuclease-free water. The diluted cDNA was then used for qPCR analysis for use in qPCR analysis using AzuraQuant Green Fast qPCR Mix (AzuraQuant; Raynham, MA; catalog no: AZ-2101). The program used for performing qPCR was as follows: Enzyme activation at 95°C for 2 minutes, followed by denaturation at 95°C for 5 seconds, followed by annealing with gene-specific primers (Table 4) at 60°C for 30 seconds for 40 cycles. Changes in transcript levels were determined as fold change relative to control (2^−ΔΔCt method). Briefly, average Ct values from the housekeeping reference gene (β-actin) for the experimental condition were subtracted from average threshold (Ct) values of the indicated gene for each experimental condition (ΔCt). The normalized Ct value of each experimental condition was then subtracted from the normalized Ct value from the vehicle control to obtain a relative difference in transcript levels (ΔΔCt). 2 to the power of the negative value of the difference was used to obtain fold-change values.

Table 4:

List of primers used in this study

Primer	Sequence
Human IL17RD forward	5’-CTGCGGGTAGCTGAAGGTAG-3’
Human IL17RD reverse	5’-AGCTTTGGCACCTTGTATGG-3’
Human β-actin forward	5’-AGAAAATCTGGCACCACACC-3’
Human β-actin reverse	5’-GTCTCAAACATGATCTGGG-3’
Int3 -F2	5’-GCCAAGCCTTGATATGACAAAC-3’
Int3-R1	5’-TTATGAGTCATTCTCCAGCCCG-3’
GTR1	5’-GGTCTTTGAGCACCAGAGGACATC-3’

Mice and diet feeding

All experimental procedures on mice used in our experiments were performed according to protocols reviewed and approved by the MaineHealth Institute for Research (MHIR) Institutional Animal Care and Use Committee (IACUC, protocol #2104). All mice were housed in a pathogen-free environment under light (14:10 hour light:dark cycle), temperature (70°F), and humidity (55%) controlled conditions for all our experiments. We used groups of 5–8 groups of male and female mice for all our experiments. Mice were euthanized by isoflurane inhalation (Dechra Veterinary Products; Overland Park, KS) followed by cervical dislocation, consistent with the guidelines of the Panel on Euthanasia of the American Veterinary Medical Association. For experiments with C57BL/6 mice, we used 8 week-old mice from the Jackson Laboratory (catalog no: 000664).

The Kst223 (Il17rd−/−) mutant cell line was generated by gene trap technology as described by Leighton et al. [22], resulting in the insertion of the GT1TMpfs vector into the third intron of the endogenous Il17rd locus on an FVB background. The mutant mice contain a fusion of β-geo within intron 3 at nucleotide 332 [23]. These mice have been validated by our laboratory as well as by others [20, 23, 24]. The mice were backcrossed on to the C57BL/6 genetic background for at least 10 generations for our experiments. Mice were genotyped by PCR amplification of tail DNA with a three-primer combination: Int3-F2 and Int3-R1 for the wild-type band and Int3-F2 and GTR1 specific for the Kst223 insertion band as previously described [23, 24]. A representative validation of the genotype of the mice is indicated in Figure S3. Wild-type littermates were used as experimental controls (Il17rd+/+). 8–11 week old mice of the indicated genotypes were fed a western diet (WD) comprising high fat (21.2%), high cholesterol (0.21%) and no sodium cholate (Research Diets; New Brunswick, NJ; catalog no: D12079B) or standard chow diet (CD; Envigo; Indianapolis, IN; catalog no: T-2918) ad libitum for 14 weeks as indicated. The number of mice used in our experimental cohorts is given in Table 5.

Table 5:

Number of mice used for experimental analyses

			Diet
In vivo experiment	Sex	Genotype	CD	WD
Analysis of Il17rd expression	Males	C57BL6/J	6	-
	Females	C57BL6/J	6	-
Aortic leukocyte infiltration analysis	Males	Il17rd +/+	8	6
		Il17rd −/−	6	7
	Females	Il17rd +/+	8*	8
		Il17rd −/−	5	5
Aortic endothelial VCAM-1 and ICAM-1 expression	Males	Il17rd +/+	8	8
		Il17rd −/−	5	6
	Females	Il17rd +/+	8	8
		Il17rd −/−	8	8

^*One mouse developed malocclusions so was not included in the study

Primary murine embryonic fibroblasts (MEF) isolation and sample preparation:

Primary MEFs were isolated from E12.5 – E13.5 embryos obtained from timed matings between Il17rd +/− male and female mice. Embryos were genotyped by PCR amplification with the three-primer combination specific for the Kst223 insertion band as previously described [23, 24] following which MEF samples with the same genotypes were pooled together to obtain sufficient numbers of cells for proteomics analysis (n=2–3 embryos per group). The isolated MEFs were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Cytiva; Marlborough, MA; catalog no: SH30022) supplemented with 10% FBS and used at passage 5. For mass spectrometry analysis, MEFs were stimulated with IL17A (100 ng/ml) and lysed in HNTG lysis buffer containing 20 mM HEPES, 150 mM NaCl, 1% Triton X-100 (Sigma; catalog no:T8787), 10% glycerol, 1 mM Ethyleneglycol- bis β-aminoethyl-N,N,Nʹ,Nʹ-tetraacetic Acid (EGTA; Sigma; catalog no: E3889), 1.5 mM MgCl₂ (Sigma; catalog no: M8266),1mM sodium orthovanadate (Sigma; catalog no: 567540), 1mM sodium fluoride (Sigma; catalog no: 201154), 1 tablet/50ml buffer of protease inhibitor cocktail (ThermoFisher; catalog no: A32963).

Physiological measurements

Mice that were fed with WD or CD were weighed weekly. After 14 weeks of WD or CD feeding, body composition was determined by dual-energy x-ray absorptiometry (DEXA) using the PIXImus densitometer (GE Medical Lunar, Madison, WI) and Mini Spec Nuclear Magnetic Resonance (NMR, Bruker, Billerica, MA) as described in [25].

Hematological and serum analysis

For analysis of Il17rd expression in circulating leukocytes, blood was treated with 0.5M citrate buffer (Thermo Fisher, catalog no: J62918.AK) before being stained with respective antibodies for flow cytometry (see procedure below).

For obtaining differential leukocyte count, blood was collected from the mice by retroorbital bleeding technique into EDTA Microtainer tubes (BD Biosciences; Franklin Lake, NJ; catalog no: 367856) and was immediately analyzed by ProcyteDx Hematology Analyzer (Idexx, Westbrook, ME) to obtain circulating hematological parameters.

