Dendritic cell-specific SMAD3, downstream of JAK2, contributes to inflammation and salt-sensitivity of blood pressure

doi:10.21203/rs.3.rs-2908485/v1

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Dendritic cell-specific SMAD3, downstream of JAK2, contributes to inflammation and salt-sensitivity of blood pressure

https://doi.org/10.21203/rs.3.rs-2908485/v1

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Salt-sensitivity of blood pressure (SSBP), characterized by acute changes in blood pressure with changes in dietary sodium intake, is an independent risk factor for cardiovascular disease and mortality in people with and without hypertension. We previously found that elevated sodium concentration activates antigen presenting cells (APCs), resulting in high blood pressure, but the mechanisms are not known. Here, we hypothesized that APC-specific JAK2 expression contributes to SSBP. We performed bulk or single-cell transcriptomic analyses following in vitro monocytes exposed to high salt, and in vivo high sodium treatment in humans using a rigorous salt-loading/depletion protocol to phenotype SSBP after a two week anti-hypertensive drug washout period. Here we found that expression of the genes of the JAK2 pathway mirrored changes in blood pressure after salt-loading and depletion in salt-sensitive but not salt-resistant humans. Ablation of JAK2, specifically in CD11C⁺ APCs, attenuated salt-induced hypertension in mice with SSBP. Mechanistically, we found that SMAD3 acts downstream of JAK2 and STAT3, leading to increased production of highly reactive isolevuglandins and pro-inflammatory cytokine IL-6 in renal APCs, which activate T cells. This results in the production of IL-17A, IL-6, and TNF-⍺. Our findings reveal APC JAK2 signaling as a potential target for the treatment of SSBP.

Health sciences/Nephrology/Kidney diseases

Biological sciences/Physiology/Cardiovascular biology

Salt-sensitive hypertension

CD11c+ dendritic cell

JAK2

STAT3

SOCS

SMAD3

High dietary sodium consumption is rising worldwide and is associated with increased hypertension and cardiovascular disease.^1–5 However, people with salt-sensitivity of blood pressure (SSBP), defined as acute changes in blood pressure that mirror dietary sodium intake, are more vulnerable to salt-induced cardiovascular morbidity and mortality than salt-resistant individuals.^1,3,4 The mechanisms contributing to SSBP are unknown and SSBP remains an untreatable cardiovascular risk with no widely available diagnostic tool.

Our studies have found that elevated extracellular sodium enters antigen-presenting cells (APCs) via the amiloride sensitive epithelial sodium channel (ENaC) and leads to activation of NADPH oxidase leading to increased oxidation of fatty acids and production of isolevuglandins (IsoLGs). IsoLGs are highly reactive products of lipid peroxidation that adduct proteins and form neo-antigens that activate APCs. The activated APCs in turn produce pro-inflammatory cytokines that polarize T cells. The ensuing inflammatory activation results in the secretion of key cytokines including IL-17A, and TNF-⍺ leading to blood pressure elevation.⁶ The intracellular mechanisms by which elevated sodium activates APCs are still not fully understood.

The JAK-STAT pathway constitutes a rapid membrane-to-nucleus signaling module that affects every aspect of the mammalian immune system,^7–11 and has been implicated in many pathological conditions including cardiovascular disease, cancer and COVID-19 infection.^12–21 Suppression of Cytokine Signaling (SOCS3) protein inhibits the JAK-STAT pathway by inhibiting JAKs phosphorylation.²² We previously found that JAK2 plays an important role in vascular remodeling, fibrosis, and hypertension.^23–28 Dendritic cells (DCs) are the most potent and professional APCs that play an essential role in the development and maintenance of immune responses and tolerance through the JAK-STAT pathway.⁷ Immune cells including DCs exhibit the highest expression of JAK2,²⁹ and in steady state, the JAK2-STAT3 pathway is essential for the maturation of DCs in mice.⁷ However, the role of APC-specific JAK2 in the pathogenesis of salt-sensitive hypertension is not known.

In the current studies, we hypothesized that the rapid signaling cascade of JAK2 specifically in APCs contributes to salt-induced blood pressure in salt-sensitive people. To test this hypothesis, we performed bulk and single cell transcriptomic analysis with in vitro and single-cell high dietary sodium treatment in humans using a rigorous salt-loading/depletion protocol to phenotype for salt-sensitivity of blood pressure. Mice lacking JAK2 in CD11C⁺ APCs were also used to investigate the role of JAK2 in salt-sensitive blood pressure.

Human studies

All participants provided written consent before enrollment. All studies were approved by the Institutional Review Board of Vanderbilt University Medical Center, and all procedures were performed in accordance with the Declaration of Helsinki. We studied two cohorts, in cohort one (Demographic information in Table 1 and previously published),^30,31 we isolated monocytes for in vitro bulk RNA sequencing analysis as we have previously published.³¹ Briefly, heparinized blood (40 mL) was obtained from volunteers, and peripheral blood mononuclear cells (PBMCs) were isolated using a Ficoll-gradient protocol. Monocytes were further isolated from these cells by magnetic labeling and negative selection using the Miltenyi monocyte isolation kit (Miltenyi Biotec 130-091-151) and cultured in 12-well plates at 1 × 106/mL density in either control RPMI media (150 mMol/L Na⁺) or media containing 190 mMol/L Na⁺. RPMI media 1640 (Gibco) was supplemented with 10% FBS, 1% pen/strep, 1% HEPES, and 2-Mercaptoethanol (0.05 mM). Isolated monocytes were treated with normal and high salt in vitro, and bulk RNA sequencing was performed.

Table 1

Demographic characteristics of the participants for Cohort 1.
	(n = 11)
Age, y	34.7 ± 11.8
Female sex, n (%)	11 (100)
Black, n (%)	1 (9)
SBP, mmHg	110.9 ± 16.7
DBP, mmHg	69.0 ± 6.8
HTN, %	9
BMI, kg/m²	28.6 ± 17.5
Mean ± SD. BMI denotes body mass index; DBP, diastolic blood pressure; HTN, hypertension; and SBP, systolic blood pressure.

