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

Abstract

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.

The Antioxidative Effects of Flavones in Hypertensive Disease

“…Its phosphorylation and subsequent activation are regulated by a variety of pathways upstream [15]. IL-6 signaling, via the JAK2/STAT3 pathway, results in Smad3 phosphorylation, which leads to the upregulation of NADPH oxidase components [15,16]. The canonical TGF-β1 signaling pathway, which is also involved in the activation of the pro-inflammatory transcription factor NF-κB, is dependent on Smad3 phosphorylation [17].…”