For analysis of circulating cholesterol and IL17, mice were fasted for 4 hours. Whole blood was centrifuged at 2500 rpm for 15 minutes, and the supernatant was collected to obtain serum. Levels of circulating cholesterol levels were measured using the Total cholesterol kit (Cell BioLabs; San Diego, CA; catalog no: STA-384) according to the manufacturer’s instructions. Circulating levels of IL17A were determined using the R&D Quantikine^™ HS Mouse IL-17 Immunoassay (R&D Systems, Minneapolis, MN, catalog no: MHS170) according to the manufacturer’s instructions. All hematological and serum assays were performed in singlicate.

Tissue harvest

Post euthanasia, the peritoneal and thoracic cavities of the mouse were exposed, and the left ventricle of the heart was perfused with PBS. The organs of the respiratory (lungs, trachea) and the digestive (stomach, intestine, and liver) systems were removed from the thoracic and abdominal cavities to expose the heart, diaphragm and kidneys. The aorta and aortic branches (brachiocephalic and carotid arteries) were then dissected from the heart until the aortic bifurcation according to the procedure described in [26]. The harvested aorta was subjected to flow cytometry according to the procedure described below.

Flow cytometry analysis

For in vivo aortic flow cytometry, the aorta was dissected until the thoracoabdominal branch, and the surrounding perivascular and periaortic lymph nodes were removed. The aorta was then subjected to flow cytometry analysis according to the procedure described in [27]. Briefly, the aorta was digested using an enzyme cocktail mixture of 10 mg/ml collagenase type II (Worthington Biochemical, Lakewood, NJ, catalog no: LS004174) and 5 mg/ml Dispase Type II (Sigma, catalog no: D4693) for 30 min at 37°C. For our experiments involving analysis of in vivo aortic EC, we combined samples from 2 mice to account for the low number of endothelial cells. The cell suspension was then incubated with anti-mouse CD16/CD32 (BD Biosciences, catalog no: 553140) to block Fc receptors, followed by staining with the indicated antibodies in FACS buffer (PBS + 2% BSA + 2mM EDTA) for 20 minutes. The cells were then stained with 4,6-diamidino-2-phenylindole (DAPI; 0.04 μg/ml; Thermo Fisher; catalog no: 62248) for assessment of cell viability before being analyzed by flow cytometry for the indicated markers. Data were collected using the MACSQuant Analyzer 10 (Miltenyi; Gaithersburg, MD) and analyzed using the FlowJo software (BD Biosciences). For analysis, viable singlet cells were gated into immune and non-immune cells by quantifying the number of CD45^pos and CD31b^pos cells (antibody list in Table 6). The mean expression level (ΔMFI) of ICAM-1, VCAM-1, IL17RA, and IL17RC was obtained in CD31^pos cells in the non-immune cell fraction by subtracting the MFI of unlabeled cells from the MFI of the labeled cells.

Table 6:

List of antibodies used for flow cytometry analysis

Antibody	Target species	Clone	Company
IL17RD	Mouse	309822	R&D
IL17RA	Mouse	448	R&D
IL17RC	Mouse	2270	R&D
CD45	Mouse	30-F11	Biolegend
CD11b	Mouse	M1/70	Biolegend
Ly6G	Mouse	1A8	Biolegend
Ly6C	Mouse	HK1.4	Biolegend
MHCII	Mouse	M5/114/15.2	Biolegend
F480	Mouse	BM8	Biolegend
CD11c	Mouse	N418	eBioscience
VCAM-1	Mouse	429	Biolegend
ICAM-1	Mouse	YN1/1.7.4	Biolegend

For immune cells in the aorta and blood, the myeloid cell population was obtained in the CD45^pos cells by gating the number of CD11b^pos cells. The number of neutrophils in the aorta was quantified by gating the number of Ly6G^pos within the CD11b^pos myeloid cells. The Ly6G^neg cell population was then gated for Ly6C^pos cells to obtain the number of monocytes infiltrated into the aorta. Cells expressing MHCII, F4/80, and CD11c were defined as macrophages. For analysis of Il17rd expression in blood, viable singlet cells were gated into CD11b^pos myeloid cells. A flowchart showing the gating strategy is shown in Figure S2.

Proteomics analysis and data acquisition

Liquid chromatography/mass spectrometry (LC/MS) was conducted according to previously established methods [28, 29]. All solvents used for LC/MS were mass spectrometry or equivalent grade. Briefly, cellular lysates from each experimental condition were resuspended in 400 μl of solution containing 8M Urea in 50mM Tris-HCl pH 8.0 containing phosphatase and protease inhibitors (Roche) followed by sonication for 3 × 10 seconds (Branson Sonifier 250, Branson Ultrasonics), and cooled on ice [28, 29]. Following disulfide reduction and capping with dithiothreitol (DTT) and iodoacetamide, the samples were digested with trypsin overnight (Promega, Trypsin Protease, MS Grade, sequencing grade). The tryptic digest samples were purified with C18 Spin Columns. (ThermoFisher Scientific).

Peptide separation for LC/MS was accomplished using Dionex Ultimate (RSLC_3000) chromatography system using C18 reverse phase chromatographic columns (Thermo Scientific) [28, 29]. Each sample was loaded onto an analytical reverse-phase C18 nanocolumn (40 cm length, 75 μm ID) packed with Reprocil Pur C18,1.9μm and resolved by an increasing acetonitrile gradient over 100 min at a flow rate of 220 nL/min. A gradient of increasing buffer B (80% acetonitrile, 19.9% water, 0.1% formic acid) from 10–35% from 91–95 minutes; to 35–95% from 95–98 minutes, to a steady concentration of 95% of buffer B from 98–100 minutes, followed by a decrease in buffer B from 95–2%. Buffer A: 98% water, 1.9% acetonitrile, 0.1% formic acid; Buffer B: 80% acetonitrile, 19.9% water, 0.1% formic acid.

Tryptic peptides were analyzed on a Sciex TripleTOF 5600 mass spectrometer. The mass spectrometer was operated using data dependent acquisition (DDA) to create a spectral ion library. A Sequential window acquisition of all theoretical spectra (SWATH) was implemented for relative quantitation as previously described [30]. For both DDA and SWATH workflows, the mass spectrometer used an ion spray voltage floating (ISVF) of 2400 V, curtain gas (CUR) 25 PSI, interface heater temperature (IHT) 150 °C, ion source gas 1 of 6 PSI and a declustering potential (DP) 100 V. All data acquired used a high resolution MS scan from 350–1500 m/z to select the 50 most intense ions prior to MS/MS analysis using collision induced dissociation (CID). Charge states 2–5 were selected with an exclusion time of 15 seconds and an accumulation time of 250ms for time-of-flight (TOF) MS, and 50ms for TOF MS/MS, cycle time 2.8 seconds [28, 29].