In cohort two, we performed single-cell Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) to profile transcriptomes in immune cells in hypertensive patients and phenotyped for salt-sensitivity of blood pressure employing an established protocol of rapid salt load and depletion as depicted in Fig. 2A (Demographic information in Table 2, also previously published).³⁰

Table 2

Demographics of participants for salt-sensitivity phenotyping in cohort 2.
	B	SL	SD
SBP (mmHg)	137.7 ± 3.1	141.3 ± 2.9	138.7 ± 2.9
DBP (mmHg)	85 ± 2.2	86.1 ± 2.3	87.0 ± 2.4
MAP (mmHg)	104.7 ± 2.1	105.6 ± 2.2	104.7 ± 2.1
Urinary Na⁺ Excretion (mmol/day)	150.4 ± 13.7	350.8 ± 27.9	73.5 ± 13.9
Urinary K⁺Excretion (mmol/day)	59.2 ± 7.7	67.7 ± 7.8	40.4 ± 2.9
Urinary Na⁺ /K⁺Ratio	2.9 ± 0.3	6.8 ± 1.6	1.9 ± 0.3
PRA (ng/L)	9.3 ± 3.1	9.6 ± 2.5	16.7 ± 5.4
Aldosterone (ng/dL)	12.1 ± 2.2	7.7 ± 1.8	13.1 ± 2.1
Plasma EET (pmol/ml)
8–9	5.2 ± 0.4	5.8 ± 0.48	5.7 ± 0.5
11–12	4.0 ± 0.5	4.8 ± 0.6	3.6 ± 0.3
14–15	7.9 ± 1.2	7.4 ± 0.7	6.4 ± 0.5
Total	17.1 ± 2.0	18.0 ± 1.3	15.7 ± 1.1
Urine EET (pmol/24h)
8–9	62.0 ± 14.6	72.8 ± 22.4	63.0 ± 14.0
11–12	56.6 ± 9.1	100.3 ± 27.5	67.5 ± 13.0
14–15	224.1 ± 78.9	368.4 ± 128.2	192.4 ± 51.9
Total	342.7 ± 97.6	541.6 ± 165.0	322.9 ± 71.8

Inclusion/Exclusion Criteria

We included patients between 18 and 65 of age either with systolic blood pressure (SBP) > 140 mmHg or diastolic blood pressure > 90 mmHg or on antihypertensive treatment. Patients were excluded if they had diabetes mellitus, confirmed, or suspected renal, renovascular, or endocrine causes of secondary hypertension, with infectious or inflammatory disease (i.e., active infection or connective tissue disorder), have active cancer, history of an acute cardiovascular event within six months of the study, on treatment to increase BP (e.g., selective serotonin reuptake inhibitors and serotonin and norepinephrine reuptake inhibitors, adrenergic agonists for attention deficit hyperactivity disorder, chronic use of decongestants or non-steroidal anti-inflammatory drugs) or alter the immune response (e.g., direct immunomodulators, immunosuppressants, glucocorticoids), and if they were pregnant. We collected demographic and clinical data directly from the participants and by clinical chart view.

Bulk-RNA sequencing and bioinformatics

High-quality RNA was isolated from the samples using RNeasy Midi Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. Quality control of the RNA samples was performed by Vanderbilt Technologies for Advanced Genomics (VANTAGE) core ensuring high RNA Integrity Number (RIN) value. Polyadenylated RNA sequencing was performed at the VANTAGE core using the Illumina Tru-Seq RNA sample prep kit. Pair-end sequencing was done on the Illumina HiSeq2500. Using the R package, the FASTQ data files from the paired-end sequencing analysis were aligned with TopHat 2 for each sample against the human GRCh38 reference genome assembly. Quality control for the RNA-Seq was performed during the following stages: 1) RNA quality; 2) raw read data (FASTQ); 3) alignment; 4) gene expression. Quality control for the raw data and alignment was conducted using QC3, and the MultiRankSeq method was used for expression analysis. False discovery rate (FDR < 0.05) was used to correct for multiple hypothesis testing. Expression heatmaps were created using the online available tool Heatmapper (http://www.heatmapper.ca/expression/). Volcano plots were created using the online tool VolcaNoseR (https://huygens.science.uva.nl/VolcaNoseR/). The fold change for the volcano plot was ≥ 1.1. Enrichment pathway analysis was performed based on the Reactome database using the online tool Enrichr (https://maayanlab.cloud/Enrichr/#). Color wheels were created using Excel.

We got 47,757 transcripts after sequence alignment to the mouse genome. From this, 39,898 transcripts were removed which were either pseudogenes or transcripts with 0 values leaving 7,859 genes for further analysis. Among these 7,859 genes, 6,560 genes were scored as differentially expressed (DE) genes with adjusted p-value ≤ 0.05 and fold change ≥ 1.1. Among the DE genes 3,010 genes were up-regulated (Supplemental Table X), whereas 3,550 genes were downregulated (Supplemental Table Y). We performed enrichment pathway analysis with the up-regulated and down-regulated DE genes separately based on the Reactome database using the online tool Enrichr (https://maayanlab.cloud/Enrichr/#). The significant pathways obtained from this pathway analysis are enlisted in Supplemental Table A (up-regulated pathways) and Supplemental Table B (down-regulated pathways). Based on this pathway analysis we calculated the contribution of these pathways in 10 major cellular biological processes: (1) inflammation, (2) stress, (3) cell cycle, replication, transcription, and epigenetics, (4) growth and development (5) cell-cell interaction and motility, (6) transporters, (7) metabolism, (8) proteasomal degradation, (9) protein synthesis and modification and (10) extracellular matrix remodeling pathways (Fig. 1B).

Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq)

Cell hashing and CITE-Seq analysis was performed as previously described.³⁰PBMCs were isolated as described by the manufacturer’s protocol (Fisher Scientific, Cat# 14-959-51D) in BD Vacutainer® CPT™ Mononuclear Cell Preparation tubes. Briefly, PBMCs were stained with antibody-oligonucleotide conjugates and sample-specific hashtags were included to allow for sample multiplexing. The complex was fed into the microfluidic system along with custom beads conjugated with poly dT and nucleotide sequences designed to capture the hgRNA barcodes. Oil was included to create a small vesicle of a single bead and a single cell with an antibody-oligonucleotide complex bound to it. The cell was lysed, and intracellular cDNA and Antibody Derived Tags (ADTs) were formed using reverse transcription of the mRNA to cDNA in the VANTAGE facility.

