Bulk RNAseq of ex vivo salt treated isolated human monocytes

Salt sensitivity of blood pressure (SSBP) is associated with persistent immune activation after sodium normalization, suggesting immune “salt memory.” Prior work identified that sodium entry into antigen-presenting cells (APCs) via epithelial sodium channels triggers NLRP3 inflammasome activation through the formation of isolevuglandins (IsoLGs), highly reactive lipid aldehydes. IsoLG scavenging with 2-hydroxybenzylamine (2-HOBA) reduces NLRP3 expression and attenuates salt-induced hypertension, implicating IsoLGs as upstream immune regulators. This study tested whether IsoLGs also act as epigenetic modifiers by covalently modifying histone H1, thereby altering chromatin accessibility at inflammatory loci such as NLRP3. PBMCs from 10 hypertensive adults classified as salt-sensitive (SS) or salt-resistant (SR) were collected at baseline, after salt loading, and after depletion. Integrated single-cell RNA-seq and ATAC-seq revealed that SS monocytes exhibited salt-induced enhancer activation and increased chromatin accessibility at the NLRP3 locus. IsoLG–protein adducts correlated with blood pressure changes and were most prominent in CD14? monocytes. In murine APCs, high salt induced IsoLG adduction of histone H1, attenuated by 2-HOBA. Knockdown of H1F0 reduced high salt–induced NLRP3 expression, supporting a role for H1.0 in transcriptional regulation. These findings identify IsoLG modification of histone H1 as a potential mechanism linking extracellular sodium exposure to persistent chromatin remodeling and sustained inflammatory gene expression in SS individuals. IsoLG–histone adducts may represent novel therapeutic targets for immune-driven hypertension. Overall design: Heparinized blood was obtained from 11 volunteers, and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient. Briefly, collected blood was diluted 4-fold with PBS+2mM EDTA buffer then carefully layered over 15 mL of Ficoll-PaqueTM in a 50 mL conical tube. After centrifugation at 400xg for 30 minutes at 20°C, carefully aspirate the top plasma layer. Next, carefully remove the next layer containing PBMCs and tranfer to another 50 mL conical tube. Fill with buffer and centrifuge again at 300xg for 10 minutes at 20°C. Resuspend the cell pellet and proceed to monocyte isolation. Isolation of monocytes was achieved by magnetic labeling and negative selection using the Miltenyi monocyte isolation kit (Miltenyi Biotec, Cat# 130-091-151), according to manufacturer's protocol using LS columns. Monocytes were then cultured in 12-well plates at 1 x 10^6 /mL density in either normal salt RPMI media (150 mM) or high salt RPMI media (190 mM) for 72 hr. RPMI media 1640 (Gibco) was supplemented with 10% FBS, 1% pen/strep, 1% HEPES, and 2-Mercaptoethanol (0.05 mM). For total RNA isolation, we used the RNEasy Midi Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. Targeted analysis of polyadenylated transcripts was performed using the Illumina Tru-seq RNA sample prep kit, and paired-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 RNAseq data occurred 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 were performed using QC3 , and the MultiRankSeq method was used for expression analysis. Raw data false discovery rate (FDR < 0.05) was used to correct for multiple hypothesis testing.