Statistical analysis

Data were tested for normality using the Shapiro-Wilk test. Data sets with the normal distribution of variables were analyzed for statistical significance using Student’s unpaired t-test for comparisons between 2 experimental groups or two-way analysis of variance (ANOVA) with Sidak’s post-hoc test and are presented as Mean ± Standard Deviation (S.D) for more than 2 experimental groups. Data sets which were significantly departed from normality by the Shapiro-Wilk test were tested for statistical significance using the Mann-Whitney test for comparisons between 2 experimental groups or two-way analysis of variance (ANOVA) with Sidak’s post-hoc test after logarithmic transformation of variables and represented as Median ± Interquartile range (IQR). A p-value of less than 0.05 was considered significant for all our statistical tests. For LC/MS analysis, data were searched against the reviewed mouse UniProt database (37,201 proteins) using the ProteinPilot software (Sciex, Version 5.0.5) with the Paragon algorithm. Peptides used for protein identification met a minimum confidence threshold of greater than 99%. Spectral alignment and targeted extraction of SWATH data were performed using the SWATH Processing Micro App in PeakView (Sciex, Version 2.2.0) using the reference DDA ion library. The data was imported into MarkerView (Sciex, Version 1.2.1) where the data was ratio-normalized. The groups were compared by principle component analysis (PCA) following most likely ratio (MLR)-based normalization, and compared for significance using Fisher’s modification of Student’s t-test [31]. Additional data, including PCA test without MLR normalization, are available in Supplemental data.

RESULTS:

Expression of IL17RD in human aortic endothelial cells.

We first determined the expression of IL17RD in Human Aortic Endothelial Cells (HAECs) and in the THP-1 human monocytic cell line using RT-qPCR. Because IL17RD is highly expressed in human umbilical vein endothelial cells (HUVECs) [32, 33], the total RNA isolated from these cells was used as a positive control. As shown in Figure 1, we observed that HAECs express significantly higher levels of IL17RD compared to HUVECs, whereas THP-1 cells express IL17RD at 20% lower levels than HUVECs. Thus, our results demonstrate high levels of IL17RD mRNA expression in primary HAECs

Figure 1: Analysis of Il17rd expression in human endothelial cells and THP-1 cells by RT-qPCR.

HUVEC and HAEC cells at passage 5 were grown to confluence and harvested. THP-1 cells were harvested at a concentration of 0.3–0.9 × 10⁶ cells/ml. Total RNA was isolated from the cells, and was reverse transcribed for analysis of IL17RD expression by RT-qPCR. Graphs representing quantification of IL17RD mRNA normalized to β-actin in HUVEC, HAEC, and THP-1 cells. **p<0.005, ***, p<0.0005

Role of IL17RD in IL17A–induced adhesion of monocytes

We then tested the role of IL17RD in the modulation of IL17A-induced aortic EC-monocyte interaction by employing an RNAi-mediated IL17RD inactivation approach. To suppress IL17RD levels in HAECs, we transfected 50–60% confluent HAECs with IL17RD siRNA or NT scrambled control siRNA and assessed IL17RD mRNA levels 72 hours after transfection. As shown in Figure 2a, transfection of HAECs with IL17RD siRNA resulted in an 83% decrease in IL17RD mRNA expression compared to HAECs transfected with NT siRNA.

Figure 2: siRNA-driven down-regulation of IL17RD in human endothelial cells suppresses the adhesion of THP-1 cells.

HAECs were grown to 50–60% confluence and transfected with NT or IL17RD siRNA for 6 hours (a) Graphs representing quantification of IL17RD mRNA expression by RT-qPCR in HAECs transfected with siNT or siIL17RD demonstrating efficiency of siRNA mediated knockdown after 72h (b) Quantification of adhesion of fluorescently labeled THP-1 cells to confluent monolayer of vehicle (0.2% BSA) or IL17A treated siNT or siIL17RD transfected HAECs after IL17A treatment for 24 hours. Data are representative of n=5 replicates.(c) Representative fluorescence micrograph images of experimental wells from (b) taken at original 40X magnification. Scale bars depict 750 μm. Data were analyzed by two-way ANOVA with Sidak’s post-hoc test. Results in panel (a) are presented as Mean ± S.D., results in panel (b) are presented as Median ± I.Q.R. For results in panel (b), data was analyzed with two-way ANOVA using absolute values, log transformed values did not demonstrate statistical significance in the NT group. *p<0.05, **p<0.005

Next, we assessed the role of IL17RD in the modulation of IL17A-induced aortic EC-monocyte interaction, specifically with respect to the adhesion of monocytes onto the endothelial monolayer [12]. For this purpose, we used THP-1 cells as a human monocytic cell line model that does not express IL17RD (Fig. 1a). We then stimulated siNT or siIL17RD transfected HAECs with IL17A or vehicle control in vitro for 24 hours and added THP-1 cells fluorescently labeled with Calcein Orange-Red for 1 hour. As shown in Figures 2b and 2c, siIL17RD transfected HAECs had significantly decreased the adhesion of THP-1 cells to the endothelial monolayer compared to siNT transfected HAECs in response to IL17A stimulation. Thus, our results indicate that IL17RD promotes IL17A induced adhesion of THP-1 cells to the endothelial monolayer in vitro.

IL17A does not stimulate VCAM-1 and ICAM-1 expression in HAECs.

Next, we investigated whether IL17RD regulates EC activation downstream of IL17A. In order to determine this, we analyzed the expression of endothelial cell adhesion molecules, specifically VCAM-1 and ICAM-1 in siNT or siIL17RD transfected HAECs stimulated with IL17A for 24-hours [12, 13]. As shown in Figure 3a–c, immunoblot analyses of NT and IL17RD transfected HAECs showed that stimulation of HAECs with IL17A did not induce any difference in the expression of VCAM-1 and ICAM-1, and further suppression of IL17RD did not affect the expression of VCAM-1 and ICAM-1 upon treatment with vehicle or IL17A. Thus, our results suggest that IL17RD promotion of IL17A induced THP-1 adhesion to HAECs is not dependent of upregulation of VCAM-1 and ICAM-1 expression.

Figure 3: IL17RD promotes monocyte adhesion in HAECs in an ICAM-1- and VCAM-1-independent manner.

(a) Representative immunoblots of protein expression of endothelial markers VCAM-1 (120 kDa) and ICAM-1 (92 kDa) in siNT or siIL17RD transfected HAECs treated with either vehicle control (0.2% BSA) or IL17A (100 ng/ml) for 24 hours. Figure (a) is representative of n=3 independent experiments. (b,c) Graphs representing densitometric quantification of n=3 independent experiments normalized to β-actin (45 kDa). Results in panel (b) are presented as Median ± I.Q.R, results in panel (c) are presented as Mean ± S.D. Data were analyzed using two-way ANOVA with Sidak’s post-hoc test.