10x Genomics Cell Ranger 6.0.2 was used to quantify genes. In-house scripts were used to demultiplex sample-specific hashtags, and the hashtag abundance cutoff of positive cells was determined by modified R package cutoff[https://github.com/shengqh/cutoff]. Each cell was classified as singlet with specific hashtag, doublet or negative and genotype-based demultiplex result from Souporcell were integrated with this hashtag-based demultiplex result.³² Clustering analysis was performed using Seurat³³ with resolution 1.0. and the cell type of each cluster was classified based on cell activity database^33,34 and manually refined according to cell-specific marker genes. We used edgeR³⁵ to identify differential expressions across conditions and the WebGestaltR³⁶ package was used to perform Genome Ontology and KEGG pathway over-representation analysis on differentially expressed genes. We performed gene enrichment analysis using the GSEA³⁷ package.

Animals and Blood Pressure Monitoring

All animal procedures were approved by Vanderbilt University’s Institutional Animal Care and Use Committee, and the mice were housed and cared for in accordance with the Guide for the Care and Use of Laboratory Animals, US Department of Health and Human Services. We purchased C57BL/6J TgCD11c^cre (CD11cCre^+/+) mice from Jackson Laboratories (Bar Harbor, ME, USA), approximately 10–12 weeks (about 3 months) of age. JAK2^fl/fl mice were developed and generously gifted by Dr. Andrea Krempler. JAK2^fl/fl mice on the FVB background³⁸ were backcrossed for over 10 generations to the C57BL/6 background. JAK2 was deleted in CD11c⁺ cells by crossing TgCre/CD11c with JAK2^fl/fl mice (JAK2^CD11cKO). To induce salt-sensitivity of blood pressure in otherwise salt-resistant C57BL/6J mice³⁹, mice were randomly selected to initially receive G-nitro-L-Arginine-Methyl Ester (L-NAME, 0.5 mg/mL) in drinking water for 2 weeks followed by a washout period when mice were given regular water and chow ad libitum before they fed a 4% high-salt diet (Envigo, cat # TD.03095) for 3 weeks. Blood pressure was monitored using both noninvasive tail-cuff and invasively using state-of-the-art radiotelemetry. After the radiotelemetry surgery, mice were allowed to recover for 10 days before feeding them L-NAME and a high salt diet.

Metabolic Cage Studies

After the L-NAME and high salt regimen, mice were injected with saline (10% of their body weight) and placed in a metabolic cage for four hours. The urine was collected, volumes were measured, and both groups of mice were compared. At the end of the blood pressure monitoring period and metabolic cage experiment, mice were sacrificed using CO₂ inhalation.

Isolation of Mouse Tissues

After euthanasia, we sprayed 70% ethanol on the chest of mice. The skin and chest wall were cut carefully to expose the heart. A small incision was made in the right atrium using a 23-gauge needle, and saline was injected into the apex of the left ventricle. We perfused mice until all organs had blanched. The spleen, kidney, and aorta were collected, digested, and used for further analyses.

Catecholamine Measurement

Before perfusing the mouse, we collected about 500 µl blood from the heart ventricle using a 1 ml syringe in Eppendorf tube with 10 µl EGTA-glutathione. Plasma was obtained by centrifugation at 2100 × g for 10 minutes at room temperature (RT). Plasma was aliquoted in Eppendorf tubes, flash frozen in liquid nitrogen, and then stored at -80°C. Plasma epinephrine and norepinephrine were measured by HPLC via an electrochemical detection as previously published. Plasma is absorbed onto alumina at a pH of 8.6, eluted with dilute perchloric acid, and auto-injected onto a c18 reversed-phase column. An internal standard (dehydroxylbenzylamine; DHBA) was included with each extraction to monitor recovery, and standard curves for both epinephrine and norepinephrine are run. Results are quantitated through a chromatography data station.^40,41 Catecholamines assays were performed by the Vanderbilt University Medical Center Hormone Assay and Analytical Services Core.

Method to attach the Alexa Fluor 488 fluorophore to the D-11 Antibody

To conjugate the D-11 antibody (D11 ScFv) to the Alexa Fluor 488 fluorophore, we used the Alexa Fluor 488 Antibody labeling kit (Thermofisher, Cat# A-20118). We added 100 µL of D-11 antibody to the vial of Alexa Fluor 488 dye. The vial was inverted and incubated for 1 hour at RT in the dark. Next, a purification column was prepared. We added 750 µL of resin mix to the column and centrifuged at 1100 x g for 3 minutes. The D-11 and Alexa Fluor 488 vial was added dropwise to the top of the resin bed and the column was then centrifuged at 1100 x g for 5 minutes. The conjugated D-11 antibody was collected from the eluent. We used this D-11 Alexa Fluor 488 tagged-antibody to detect IsoLG protein adducts.

Generation of single-cell suspensions from mouse organs

After harvesting, we placed the kidney in a dissociation tube containing 3 mL of kidney digestion solution (1 mg/mL of collagenous D, 0.1 mg of DNase, and 3 mL of PBS). The tube was then inserted into a semi-automated dissociator device for two minutes and then transferred to the incubator for continuous rotation in a 37°C for 20 minutes to digest. After digestion, we centrifuged the tubes at 400 x g for 10 minutes at 4°C. The supernatant was aspirated, and the pellet was resuspended in a 36% Percoll solution (Sigma-Aldrich). The cell suspension was gently layered on a 72% Percoll solution in a different tube to create a density gradient to collect the immune cells. We centrifuged the tubes at 2400 rpm, acceleration 9, and deceleration 1 for 15 minutes at 4°C. The thin “junk layer” or “gooey material” was gently aspirated to remove debris. We collected the upper part of the gradient till the "buffy coat" was reached (it may appear off-white with a reddish tinge [red blood cells]) in a 15 mL conical tube and washed with 15 mL of PBS by centrifuging at 350 x g for 10 minutes at 4°C. The supernatant was aspirated, and the pellet was used for staining for flow cytometry analysis.

