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Background: High salt consumption is associated with increased cardiovascular risk and higher morbidity and mortality in salt-sensitive hypertensives than in salt-resistant normotensives. Salt sensitivity of blood pressure (SSBP) is an independent predictor of death due to cardiovascular disease. Although the role of SMAD3 has been extensively studied in kidney fibrosis during renal artery stenosis and other cardiovascular disorders, the role of this pathway in immune cells contributing to SSBP is yet to be defined. Hypothesis: We hypothesized that antigen-presenting specific SMAD3, downstream of JAK2, mediates IsoLG-protein adducts formation, T cell activation, and inflammation and contributes to SSBP. Method. We enrolled two cohorts of participants. We isolated monocytes from cohort one, treated them with normal or high salt, and performed RNA-seq analysis. We used an inpatient salt load and salt depletion protocol to phenotype for salt-sensitive and salt-resistant participants in cohort 2 and performed CITE-Seq analysis. In additional experiments, we generated dendritic cell (DC)-specific JAK2 knockout mice (DCJAK2KO) and performed molecularly and flow cytometric immune phenotyping along with both noninvasive tail-cuff and state-of-the-art radio telemetry blood pressure and heart rate (HR) monitoring in the L-NAME/high salt model of salt sensitivity. We used immunohistochemistry and Fluorescent In Situ Hybridization for spatial and differential infiltration of DCs, and expression of JAK2, SMAD3, ENAC-γ, and IL-6 in DCs as well as fibrosis and macrophages in the kidney. Results: Both bulk and single-cell transcriptomic analyses of human myeloid antigen-presenting cells revealed that high salt treatment in vitro and in vivo upregulates genes of the JAK-STAT-SMAD pathway and downregulates downstream regulators, including the suppressor of cytokine signaling (SOCS) genes. DCJAK2KO mice exhibited attenuated salt-sensitive hypertension ( Systolic blood pressure, 121.6 vs. 138.5, SEM±3.3, n=6) and reduced HR compared to the wildtype littermates during L-NAME/high salt regimen. This was associated with reduced phosphorylation/activation of SMAD3 in total leukocytes ( 982.6 vs 434.7, SEM±107.3), DCs (63.6 vs 18.8, SEM±10.73), and monocytes ( 17.5 vs 106.2, SEM±34.1). Inflammatory markers, IsoLG-protein adducts ( 7.1 vs 25.9, SEM±3.6), IL-6 (6.8 vs 26, SEM±5.9), and TGF-β1 ( 38 vs 91.8, SEM±26.4) in DCs were significantly attenuated. Similarly, these markers were downregulated in total leukocytes and monocytes. The CD8a+ central memory T (TCM) and effector memory T (TEM) cells exhibited lowered IL-17A ( 14.6 vs 26.3, SEM±5.1; 5.7 vs 38.5, SEM±13.96) and IFN-γ ( 26.3 vs 1.104.7, SEM±59.01; 11.6 vs 52.3, SEM±15.6) expression. DCJAK2KO mice also showed attenuated infiltration of total leukocytes, DCs, monocytes, and lymphocytes in the kidney. Conclusion: These results indicate that DC-specific SMAD3 downstream of JAK2 plays an essential role in SSBP. 1K01HL130497-01 (Kirabo, PI); 1R01HL144941-01A1 (Kirabo, PI); 1R03HL155041-01 (Kirabo, PI); 5R01HL147818-22 (Kleyman & Kirabo, MPI); AHA 903428 (Ishimwe). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Background: Salt-sensitivity of blood pressure puts both normotensive and hypertensive individuals at a higher risk for cardiovascular morbidity and mortality. Isolevuglandins (IsoLGs) have been shown to be increased in multiple oxidative-stress associated diseases, including salt-sensitive hypertension. We had previously reported that mice who received adoptive transfer of DCs exposed to high salt exhibited a prohypertensive response, which was abolished when these DCs were pretreated with IsoLG specific scavenger, 2-HOBA. Additionally, we found that scavenging of IsoLGs using 2-HOBA in a mouse model of salt-sensitive hypertension blocked NLRP3 inflammasome in dendritic cells (DCs). Whether IsoLGs directly lead to NLRP3 inflammasome activation remain unknown. IsoLGs have been shown to adduct to histones, specifically H4 histones. Histones are responsible for regulating chromatin structure. However, it is unknown how IsoLG-modified histones affect gene transcription. We tested the hypothesis that elevated sodium in APCs leads to IsoLG-adducted H4 histones increasing transcriptional access to NLRP3 inflammasome components contributing to salt-sensitive hypertension. Methods: In vitro studies were performed on isolated monocytes from 11 hypertensive individuals treated with either normal salt (150mM) or high salt (190mM) and submitted for RNA sequencing. In vivo studies were performed on 19 hypertensive subject enrolled in an acute inpatient protocol to assess response to salt loading (460 mmoL/24 hours) and salt depletion (10 mmoL/24 hours, and furosemide 40 mg x 3). IsoLG-containing DCs were identified using flow cytometry as CD45+MerTK-HLADR+CD1c+. Isolated PBMCs from a salt sensitive subject (ΔSBPSD-SL = -12.9) and a salt resistant subject (ΔSBPSD-SL = 0.35) were analyzed using assay for transposase-accessible chromatin with high-throughput sequencing (ATACseq). Results: Using RNA sequencing data of isolated monocytes and the EpiFactors database, I identified differentially expressed genes related to histones and histone modification. This analysis demonstrated an overall upregulation in histone modification after high salt visualized in a heatmap and PCA plot, specifically upregulation of gene HIST4H4 which encodes the Histone H4 (25.4 ± 4.6 vs 65.5 ± 6.5, [n=11, q=1.5 x 10-3, paired analyses and adjusted P values (q), false discovery rate (+] increase in IsoLG-adducts may epigenetically regulate NLRP3 inflammasome components in SSBP. No Disclosures. Funding Sources: K01HL130497, R01HL147818, R03HL155041, R01HL144941 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.