IL17A does not significantly induce activation of the NFκB and MAPK pathways in HAECs.

We then sought to determine whether IL17RD modulates IL17A-induced activation of the canonical MAPK and NF-κB signaling pathways [17]. To investigate this, we determined IL17A-induced activation of known IL17RD signaling effector pathways in siNT or siIL17RD treated HAECs, including ERK [14] Akt [14, 34], JNK [35], p38 [17, 18] and NF-κB pathways [36, 37]. As shown in Figure 4a–g, immunoblot analyses showed that IL17A stimulation did not induce a significant increase in the activation of the ERK, p38, JNK, Akt, and NF-κB pathways in HAECs. Further, depletion of IL17RD did not affect the activation of either pathway, suggesting that IL17RD promotion of IL17A-induced THP-1 cell adhesion is not dependent on IL17-mediated activation of canonical MAPK and NF-κB signaling pathways in HAECs.

Figure 4: Effect of IL17RD inactivation on the activity of downstream signaling cascades in human endothelial cells stimulated with IL17A.

(a) Representative immunoblots depicting the activation of ERK (42, 44 kDa), p38 (40 kDa) and JNK (46,54 kDa) MAPK pathways and (b) PI3K/Akt (60 kDa) and NF-κB (65 kDa) pathways in siNT or siIL17RD transfected HAECs treated with IL17A (50 ng/ml) for the indicated time points. Figures (a, b) are representative of n=3 independent experiments. (c-g) Graphs representing densitometric quantification of blots from n=3 independent experiments normalized to total ERK (42,44 kDa), total p38 (40 kDa), total JNK (46,54 kDa), total Akt (60 kDa) and total NF-κB (65 kDa) as indicated on each graph. Graphs (c-g) represent quantification of n=3 replicates. Results in panels c and d are presented as Median ± I.Q.R, results in panels e,f and g are presented as Mean ± S.D. Data were analyzed using two-way ANOVA with Sidak’s post-hoc test.

Il17rd is expressed in aortic endothelial cells in vivo.

Next, we sought to investigate the in vivo relevance of our findings of Il17rd promotion of monocyte attachment. For this purpose, we initially characterized Il17rd expression in aortic ECs and in circulating blood myeloid cells in male and female C57BL/6 mice as per the gating strategy in Fig S2. As shown in Figure 5a, we observed expression of Il17rd in aortic endothelial cells, whereas we did not detect any expression of Il17rd in CD45^pos CD11b^pos myeloid cells in the circulating blood (Representative histogram in Figure S4). We obtained similar results in females, as shown in Figure 5b, suggesting that Il17rd expression in vivo is independent of biological sex. Collectively, our findings establish that Il17rd is expressed in aortic endothelial cells in vivo.

Figure 5: Il17rd is expressed in aortic endothelial cells in vivo.

Single cell-suspensions of aortas and blood leukocytes were obtained from 8-week-old male and female C57BL/6J mice (n=6 mice/group). Analysis of cell surface Il17rd expression in circulating blood and aortic cell suspensions of by flow cytometry. Quantification of Il17rd expression on the cell surface of: (a) CD31^pos Endothelial cells (b) CD45^posCD11b^pos immune cells. Each data point is representative of aortic ECs combined from 2 mice. Results are presented as Mean ± S.D.; nd - not detected

Loss of Il17rd decreases weight gain and proportion of body fat in male mice upon 14-week WD feeding.

To evaluate in vivo extent of leukocyte infiltration and in vivo aortic EC expression of VCAM-1 and ICAM-1, we used 8–10 week-old mice male and female global Il17rd null mice [22] or littermate wild-type (Il17rd +/+) mice and fed them with WD or CD for 14 weeks. We initially assessed physiological parameters in these mice. As shown in Fig 6a, male Il17rd−/− mice were characterized by significantly lower body weight upon 14 weeks of WD feeding as compared to male Il17rd+/+ mice, whereas female Il17rd−/− mice showed no differences in body weight compared to Il17rd+/+ mice upon 14 weeks of WD feeding as shown in Fig 6b. The cohorts of Il17rd−/− and Il17rd+/+ mice that were maintained on CD showed no difference in body weight upon 14 weeks of feeding either in males as shown in Fig 6c or in females as shown in Fig 6d. Thus, our results suggest that Il17rd promotes weight gain upon WD feeding in male mice. Next, we analyzed body composition in our experimental groups. As shown in Fig 6e, in male mice, WD feeding for 14 weeks increased the amount of body fat in Il17rd+/+ mice as compared to CD fed mice consistent with previous literature [38], whereas Il17rd−/− mice were resistant to increase in body fat upon WD feeding as compared to CD-fed mice. Further, Il17rd−/− mice had significantly decreased body fat as compared to WD fed Il17rd+/+ mice. In females, WD feeding did not affect the amount of body fat in Il17rd+/+ mice upon WD feeding [38], and deletion of Il17rd did significantly affect the amount of body fat upon CD or WD feeding as shown in Fig 6f. We then analyzed the amount of lean mass in our experimental cohorts. As shown in Figures 6g and 6h, 14-week WD feeding did not affect the amount of lean mass in Il17rd+/+ mice or Il17rd−/− mice as compared to CD-fed mice in males or in females. Thus, our results suggest that Il17rd promotes weight gain and accumulation of body fat in male mice upon 14 week WD feeding.

Figure 6: Loss of Il17rd decreases weight gain and proportion of body fat in male mice upon 14 week WD feeding.

Weekly weight of 8–10 week old (a) male and (b) female Il17rd−/− and Il17rd+/+ mice upon 14-week WD feeding. Weekly weight of 8–10 week old (c) male and (d) female Il17rd−/− and Il17rd+/+ mice upon 14 week of WD or CD feeding. Amount of body fat in (e) males and (f) female Il17rd−/− and Il17rd+/+ after 14 weeks WD or CD feeding. Amount of lean mass in (g) males and (h) female Il17rd−/− and Il17rd+/+ after 14 weeks of WD or CD feeding. Data were analyzed using two-way ANOVA with Sidak’s post-hoc test. Results in panels (a-d, g, h) are presented as Mean ± S.D., results in panels (e,f) are presented as Median ± I.Q.R *p<0.05, **p<0.005. n=5–8 mice per sex per genotype.

Effect of loss of Il17rd on the levels of circulating IL17A and cholesterol in male and female mice upon WD feeding.

We first analyzed serum levels of circulating IL17A in our experimental groups. In males, WD feeding induced an increase in serum levels of IL17A in Il17rd+/+ mice as compared with Il17rd+/+ mice fed a CD, consistent with previous findings [39]. However, deletion of Il17rd did not significantly affect WD-induced upregulation in serum IL17A levels as shown in Figure 7a. In females, WD feeding did not induce any differences in serum levels of IL17A in either Il17rd+/+ or Il17rd−/− mice experimental groups, as shown in Figure 7b, indicating that WD-induced upregulation of IL17A is dependent on biological sex.