We performed the same steps above for the aorta; however, the aorta was not processed using the automatic dissociator. Aortas were placed in a 1.5mL Eppendorf tube containing 1 mg/mL of collagenase A and B, 0.1 mg of DNase, and 1 mL of RPMI media, and was minced with scissors in the solution for 2 minutes. Aortas were incubated under continuous rotation for 30 minutes at 37°C.

The spleen was smashed through a 40 µm filter on a conical tube and washed with 15 mL cold PBS. The solution left in the conical tube was then centrifuged at 300 x g for 10 minutes at 4°C, the supernatant was aspirated. Then, the samples were washed with 10 mL of cold PBS and spun down again at 300 x g for 10 minutes at 4°C, and the pellets were used for surface staining.

Splenocyte Isolation and Western Blot

After euthanizing mice, we harvested spleens and placed them in dissociation buffer in gentle MACS™ C tubes (Miltenyi Biotec, Cat# 130 − 107 093–237). Spleens were then dissociated using a syringe plunger and spleen homogenates were passed through a 40 µm cell strainer and washed with Dulbecco’s PBS (dPBS) to create a single-cell suspension for further flow cytometric analysis. In other experiments, splenocytes in a single-cell suspension were plated in 24-well plates at a density of 1x10⁶/mL in either normal salt RPMI (150 mM, Na⁺) or high salt RPMI (190 mM, Na⁺) for 48 hours. GibcoTM RPMI 1640 media (ThermoFisher, Cat# 11875119) was supplemented with 10% FBS, 1% pen/strep, 1% HEPES, and 2-Mercaptoethanol (0.05 mM). Protein was estimated with the BCA method, and Western blot samples were prepared as described previously.⁴²

Flow Cytometry

The single cell suspensions from the kidney, aorta, and spleen were transferred into separate polystyrene FACS tubes and washed with 1 mL of cold PBS with centrifugation at 350 x g for 7 minutes at 4°C. We stained cells with Zombie NIR fixable stain (Biolegend, Cat# 423106) for 15 min at 4°C to identify live and dead cells. In the meantime, a master mix was prepared for all the antibodies used for the surface staining while protecting from light (1:100 ratio in MACS buffer). The information for the antibodies used is available in Table S1. After live-dead staining, the supernatant was aspirated, and 100 µL of the master mix was added into each FACS tube, vortexed, and incubated for 20 minutes at 4°C protected from light. After the incubation, we immediately added FIX & PERM medium A (Invitrogen, Cat# GAS001S100), vortexed, and incubated for an additional 20 min at RT protected from light. After incubation, we again washed samples with 1 mL DPBS by centrifuging at 350 x g for 7 minutes. Samples were stained for intracellular markers shown in Table S1. A master mix with intracellular antibodies was prepared in FIX & PERM medium B (Invitrogen, Cat# GAS002S100) in a 1:100 ratio. After surface staining and washing the samples, a 100 µL of the master mix was added into each FACS tube, vortexed, and incubated for 30 minutes at RT protected from light. vortexed, and incubated for an additional 20 min at RT. The supernatants were aspirated, 100 µL of MACS buffer was added to each tube, and samples were run using Cytek Aurora™. We used FlowJo 10.8.1 for flow cytometry data analysis.

Immunohistochemistry and Masson’s Trichrome Staining

Both anti-F4/80 immunolabeling and Masson’s trichrome staining were performed and analyzed by a blinded observer. Kidneys were fixed in 10% neutral buffered formalin, routinely processed and paraffin-embedded, then cut into five µm sections by the Translational Pathology Shared Resource core at Vanderbilt University Medical Center. Renal immune cell infiltration was analyzed in kidney sections obtained from DC^JAK2KO and DC^JAK2WT mice after L-NAME/high salt (HS) regimen. All steps besides dehydration, clearing and cover-slipping were performed on the Leica Bond-Max IHC auto-stainer. Slides were deparaffinized and heat-induced antigen retrieval was performed using the Epitope Retrieval 2 solution for 10 minutes. Slides were incubated with a monoclonal rabbit anti-mouse F4/80 antibody at a 1:250 dilution for 60 minutes. The Bond Polymer Refine Detection system was used for visualization. Slides were then dehydrated, cleared, and cover-slipped. Mason’s trichrome staining was performed on the Gemini autostainer. The perivascular fibrosis index was assessed at the level of the arcuate arteries and their arteriolar branches throughout the renal cortices on a semiquantitative scale of 0–3.

Fluorescence In-Situ Hybridization

The Fluorescence In-Situ Hybridization (FISH) was performed as per our established protocol published earlier.⁴² We employed RNAscope Multiplex Fluorescent Reagent Kit v2 (Cat# 323110 Advanced Cell Diagnostics [ACD] Bio-Techne, Newark, CA) and probes designed for CD11c, JAK2, ENAC-γ, and IL-6 were used. Briefly, we perfused kidneys with 10% neutral buffered formalin solution, dehydrated them in a graduated ethanol series, and embedded them in paraffin at the Vanderbilt Pathology Laboratory Services for further processing. Kidney sections were cut to a thickness of 5 mm and were placed on transparent slides. We deparaffinized kidney sections using Histo-Clear Solution, dehydrated them in absolute ethanol at RT, and then blocked endogenous peroxidase with RNAscope Hydrogen Peroxide. We retrieved the tissues by boiling them in the target retrieval solution (ACD Bio-Techne) between 100 and 104°C for 15 minutes and then treated them with Protease Plus at 40°C for 30 min. Target probes (Mm-ITGAX-C1, reference number 311501; Mm-SCNN1g-C2, reference number 422091 or Mm-IL-6-C2, reference number 315891; Mm-JAK2-C3, reference number 320269) were hybridized for 2 hours at 40°C, followed by a series of signal amplifications (amplifications 1–3), and, in between, washing with RNAscope Wash buffer (twice for 2minutes). We assigned ITGAX (CD11c), ENaC-γ (SCCN1G), and JAK2 mRNA probes to channels HRP–channel 1 (HRP- C1), HRP-C2, and HRP-C3, respectively. Similarly, for a second set of probes ENaC-γ (SCCN1G) was replaced with IL-6 mRNA probes and was assigned to channels HRP–channel 1 HRP-C3. The HRP-C1 signal was developed using RNAscope Multiplex FL v2 HRP-C1 with Opal 520 fluorophore (Cat# FP1487001KT, Akoya Biosciences), blocked with RNAscope Multiplex FL v2 HRP blocker, and washed with RNAscope Wash buffer in between. Similarly, HRP- C2 and HRP-C3 signals were developed using Opal 570 (Cat# FP1488001KT, Akoya Biosciences) and Opal 650 (Cat# FP1496001KT, Akoya Biosciences) fluorophores, respectively. We assigned HRP-C1, HRP-C2, and HRP-C3 to CD11c, ENaC-γ or IL-6, and JAK2 probes, respectively. We performed all hybridization steps at 40°C in a HybEZ Hybridization System (ACD Bio-Techne). After the completion of the RNAscope assay, tissue sections were counterstained with 49,6-diamidino-2- phenylindole (DAPI); incubated for 30 seconds at RT; mounted onto slides using VectaMount mounting medium (H-500; Vector Laboratories, Burlingame, CA); and the slides were then dried, overnight, in the dark, at RT. We viewed and imaged the sections using confocal microscopy (Zeiss LSM 880) at Vanderbilt Cell Imaging Shared Resource (CISR) Core. We employed Fiji to analyze and process the images.