Figure 7: Effect of loss of Il17rd on the levels of circulating IL17A and cholesterol levels in male and female mice upon WD feeding.

Serum levels of IL17A in 8–11 week old (a) male and (b) female Il17rd−/− and Il17rd+/+ mice fed a WD or CD for 14 weeks. Levels of total serum cholesterol in (c) male and (d) female Il17rd−/− and Il17rd+/+ mice fed a WD or CD for 14 weeks. Data were analyzed by two-way ANOVA with Sidak’s post-hoc test. For results in panel (a), data was analyzed with two-way ANOVA using absolute values, as log transformed values did not demonstrate statistical significance in the Il17rd+/+ group. Results in panels (a,b,c) are presented as Median ± I.Q.R, results in panel (d) are presented as Mean ± S.D. *p<0.05, **p<0.005, ***p<0.0005. n=5–8 mice per sex per genotype.

We then analyzed serum levels of circulating cholesterol as previous studies have shown that cholesterol is essential for the differentiation of Th17 cells and induction of IL17 signaling in vivo [40]. As shown in Fig 7c–d, total cholesterol levels were elevated in male Il17rd+/+ and Il17rd−/− mice upon WD feeding as compared to CD, and the levels were not significantly different between the two groups. Whereas in females, the levels of cholesterol were significantly decreased in WD-fed Il17rd−/− mice as compared to WD-fed Il17rd+/+ mice. Collectively, our results suggest that Il17rd upregulates circulating levels of IL17 in male mice upon WD feeding, whereas females show no significant upregulation of WD-induced IL17 signaling in vivo.

We first analyzed serum levels of circulating IL17 in our experimental groups. In males, WD feeding induced an increase in serum levels of IL17A in Il17rd+/+ mice as compared with Il17rd+/+ mice fed a CD, consistent with previous findings [39]. However, deletion of Il17rd did not significantly affect WD-induced upregulation in serum IL17A levels as shown in Figure 7a. In females, WD feeding did not induce any differences in serum levels of IL17A in either Il17rd+/+ or Il17rd−/− mice experimental groups, as shown in Figure 7b, indicating that WD-induced upregulation of IL17A is dependent on biological sex.

We subsequently analyzed serum levels of circulating cholesterol as previous studies have shown that cholesterol is essential for the differentiation of Th17 cells and induction of IL17 signaling in vivo [40]. As shown in Fig 7c–d, total cholesterol levels were elevated in male Il17rd+/+ and Il17rd−/− mice upon WD feeding as compared to CD, and the levels were not significantly different between the two groups. Whereas in females, the levels of cholesterol were significantly decreased in WD-fed Il17rd−/− mice as compared to WD-fed Il17rd+/+ mice. Collectively, our results suggest that Il17rd upregulates IL17 in male mice, whereas females show no significant upregulation of IL17 signaling in vivo.

Loss of Il17rd decreases myeloid cell infiltration into the mouse aorta.

We then evaluated the extent of in vivo leukocyte infiltration into the aorta in our experimental cohorts of Il17rd−/− or Il17rd−/− mice fed with WD or CD for 14 weeks. Fresh blood was harvested from the mice and was subject to hematological analysis to determine circulating blood parameters, following which we collected the aortas and assessed the extent of differential leukocyte infiltration into the aorta by flow cytometry. As shown in Figures 8a and 8b, total aortic cell counts were similar between Il17rd −/− and Il17rd +/+ mice fed with WD or CD in males as well as in females. We then analyzed leukocyte infiltration into the aorta in our experimental cohorts. As shown in Figure 8c, total circulating leukocyte counts were decreased in Il17rd−/− mice fed with a WD, whereas in females the number of circulating leukocytes was not significantly altered between male Il17rd −/− and Il17rd +/+ mice fed either a WD or CD. Analysis of leukocyte accumulation in the aorta showed that WD increased accumulation of CD11b^pos myeloid cells in the aortas of wild-type mice as has been previously described [41], whereas Il17rd deficient mice showed a significant decrease in relative accumulation of myeloid cells as shown in Figure 8d. We observed a similar phenotype in aortic infiltration of myeloid cells in females, suggesting that Il17rd regulation of leukocyte accumulation occurs independent of biological sex, indicated by Figures 8e and 8f. Next, we characterized the relative infiltration of Ly6C^pos proinflammatory monocytes. As shown in Figure 8g, in males, relative levels of circulating monocytes were not significantly different between Il17rd −/− and Il17rd +/+ mice fed either a WD or CD for 14 weeks. Further, we observed an increase in aortic infiltration of Ly6C^pos monocytes upon WD feeding in Il17rd+/+ mice [42] and further loss of Il17rd was associated with a statistically significant decrease in Ly6C^pos proinflammatory monocyte accumulation in the aorta upon WD feeding as shown in Figure 8h. Whereas the relative number of F480/MHCII/CD11c macrophages remained unchanged, as shown in Figure 8k. In females, we did not obtain any statistical significance in proinflammatory monocyte infiltration amongst the experimental groups, however we observed an increased number of F480/MHCII/CD11c macrophages as shown in Figures 8i–8l, indicating that Il17rd affects aortic recruitment of the monocyte-macrophage population upon WD feeding [42]. We also characterized the infiltration of neutrophils since previous findings have implicated IL17RD in promoting neutrophilia [17, 18]. As shown in Figures 8m and 8n, we observed that WD-feeding induced a statistically significant increase in the infiltration of Ly6G^pos neutrophil cells in the aorta in males [43], and deletion of Il17rd significantly decreased neutrophil infiltration upon WD feeding. Whereas in females, WD feeding did not affect the infiltration of neutrophils as shown in Figures 8o–8p. Summarily, our results suggest that Il17rd functions to promote IL17A-induced aortic infiltration of myeloid cells upon WD feeding in vivo.

Figure 8: Loss of Il17rd decreases myeloid infiltration into the mouse aorta.

Analysis of differential leukocyte subpopulations in circulating blood and aortic cell suspensions of 8–10 week old Il17rd −/− and Il17rd +/+ mice maintained on a WD or CD for 14 weeks (n = 5–8 mice per group per sex). Total number of cells in aortic cell suspensions in (a) males and (b) females. Number of circulating and infiltrated leukocytes in (c,d) males and in (e,f) females. Total number of circulating and infiltrated monocytes in (g,h) males and in (i,j) females. Aortic cell counts of macrophages in (k) males and (l) females. Number of circulating and infiltrated neutrophils in (m,n) males and in (o,p) females. Data were analyzed by two-way ANOVA with Sidak’s post-hoc test. Results in panels (d,g,h,k,m,n,o) are presented as Median ± I.Q.R, results in panels (a,b,c,e,f,I,j,l,p) are presented as Mean ± S.D. *p<0.05, **p<0.005, ***p<0.0005. n=5–8 mice per sex per genotype.

Loss of Il17rd does not significantly affect the aortic endothelial expression levels of VCAM-1 and ICAM-1 upon WD feeding.

We further investigated in vivo aortic EC expression levels of VCAM-1 and ICAM-1 in aortic cell suspensions of our experimental cohorts of Il17rd −/− and Il17rd +/+ mice maintained on a WD or CD for 14 weeks [44]. As shown in Figures 9a and 9b, WD-fed Il17rd+/+ and Il17rd−/− mice demonstrated no significant upregulation in aortic EC expression of VCAM-1 in males or in females. Next, we characterized the aortic EC expression of ICAM-1 in these mice. As shown in Figures 9c and 9d, expression of ICAM-1 was not significantly altered in Il17rd+/+ and Il17rd−/− upon WD feeding, which corroborates our in vitro findings that IL17 signaling mediates proinflammatory monocyte accumulation independent of upregulation in CAM expression. Thus our results suggest that WD-induced upregulation in IL17 signaling did not affect the expression of VCAM-1 and ICAM-1 in aortic endothelial cells in mice.

Figure 9: Loss of Il17rd does not affect the level of VCAM-1 and ICAM-1 expression on the aortic endothelial cells.

Analysis of cell –surface expression of VCAM-1 and ICAM-1 on CD31^pos endothelial cells from aortic cell suspensions of 8–11 week old Il17rd −/− and Il17rd +/+ mice maintained on a WD or CD for 14 weeks (n = 5–8 mice per group per sex). Analysis of VCAM-1 expression in (a) males and (b) females, ICAM-1 expression in (c) males and (d) females, and CD38 expression in (e) males and (f) females. Each data point is representative of aortic ECs combined from 2 mice. Data were analyzed using two-way ANOVA with Sidak’s post-hoc test. Results in panels (a,b,d) are presented as Median ± I.Q.R, and results in panel (c) are presented as Mean ± S.D.

Loss of Il17rd does not significantly affect the aortic endothelial expression levels of IL17RA and IL17RC upon WD feeding.

Since Il17rd has been shown to have overlapping functions with Il17rc in the context of IL17 signaling, we then characterized the expression of known receptors of IL17 signaling, including IL17RA and IL17RC in our experimental cohorts of Il17rd −/− and Il17rd +/+ mice maintained on a WD or CD for 14 weeks. As shown in Figures 10a and 10b, we observed no difference in aortic EC expression of IL17RA upon WD feeding in Il17rd+/+ mice and Il17rd−/− mice in males and in females. We then further characterized the expression of IL17RC in our experimental cohorts and observed no differences in aortic EC expression of IL17RC in our mice as shown in Figures 10c and 10d. Therefore, our results suggest that loss of Il17rd does not induce a compensatory upregulation in the expression of IL17RA and IL17RC in vivo.

Figure 10: Loss of Il17rd does not significantly affect the aortic endothelial expression levels of IL17RA and IL17RC upon WD feeding.

Analysis of expression of IL17RA and IL17RC in endothelial cells from aortic cell suspensions of 8–11 week old Il17rd −/− and Il17rd +/+ mice maintained on a WD or CD for 14 weeks (n = 5–8 mice per group per sex). Analysis of IL17RA expression in (a) males and (b) females and IL17RC expression in (c) male and (d) female mice. Each data point is representative of aortic ECs combined from 2 mice. Data were analyzed by two-way ANOVA with Sidak’s post-hoc test. Results in panels (a,b,c) are presented as Mean ± S.D., results in panel (d) are presented as Median ± I.Q.R.

Proteomic analysis of primary murine embryonic fibroblasts (MEFs) from Il17rd+/+ and Il17rd−/− mice reveals additional novel pathways and molecular mediators of Il17rd

We sought to gain further insight into the intracellular signaling pathways and downstream effector molecules that might be regulated by IL17RD in our in vivo experimental model using mass spectrometry. For this purpose, we used primary murine embryonic fibroblasts (MEFs) isolated from Il17rd+/+ and Il17rd−/− mice as a model to overcome the technical challenges associated with growing sufficient numbers of mouse primary endothelial cells required for a large scale proteomic analysis experiment. For our experimental purposes, we stimulated the Il17rd+/+ and Il17rd−/− MEFs with IL17A and analyzed the samples by mass spectrometry analysis (Figure S4). Preliminary results showed that comparatively to Il17rd+/+ MEFs the expression of Protein Kinase C alpha (PKCα) was decreased in Il17rd−/− MEFs under basal conditions, whereas the expression of tyrosine-protein kinase Janus Kinase 1 (JAK1) was significantly downregulated in Il17rd−/− MEFs upon IL17A stimulation, indicating that Il17rd might promote the activation of the respective signaling pathways (Table 7). When we further analyzed the expression of adhesion molecules, we observed that the expression of Myeloid-associated differentiation marker-1 (MYADM-1), a molecule that has been shown to enhance endothelial cell activation through ICAM-1 [45], as well as endothelin converting enzyme-1 (ECE-1) [46] were also significantly decreased in Il17rd−/− MEFs stimulated with IL17A. Further, we also detected a small although statistically significant increase in the levels of α and β-catenin in Il17rd−/− MEFs upon IL17A treatment (Table 7). In addition to this, we observed a decrease in the expression of mesenchymal markers vimentin and calponin, as well as in the expression of autophagy marker Sequestosome-1 (SQSTM1, p62) in Il17rd−/− MEFs treated with IL17A. Collectively, our results provide novel additional targets that might be regulated by IL17RD in response to IL17A.

Table 7:

List of differentially expressed molecules between primary MEFs isolated from Il17rd+/+ (Sef WT) and Il17rd−/− (Sef KO) mice stimulated with or without IL17A (50 ng/ml) and analyzed by mass spectrometry.