Statistical analysis

All data are presented here as the mean and standard error of the mean (SEM). An unpaired student t-test was used to compare two groups. Two-way ANOVA was used for experiments with three or more conditions, followed by Bonferroni repeated measure post hoc test to compare the multiple groups. We used Spearman’s rank correlation to assess the associations of continuous variables of interest and trend lines and confidence intervals (CI) were estimated with linear regression. We used GraphPad Prism Version 9.5.1 for all statistical analyses in the manuscript.

High sodium upregulates expression of the JAK/STAT/SMAD and oxidative stress pathway genes and downregulates antioxidant, anti-inflammatory, and suppressor of cytokines signaling (SOCS) genes in human monocytes

We previously reported that high salt but not equiosmolar concentration of mannitol activates human monocytes through ENaC and NADPH oxidase activation. Activated monocytes release proinflammatory cytokines such as IL-6, TNF-⍺, and IL-1β.³¹ We also showed that IL-6 activates STAT3 in monocytes isolated from hypertensive patients.²³ IL-6 and other cytokines have been shown to activate JAK/STAT pathways during various pathological conditions.⁴³ To investigate if high salt exposure activates the JAK/STAT pathway, we isolated monocytes from 11 healthy individuals and treated them with either normal (150 mMol/L) or high sodium (190 mMol/L) for 72 hours and performed bulk RNA sequencing (Fig. 1a). The demographics of the volunteers are shown in Table 1 (previously published).^30,31 Pathway analysis showed that high sodium concentration increases the expression of genes involved in inflammatory and stress-induced pathways which constitute 48% of the total upregulated pathways, while 25% were downregulated in proteasomal degradation pathway (Fig. 1b, c). The volcano plot and heatmaps indicate that genes in the JAK/STAT/SMAD pathway are upregulated while SOCS genes are downregulated (Fig. 1d, 1e, 1h). High sodium-treated monocytes exhibit significantly greater expression of JAK2, TYK2, STAT1, STAT2, STAT4, STAT5A, and STAT6 (Fig. 1f), while SOCS2, SOCS7, and CISH are significantly downregulated (Fig. 1g). Similarly, high sodium treatment significantly increased the expression of tumor necrosis factor superfamily (TNFSF) and their receptors including TNFSF10, 13, 13B, 14, TNFRSF4, 10A, 10B, 10D, 12A, and 14 (Fig. S1b, c), while antioxidant genes including superoxide dismutase 1 (SOD1), SOD2, catalase (CAT), glutathione peroxidase 1 (GPx1), GPx4, heme oxygenase 1 (HMOX1), HMOX2, peroxiredoxin 1 (PRDX1), protein disulfide isomerase family a member 6 (PDIA6), selenoprotein P (SEPP1), and thioredoxin (TXN) are significantly downregulated (Fig. S1b, d). These results demonstrate that high salt increases expression of the JAK-STAT-SMAD pathway and downregulates inhibitor SOCS genes in human monocytes. Also, high salt treatment increased the expression of oxidative and proinflammatory genes. The workflow of data analysis is shown in figure S1a.

Gene expression of the JAK/STAT/SMAD pathway in antigen-presenting cells mirrors changes in blood pressure following rapid salt-loading and depletion in salt-sensitive but not salt-resistant hypertensive patients.

To determine if gene expression of the JAK/STAT/SMAD pathway in APCs imitates in vivo changes in the blood pressure after rapid salt-load and salt-depletion among salt-sensitive hypertensive patients, we enrolled ten hypertensive patients for the inpatient protocol and determined their salt–sensitivity. The demographics of the participants are displayed in Table 2. We collected blood at baseline (day 1), after salt-loading (day 2), and salt-depletion (day 3), and isolated PBMCs. We employed cell hashing to pool the samples, and antibody-derived tags (ADTs) to identify distinct populations of immune cells (Fig. 2a-c). The Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) was performed to profile transcriptomes in immune cells (Fig. 2d). The ADTs identify distinct populations of immune cells shown in Fig. 2e. We used the CD14 marker to identify monocytes in the monocyte clusters, and major histocompatibility complexes class II (MHCII) cell surface receptors (HLA-DRA, HLA-DRB1, HLA-DRB5), a costimulatory molecule marker CD86, and CD1c to identify monocyte and DCs in the clusters (Fig. 2f). We did not observe any marked differences in the clustering of APCs including DCs and monocytes in response to salt-loading and salt-depletion (Fig. 2g). However, we found that components of the JAK/STAT/SMAD pathway are highly expressed in APCs (Fig. 2h). The results presented in Fig. 2i-j indicate that the gene transcripts of JAK2/STAT3/SMAD3 were significantly elevated in salt-sensitive patients after salt-loading and decreased after salt-depletion. In contrast, this pattern was not observed in salt-resistant patients. These findings suggest that changes in the gene expression of this pathway may reflect alterations in blood pressure specifically in salt-sensitive individuals. The salt-induced changes in the JAK2 and SMAD3 gene expressions in DCs positively correlated with the changes in pulse pressure (PP) in all participants. JAK2, STAT3, and SMAD3 also correlated with changes in FE_Na. Moreover, changes in the SMAD3 gene expression correlated with the changes in urinary sodium volume (UNaV), and fractional potassium excretion (FE_K) after salt-loading and salt-depletion (Fig. 2k).