Group	Accession	Name	p-value	Fold change
Il17rd WT to Il17rd KO	sp|P20444|KPCA_MOUSE	Protein kinase C alpha type OS=Mus musculus GN=Prkca PE=1 SV=3	0.007	1.76
	sp|P13597|ICAM1_MOUSE	Intercellular adhesion molecule 1 OS=Mus musculus GN=Icam1 PE=1 SV=1	0.011	0.55
Il17rd WT +IL17to Il17rd KO + IL17	sp|P52332|JAK1_MOUSE	Tyrosine-protein kinase JAK1 OS=Mus musculus GN=Jak1 PE=1 SV=1	2.87E-05	1.53
	sp|P13597|ICAM1_MOUSE	Intercellular adhesion molecule 1 OS=Mus musculus GN=Icam1 PE=1 SV=1	1.67E-07	0.54
	sp|O35682|MYADM_MOUSE	Myeloid-associated differentiation marker OS=Mus musculus GN=Myadm PE=1 SV=2	0.019	1.29
	sp|Q4PZA2|ECE1_MOUSE	Endothelin-converting enzyme 1 OS=Mus musculus GN=Ece1 PE=1 SV=1	0.002	1.21
	sp|P26231|CTNA1_MOUSE	Catenin alpha-1 OS=Mus musculus GN=Ctnna1 PE=1 SV=1	0.012	0.92
	sp|Q02248|CTNB1_MOUSE	Catenin beta-1 OS=Mus musculus GN=Ctnnb1 PE=1 SV=1	0.016	0.92
	sp|P20152|VIME_MOUSE	Vimentin OS=Mus musculus GN=Vim PE=1 SV=3	0.001	1.53
	sp|Q08091|CNN1_MOUSE	Calponin-1 OS=Mus musculus GN=Cnn1 PE=1 SV=1	0.006	1.24
	sp|P62737|ACTA_MOUSE	Actin, aortic smooth muscle OS=Mus musculus GN=Acta2 PE=1 SV=1	0.009	1.29
	sp|Q64337|SQSTM_MOUSE	Sequestosome-1 OS=Mus musculus GN=Sqstm1 PE=1 SV=1	0.0004	1.72

Discussion:

IL17RD was originally identified as an inhibitor of FGF signaling [15, 16] and was determined to be ubiquitously expressed in several cellular subtypes, with high levels of expression in endothelial cells [33, 47]. However, the functional role of IL17RD in endothelial cells has not yet been elucidated. Our study uses in vitro and in vivo analyses to delineate the functional significance of IL17RD in the regulation of inflammatory phenotype by focusing on its regulation of aortic leukocyte infiltration and EC activation. We report the novel findings that IL17RD is expressed in aortic ECs in vitro and in vivo. Additionally, our results demonstrate for the first time that IL17RD enhances the adhesion of monocytic cells to endothelial cells in vitro, and promotes aortic accumulation of myeloid cells, specifically proinflammatory monocytes, macrophages and neutrophils upon WD feeding in vivo.

IL17A has been previously shown to promote myeloid cell accumulation and atherosclerotic plaque formation in Apoe−/− mice. Specifically, deletion of IL17A decreased the infiltration of neutrophils, monocytes, and macrophages into the aorta in both male and female mice [11]. In our study, we observed that WD induced upregulation of IL17 levels only in males, whereas females showed no appreciable differences in the levels of IL17A. Our results suggest that in females, Il17rd promotes leukocyte infiltration by IL17-independent mechanisms, such as decreased inflammation due to attenuated levels of cholesterol [48]. Further, in our study WD feeding enhanced aortic infiltration of proinflammatory monocytes and neutrophils in males, and macrophages in females, which is different from previous findings. The biological basis of these differences in our observations might be attributable to known factors affecting inflammatory response of mice to WD-feeding such as a) differences in genetic models (Apoe−/− mice used in [11] vs BL6 mice used in our study); b) genetic factors such as the Sry gene and XX chromosome dosage [49, 50]; and c) hormonal factors such as androgens and estradiol [49, 51]; which have been previously shown to differentially regulate expression of Il17rd isoforms in gonadal tissue in previous publications [52, 53]. Thus, our results suggest that Il17rd enhances aortic WD-induced inflammation and aortic infiltration of myeloid cells in a biological sex dependent manner. Current experiments are focused on investigating whether Il17rd affects the phenotype of infiltrated monocytes (inflammatory or anti-inflammatory) and mediates aortic infiltration of other known leukocyte subpopulations such as lymphocytes [18]. Since Il17rd and Il17rc have been shown to have overlapping functions in other cellular contexts [18], we also determined the expression of Il17rc as well as Il17ra in our experimental model. However, we observed no difference in aortic expression levels of IL17RA and IL17RC in Il17rd null mice, indicating that decreased myeloid cell infiltration in these mice is specifically due to the deletion of Il17rd and not a compensatory upregulation in the expression of Il17ra and Il17rc in vivo. In this respect, our study is novel since we demonstrate IL17RD regulation of endogenous IL17A signaling as opposed to previous studies focused on administration of IL17A [17, 18].

We also report the novel observation that Il17rd promotes weight gain and fat accumulation in male mice but not in female mice upon 14 week WD feeding, highlighting a novel metabolic role for Il17rd. This aligns with previous findings that show delayed initiation of weight gain and fat accumulation in female mice upon high-fat feeding [54]. Future experiments are aimed at investigating the role of adipokines which might be involved in Il17rd mediated regulation of leukocyte infiltration into the aorta, including Leptin, Adiponectin and Resistin [55–57], as well as characterization of leukocyte accumulation in Il17rd endothelial-cell specific knockout mouse models. Thus, our study provides the basis for performing longer duration WD feeding studies to clarify the role of biological sex in Il17rd regulation of metabolism.

IL17RD has been shown to bind to IL17RA in mice and in humans to modulate the IL17A-IL17RA/IL17RC signaling axis in certain cellular contexts [18]. However, given that mouse IL17RA also shows strong binding affinity to IL17F in its heterodimeric form which contrasts with its function in humans [58], addressing the differential contribution of IL17F in our experiments with the Il17rd−/− mice remains one of the limitations of our study. In this context, future work characterizing the binding affinities of mouse and human IL17A and IL17F to IL17RD would be useful to clarify the relative contributions of these cytokines in Il17rd models of inflammation [17, 47].

EC activation encompasses molecular changes in the expression of endothelial Ig CAM molecules such as ICAM-1 and VCAM-1 [9]. Given the established role of IL17A as a mediator of ICAM-1 and VCAM-1 expression in macrovascular ECs to enhance adhesion of leukocytes to the endothelial monolayer [12, 13], and the high expression levels of IL17RD we observed in aortic ECs, our results suggest an important role for this receptor in modulating aortic EC function, validating the utility of our in vitro model. However, we observed that IL17A did not upregulate the expression of VCAM-1 and ICAM-1 in endothelial cells in our study. We postulate that these differences might arise due a number of factors including a) difference in cell type used for analysis (HUVECs vs HAECs) [13] ; b) difference in methods of analysis used (number of ICAM^pos cells in [12] vs total cellular expression levels in our study); and c) donor specific effects. Further, we did not observe any increase in aortic EC expression of VCAM-1 and ICAM-1 in vivo upon WD feeding despite enhanced IL17 levels, which is consistent with our in vitro findings.