Deletion of JAK2 specifically in myeloid antigen presenting cells prevented salt-induced hypertension and preserved the renal response to a volume load in mice To determine if expression of JAK2 in APCs plays an essential role in salt-induced inflammation and hypertension, we generated conditional JAK2 knockout mice in CD11c⁺ cells by crossing JAK2^fl/fl mice with CD11c^CRE+/− mice and littermates were used as controls (Fig. 3a). We studied both male and female mice. Western blot analysis confirmed deletion of JAK2 from isolated splenic CD11c⁺ cells (Fig. 3b-d). Deletion of JAK2 from CD11c⁺ cells had no significant effect on body weight, and weight of the heart, kidneys (left, right), and spleen (Fig. S2b-f).

Since C57BL/6 mice are salt-resistant, we treated them with L-NAME for two weeks to induce salt sensitivity, followed by two weeks washout period with regular water and chow ad libitum, and three weeks of a 4% high salt diet (Fig. S3a). We first measured blood pressure with the noninvasive tail-cuff method (Fig. 3e). Both DC^JAK2WT and DC^JAK2KO mice exhibited normal blood pressure at baseline, suggesting that ablation of JAK2 in DCs does not affect baseline blood pressure (Fig. 3e). Both groups responded similarly to L-NAME treatment and developed high blood pressure that returned to baseline when L-NAME was discontinued (Fig. 3e). Importantly, DC^JAK2KO mice were protected from developing salt-induced elevation in SBP (Fig. 3e). In addition, we employed the state-of-the-art radio telemetry method to measure SBP, diastolic blood pressure (DBP), mean arterial pressure (MAP), and heart rate (HR) in freely moving mice (Fig. 3f, g). We found that DC^JAK2KO mice were protected from salt-induced SBP, DBP, and MAP elevation after L-NAME/HS regimen (Fig. 3h top panel), corroborating the tail-cuff data. We found that both male and female DC^JAK2KO mice exhibit similar protection against developing SBP, DBP, MAP, and HR in (Fig. 3h middle and lower panels). Interestingly, we found that heart rate (HR) was also lower in DC^JAK2KO when compared to DC^JAK2WT mice. No significant difference was found in the levels of epinephrine or norepinephrine (Fig. 3i).

To determine the effect of APC JAK2 deletion on the renal response to a volume laod, mice were intraperitoneally injected with saline equivalent to10% of the mouse’s body weight and placed in metabolic cages for four hours, and urine was collected (Fig. 3j). DC^JAK2KO mice tended to have more preserved response to a volume load after L-NAME/HS regimen compared to DC^JAK2WT mice which did not reach statistical significance (Fig. 3k). In sum, these data suggest that ablation of JAK2 specifically in APCs protects mice from developing salt-induced hypertension.

Deletion of JAK2 in CD11c⁺ cells prevented phosphorylation/activation of downstream STAT3 and SMAD3

To determine if the ablation of JAK2 in CD11c⁺ APCs affects phosphorylation of downstream cascade proteins STAT3 and SMAD3 after L-NAME/HS regimen, we isolated immune cells from the kidneys and performed flow cytometry. Figure 4a shows the gating strategy to identify myeloid immune cells. We employed the fluorescence minus one (FMO) control to set the gates to identify the populations of each cell type; total leukocytes (CD45+), macrophages (MerTK+), DCs (CD11c+/MHCII + or I-A/I-E+), and monocytes (CD115+). We found that DC^JAK2KO mice exhibited significantly reduced phosphorylation levels of both STAT3 and SMAD3 in total leukocytes (CD45+), and DCs (Fig. 4b, c, e, f). The levels of p-SMAD3 were significantly downregulated without any significant changes in p-STAT3 in monocytes (Fig. 4d, g). Furthermore, western blot analysis from isolated DCs from spleens of L-NAME/HS-treated mice indicated that STAT3 phosphorylation was significantly reduced in DC^JAK2KO mice (Fig. 4h). Suppression of Cytokine Signaling 3 (SOCS3) protein inhibits the JAK2-STAT3 pathway by inhibiting JAK2 phosphorylation.²² Western blot analysis showed significantly increased expression of SOCS3 in DC^JAK2KO mice (Fig. 4i). Figure 4l summarizes the interaction between JAK2/STAT3 and SOCS3 proteins.

Deletion of JAK2 in antigen presenting cells attenuated high salt-induced renal infiltration of myeloid cells and secretion of proinflammatory cytokines

Our laboratory has previously shown immune cell infiltration in the kidney and aorta in hypertensive experimental models resulting in end-organ dysfunction and hypertension.^44,45 To determine if JAK2 deletion affects immune cell infiltration and renal fibrosis, we performed immunohistochemistry and flow cytometry. We found that DC^JAK2KO mice are protected from perivascular and glomerular fibrosis after L-NAME/HS regimen (Fig. 5a). We also found decreased infiltration of F4/80 + macrophages in DC^JAK2KO mice (Fig. 5b). To confirm these findings, we performed flow cytometry on single cell suspension of immune cells isolated from the kidneys of mice treated with and without L-NAME/HS regimen. We found that while high salt induced an increase in leukocytes (CD45+), leukocyte and monocytes infiltration was significantly lower in DC^JAK2KO mice (Fig. 5c, d). There was a similar trend in DCs, and macrophage infiltration which did not reach statistical significance (Fig. 4e, f). The levels of proinflammatory cytokine TNF-⍺, mainly secreted by macrophages, were dramatically reduced in macrophages in DC^JAK2KO compared to wildtype mice (Fig. 5g). Importantly, immunogenic protein adducts IsoLGs, and proinflammatory IL-6 cytokine levels were significantly reduced in leukocytes, DCs, and monocytes of mice with APC-specific deletion of JAK2 (Fig. 5h-m). These data suggest that ablation of JAK2 in APCs is protective against salt-induced renal inflammation and fibrosis.