Taken together, our study attempts to deepen the contextual understanding of the molecular mechanisms and regulation of functional phenotypes by IL17 signaling involving its recently identified receptor IL17RD. Our findings suggest that IL17RD functions as a molecular and cellular mediator of IL17 signaling to functionally regulate phenotypic outcomes in inflammatory disease in a cell-type-dependent manner. Our preliminary results from proteomic analysis of primary MEF samples suggest that the expression of adhesion molecule MYADM-1 might be regulated by IL17RD in response to IL17A stimulation. Mechanistically, we observed that IL17RD may upregulate the activation of the noncanonical JAK1 pathway in the MEF cells, which has been shown to promote IL17A-induced endothelial activation and leukocyte infiltration in previous studies [59], as well as of the Protein Kinase C pathway. Interestingly, we also observed in Il17rd null MEFs a decrease in the expression of the vasoconstricting factor ECE-1, which also regulates leukocyte trafficking in mouse models of vascular inflammation [46] suggesting that Il17rd might also regulate vascular tone. Further, our results indicate that Il17rd suppresses the expression of α and β-catenin in primary MEFs, which aligns with previously established role of Il17rd as an inhibitor of the Wnt signaling pathway in other cellular contexts [60]. However, in the context of endothelial cells, α and β-catenin are essential for anchoring VE-cadherin to the endothelial cell membrane and therefore our results suggest that Il17rd might also negatively regulate endothelial barrier integrity [61]. Further, our results indicate that in MEFs stimulated with IL17A, Il17rd promotes the expression of vimentin, calponin and α-smooth muscle actin (SMA) in MEFs upon IL17A stimulation in MEF cells, which are key effector molecules of the proinflammatory endothelial-mesenchymal transition (EndoMT) process [62]. In addition to this, we also found that Il17rd−/− MEFs have higher expression of autophagy mechanism component p62, which has also been shown to modulate endothelial cell function in the context of endothelial-leukocyte interaction [63]. Therefore, we suggest that in addition to the expression of adhesion molecule MYADM-1, Il17rd might also regulate endothelial cell functions such as vasomotor tone, autophagy, as well as EndoMT to promote transendothelial leukocyte trafficking [61–63].

Taken together, our results highlight the diagnostic potential of assessing endothelial IL17RD expression during cardiometabolic diseases associated with elevated IL17A levels, such as atherosclerosis, obesity and type 2 diabetes, and provide a clinical basis for future research focused on designing peptides targeting IL17RD expression in endothelial cells. Future research can be focused on characterizing the role of IL17RD in other organ and tissues involved in the regulation of inflammation, such as liver and adipose [52]. Our work also lays the foundation for future studies investigating the therapeutic utility of IL17RD as a diagnostic biomarker in other IL17A-IL17RD regulated inflammatory diseases associated with enhanced endothelial cell activation and cardiovascular comorbidities such as psoriasis [18, 64, 65]. However, in order to prevent off-target phenotypic effects that might arise due to clinical targeting of IL17RD, it is important to identify whether IL17RD modulates signaling downstream of IL17 cytokines through yet unidentified receptor complexes such as with IL17B and IL17D [66], as well as determine whether IL17RD interacts with other cognate receptors of the canonical IL17 signaling superfamily such as IL17RB and IL17RE via its SEFIR domain [17, 47]. In this respect, it is also important to perform structural and 3D crystallographic studies of IL17RD to aid in identification of other signaling adaptors and interacting partners that might lead to lead to dysregulated signaling [47]. To summarize, our study provides impetus to future research addressing the gaps in our understanding of IL17 biology with a focus on studying the interaction of IL17A with IL17RD.

Highlights:

1. IL17RD promotes IL17A induced THP-1 adhesion to human endothelial cells.

2. IL17RD promotion of THP-1 adhesion is not dependent on VCAM-1 and ICAM-1.

3. IL17RD promotion of THP-1 adhesion is not dependent on canonical IL17A pathways.

4. Il17rd promotes aortic accrual of myeloid cells upon western diet feeding in mice.

5. Il17rd mediated myeloid accrual is not dependent on endothelial VCAM-1 and ICAM-1.

List of abbreviations

ANOVA

Analysis of Variance

ATCC

American Type Culture Collection

BSA

Bovine Serum Albumin

CAM

cell adhesion molecules

CD

Chow diet

CID

collision induced dissociation

CUR

curtain gas

DAPI

4′,6-diamidino-2-phenylindole

DDA

data dependent acquisition

DEXA

dual-energy x-ray absorptiometry (DEXA)

DMEM

Dulbecco’s Modified Eagle Medium

DP

declustering potential

dPBS

Dulbecco’s Phosphate Buffered Saline

DTT

Dithiothreitol

EBM-2

Endothelial Basal Media-2

ECE-1

endothelin converting enzyme-1

EGM-2

Endothelial Cell Growth Medium-2

EndoMT

endothelial-mesenchymal transition

ERK

Extracellular Regulated Kinase

FBS

Fetal Bovine Serum

FGF

Fibroblast Growth Factor

FGFR

FGF receptor

HAEC

Human Aortic Endothelial cells

HEPES

((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)

HUVEC

Human Umbilical Vein Endothelial cells

HRP

Horseradish peroxidase

IHT

interface heater temperature

Ig

Immunoglobulin

ICAM-1

Intercellular adhesion molecule-1

IL17A

Interleukin-17 A

IL17RA

Interleukin-17 Receptor A

IL17RC

Interleukin-17 Receptor C

IL17RD

Interleukin-17 Receptor D

I.Q.R

Interquartile range

ISVF

ion spray voltage floating

JAK1

Janus Kinase 1

JNK

c-Jun N-terminal kinase

LC/MS

Liquid chromatography/mass spectrometry

MAPK

Mitogen activated protein kinase

MEF

Murine Embryonic Fibroblasts

MLR

Most Likely Ratio

MYADM

Myeloid-associated differentiation marker

NT

non-targeting scrambled

NF-κB

Nuclear Factor kappa light chain enhancer of activated B cells

NMR

Nuclear Magnetic Resonance

PCA

Principle Component Analysis

PI3K

Phosphoinositide 3 kinase

PKCα

Protein Kinase C alpha

RPMI

Roswell Park Memorial Institute

S.D.

Standard Deviation

Sef

Similar expression to FGF

SEFIR

Sef/IL17R

SQSTM1

Sequestosone-1

SMA

smooth muscle actin

SWATH

Sequential window acquisition of all theoretical spectra

TEM

transendothelial migration

Th17 cells

type 17 T-helper T-cells

TOF

time-of-flight

VCAM-1

Vascular cell adhesion molecule

WT

Wild Type

WD

Western Diet

Availability of supporting data:

The authors will share the supporting data available upon reasonable request. Mass spectrometry data have been deposited into the Proteomics Identifications Database (PRIDE).