Deletion of JAK2 in antigen presenting cells attenuated high salt-induced renal infiltration of T lymphocytes and secretion of proinflammatory cytokines

We previously found that APCs activate T cells by presenting immunogenic IsoLGs, creating a proinflammatory milieu leading to hypertension.^{45 46,47} To determine if ablation of JAK2 in APCs affects lymphocyte infiltration and activation, we used flow cytometry to analyze single cell suspensions isolated kidneys of mice treated with and without L-NAME/HS regimen. The gating strategy to identify total leukocytes (CD45+), total lymphocytes (CD3+), T helper, (CD4+), cytotoxic T cell (CD8a+), and effector memory T, (T_EM; CD44+, CD62L-), and central memory T (T_CM; CD44+/CD62L+) cells is shown in Fig. 6a. Our data analysis showed a marked decrease in total T lymphocytes (CD3+), and CD8a + T cells (Fig. 6b, c) in the kidney of DC^JAK2KO. CD4 + T cell populations did not change between DC^JAK2WT and DC^JAK2KO (Fig. 6c). We previously reported that L-NAME/HS treatment increases the formation of memory T cells (T_EM) in the kidney, and these cells are the predominant source of cytokine IL-17A release in the kidney.³⁹ Our data showed that T_EM and T_CM were significantly reduced in CD4 + and CD8a + T cell populations in DC^JAK2KO mice (Fig. 6d, e). Importantly, the levels of proinflammatory cytokines TNF-⍺, IL-17A, and IL-6 were markedly reduced in T_EM of the CD8a + population in DC^JAK2KO mice (Fig. 6f-h). In parallel, the levels of TNF-⍺, IL-17A, and IL-6 were markedly reduced in T_CM of the CD8a + population (Fig. 6i-k). We did not find any significant differences in the levels of these proinflammatory cytokines including IFN-γ in T_EM or T_CM of the CD4 + positive cells (Fig. S4a-h). The levels of IFN-γ in T_EM or T_CM of the CD8a + positive cells were also not different between DC^JAK2WT and DC^JAK2KO mice. Taken together, these data suggest that ablation of JAK2 from APCs is protective against infiltration of T lymphocytes, formation of CD8a + T_EM and T_CM, and cytokine production.

Deletion of JAK2 in antigen presenting cells attenuated high salt-induced IL-6 and ENaC-γ expression

Previous studies from our lab and others indicate that cytokines including IL-6 modulate expression and activity of the epithelial sodium channel (ENaC), and other sodium transporters.^30,48,49 Moreover, we recently reported that ENaC-γ activation in DCs promotes inflammation and salt-sensitive hypertension.³⁰ IL-6 is produced by multiple immune cells including monocytes, DCs, lymphocytes, and endothelial cells; it activates JAK/STAT pathway and increases the expression of proinflammatory cytokines.^50,51 Importantly, STAT3 increases the expression of IL6 and results in a feedforward autocrine feedback loop.^51,52 To understand if ablation of JAK2 in DCs spatially affects IL6 and ENaC expression, we performed Fluorescence In Situ Hybridization (FISH) in the kidney. FISH data demonstrated that DC^JAK2KO mice exhibit dramatic attenuation of IL-6 in the kidney after L-NAME/HS regimen (Fig. 7b right), supporting our flow cytometry data in Fig. 5h, j, k, m, and Fig. 6h and k. Interestingly, the expression of IL-6 in DC^JAK2WT was more prominent in the cortex than the medulla; and within the cortex, this expression was more confined to proximal convoluted tubules (PCTs), and distal convoluted tubules (DCTs) in the kidney of DC^JAK2WT (Fig. 7b, left). The ENaC expression is confined to distal tubules (DTs) and collecting ducts (CD). We found that DC^JAK2KO mice showed reduced expression of ENaC-γ in DTs after L-NAME/HS regimen (Fig. 7f, right). We used the CD11c marker to identify myeloid DCs in the kidney and found a marked reduction of CD11c + DCs in the kidney of DC^JAK2KO mice (Fig. 7a, e), confirming the flow cytometry data in Fig. 5f. The overall expression of JAK2 was markedly reduced in the kidney of DC^JAK2KO mice compared to DC^JAK2WT mice (Fig. 7c, g). Collectively, these data suggest that ablation of JAK2 in APCs reduces salt-induced spatial expression of IL-6 and ENaC-γ in the kidney.

In this study, we demonstrate that expression of JAK2 specifically in CD11c⁺ APCs promotes salt-sensitive hypertension via a pathway involving downstream phosphorylation and activation of STAT3 and SMAD3 leading to increased formation of IsoLGs and inflammation (Fig. 8). Elevated sodium induced changes in the expression of the JAK2/STAT3/SMAD3 pathway genes in circulating human APCs and mirrored changes in blood pressure after salt-loading and salt-depletion in salt-sensitive but not salt-resistant people. Ablation of JAK2, specifically in CD11⁺ APCs, attenuated salt-sensitive hypertension in mice. These findings link circulating APC JAK2 signaling to salt sensitivity of blood pressure and promise treatment and feasible diagnostic options for this independent cardiovascular risk factor.

In 2016 the American Heart Association released a statement in which salt sensitivity of blood pressure was defined as changes in blood pressure that parallel changes in salt balance. In humans, blood pressure responses to salt loading or depletion are normally distributed. The current diagnosis which uses arbitrary cutoffs in response to salt-loading/depletion-protocols is not feasible in the clinic research investigating therapeutic options, yet there is clear evidence that salt-sensitive people (about 25% of the normotensive and 50% of the hypertensive population) have higher cardiovascular risk than their salt-resistant counterparts.⁵³ Thus, it is imperative to identify feasible diagnostic tools to facilitate therapeutic drug discovery. Our current finding that components of the JAK2/STAT3/SMAD3 in circulating human APCs sense and change according to exogenous sodium has important implications for the diagnosis given the accessible nature of blood and these molecules which may be used as biomarkers of salt-sensitivity in humans.

The acute dynamic blood pressure regulation in salt-sensitive normotensive and hypertensive individuals is fundamentally different from the chronic blood pressure development in salt-resistant patients and not supported by the infinite gain of the pressure-natriuresis curve concept proposed by Guyton and colleagues. Guyton and colleagues proposed that a sustained hypertensive response to salt would require impaired renal natriuresis.⁵⁴ While this is true for chronic salt-induced renal damage and blood pressure elevation, other studies have found that salt retention or plasma volume expansion is not different in salt-sensitive versus salt-resistant humans.⁵⁴ Animal studies have corroborated this phenomenon.⁵⁵ Thus, further research is warranted to understand the extrarenal mechanisms contributing to salt sensitivity of blood pressure.

Interestingly, we find that ablation of JAK2 in APCs reduced the phosphorylation of STAT3 and SMAD3 and renal immune cell infiltration. Moreover, the dynamic changes in JAK2 and SMAD3 according to salt-loading/depletion correlated with fractional sodium excretion, andan important role of immune cell activation in the kidney in salt-sensitive hypertension. We and others have shown that oxidative stress and proinflammatory cytokines induce immune cell activation and infiltration in the kidney.^45,56,57 We previously found that APCs accumulate in the cortico-medullary junction of the kidney in people with hypertension and that kidney injury results in a marked accumulation of sodium in the kidney.³¹ Thus, further studies are needed to determine how the kidney contributes to immune cell activation leading to salt sensitivity.

The mechanism/s of salt-induced oxidative stress and cytokine production are poorly understood. The JAK/STAT pathway is considered one of the main pathways to modulate oxidative and proinflammatory pathways and associated genes.^{17,27,58−61} SMAD3 has been shown to activate Nox4/Nox2 gene expression in kidney myofibroblasts and increase oxidative stress.⁶² The suppressors of cytokine signaling (SOCS) family of proteins are negative feedback inhibitors of the JAK/STAT pathway.^63,64 Our findings indicate that high salt exposure to monocytes downregulates SOCS family members such as SOCS2 and Cytokine-inducible SH2 (CISH). We have previously shown that APCs including DCs, and monocytes drive salt-induced inflammation and blood pressure elevation in humans and experimental animal models.^30,31,45,65 In addition, our data suggest that monocyte exposure to high salt increases the expression of the genes involved in proinflammatory and oxidative stress and reduces the expression of antioxidant and anti-inflammatory genes. Our studies in mice with APC specific ablation of JAK2 confirmed these studies and further revealed downstream formation of immunogenic IsoLG-protein adducts that we have previously shown to induce inflammation and blood pressure elevation.

Previous human studies have shown that JAK2 mutations are associated with arterial and pulmonary hypertension.^66–72 JAK2 has been implicated in contributing to hypertension,^73,74 However, despite being one of the main signaling pathways that leads to modulating inflammatory and oxidative stress, the role of JAK/STAT/SMAD and related genes is understudied in the context of hypertension or SSBP. Our study has multiple strengths. We employed ex vivo, and in vivo approaches to show the essential role of the JAK-STAT-SMAD pathway in SSBP. For in vivo approaches we enrolled hypertensive patients and phenotyped them for salt-sensitivity using a 3-day inpatient protocol in a tightly controlled environment. Further, we performed a state-of-the-art CITE-Seq approach to observe the changes in gene expression in different immune cells after salt-load and depletion. To understand the mechanism, we generated a transgenic mouse model and measured blood pressure with state-of-the-art radio telemetry in conscious free moving mice. To avoid any sex bias, we used an equal number of male and female mice and showed that gender does not play a role in final outcomes. Most importantly, to our knowledge, this is the first study showing that the JAK/STAT/SMAD pathway of antigen-presenting cells contributes to salt sensitivity The relatively low number of participants in our inpatient study is a potential limitation of our study. Since we knock out JAK2 from DCs in mice, it will be difficult to dissect the effect of DC versus systemic JAK2 inhibition on inflammation and hypertension.

Taken together, our data showed that JAK/STAT/SMAD pathway in APCs plays an important role in salt-sensitive hypertension by activating oxidative and inflammatory pathways in the kidney. Current methodologies for phenotyping humans for salt sensitivity are laborious, cumbersome, and costly, constituting a significant barrier to conducting extensive studies for treatment discovery.⁵³ Activation/phosphorylation of the JAK2/STAT3/SMAD3 pathway and IsoLG in circulating APCs may not only be a biomarker but also a potential target for the treatment of salt-sensitivity.

Author contributions:

A.K., T.K., M.S., P.P.S., and K.W. conceived and designed the research; M.S. L.A.A, A.P.M., L.A.E., J.H.P., J.A.I., N.D.L.V., and P.D.K. performed the experiments; C.F.D., T.A. helped perform Western blot and prepare figures 1 and 2; M.S. L.A.A, A.P.M., C.B., L.A.E., Q.S., J.H.P., J.A.I., C.L.L., F.E., C.W., N.D.L.V., and S.P. analyzed data; M.S. and A.K. interpreted the results of experiments; M.S. L.A.A., A.P., C.B., Q.S., J.H.P., C.L.L., F.E., J.Y., and M.K.G. prepared figures. M.S. and L.A.A. drafted the manuscript; M.S. and A.K. edited and revised the manuscript; A.K. approved the final version of the manuscript. All authors have read and agreed to the current version of the manuscript.

Funding:

This study was supported by the National Institutes of Health grants R01HL147818 (AK and TK), T32HL144446 (AP), R03HL155041 (AK), R01HL144941 (AK), Doris Duke CSDA 2021193 (CNW), K23 HL156759 (CNW), Burroughs Wellcome Fund 1021480 (CNW), the Vanderbilt CTSA grant UL1TR002243 from NCATS/NIH, NIH grants DK059637 and DK020593 to Vanderbilt University Medical Center Hormone Assay and Analytical Services Core, R01HL138519 (AKH), R56AG068026 (ERG).

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Dendritic cell-specific SMAD3, downstream of JAK2, contributes to inflammation and salt-sensitivity of blood pressure

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