DC ENaC-Dependent Inflammasome Activation Contributes to Salt-Sensitive Hypertension

Abstract

Background:

Salt sensitivity of blood pressure is an independent predictor of cardiovascular morbidity and mortality. The exact mechanism by which salt intake increases blood pressure and cardiovascular risk is unknown. We previously found that sodium entry into antigen-presenting cells (APCs) via the amiloride-sensitive epithelial sodium channel ENaC leads to the formation of isolevuglandins (IsoLGs) and release of pro-inflammatory cytokines to activate T cells and modulate salt-sensitive hypertension. In the current study, we hypothesized that ENaC-dependent entry of sodium into APCs activates the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome via IsoLG formation leading to salt-sensitive hypertension.

Methods:

We performed RNAseq on human monocytes treated with elevated sodium in vitro and Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) analysis of peripheral blood mononuclear cells (PBMCs) from participants rigorously phenotyped for salt sensitivity of blood pressure using an established inpatient protocol. To determine mechanisms, we analyzed inflammasome activation in mouse models of deoxycorticosterone acetate (DOCA)-salt-induced hypertension as well as salt-sensitive mice with ENaC inhibition or expression, IsoLG scavenging, and adoptive transfer of wildtype dendritic cells into NLRP3 deficient mice.

Results:

We found that high levels of salt exposure upregulates the NLRP3 inflammasome, pyroptotic and apoptotic caspases, and IL-1β transcription in human monocytes. CITE-seq revealed that components of the NLRP3 inflammasome and activation marker IL-1β dynamically vary with changes in salt loading/depletion. Mechanistically, we found that sodium-induced activation of the NLRP3 inflammasome is ENaC and IsoLG dependent. NLRP3 deficient mice develop a blunted hypertensive response to elevated sodium, and this is restored by the adoptive transfer of NLRP3 replete APCs.

Conclusions:

These findings reveal a mechanistic link between ENaC, inflammation, and salt-sensitive hypertension involving NLRP3 inflammasome activation in APCs. APC activation via the NLRP3 inflammasome can serve as a potential diagnostic biomarker for salt-sensitivity of blood pressure.

Graphical Abstract

Introduction

Hypertension is an important contributor to the development of morbidity and mortality¹ and is a major modifiable risk for developing renal and cardiovascular disease.² Regulation of blood pressure requires a healthy balance between salt and body fluid volume. The World Health Organization recommends at most 5 grams (g) per day of daily salt intake (i.e. 2 g of sodium). However, the global salt intake ranges from 8 – 10g per day.³ A reduction in dietary sodium (Na⁺) decreases hypertension and related cardiovascular events.^{4, 5} Excess salt consumption is particularly of major concern among people with salt-sensitivity of blood pressure (SSBP). SSBP is a pathophysiological phenotype defined as exaggerated blood pressure response to salt loading and salt depletion that affects 25% of the general population and 50% of people with hypertension.⁶

Over the past several years, it has become increasingly evident that a high salt diet (HSD) induces autoimmunity or inflammation; however, the physiological mechanisms are not well understood. Both the innate and adaptive immune systems play a role in the pathogenesis of salt-sensitive hypertension.⁷ Murine macrophages and T cells exposed to high NaCl concentration induce a pro-inflammatory state.^8–13 We recently found that excess extracellular sodium activates epithelial sodium channel (ENaC)-dependent production of the pro-inflammatory cytokine IL-1β in antigen-presenting dendritic cells (DCs) to mediate salt-sensitive hypertension through the production of interleukin 17-A (IL-17A) by polarized T cells.^14–16 Experimental evidence in both humans and mice has suggested that pro-inflammatory cytokines such as IL-1β may potentiate hypertension by altering immune, endothelial, and renal responses.¹⁷ Although it is well known that hypertensive patients have increased circulating levels of IL-1β, only recently has the inflammasome, a major source of IL-1β, been implicated in the development of salt-sensitive hypertension,¹⁸ and the specific cellular source is not known.

The NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a major source of circulating IL-1β. NLRP3 consists of the central nucleotide-binding and oligomerization domain (NOD), pyrin domain, and a carboxy-terminal leucine-rich repeat (LRR) domain, oligomerizes with the apoptosis-associated speck-like protein (ASC) containing a carboxyterminal caspase recruitment domain (CARD) to proteolytically cleave pro-caspase-1 into the bioactive caspase-1. Caspase-1 activation results in the subsequent maturation and secretion of IL-1β.^{19, 20} The inflammasome is a well-characterized mediator of inflammation in multiple chronic inflammatory diseases including gout,²¹ rheumatoid arthritis,²², and atherosclerosis²³ and recent studies suggest that inflammasome activation is at least partially responsible for the development of high blood pressure and associated kidney inflammation in mouse models of hypertension.^{24, 25}

To date, it is not known whether inflammasome activation in myeloid APCs plays a role in the pathogenesis of salt-sensitive hypertension. In the present study, we performed experiments in a rigorously phenotyped cohort of humans as well as mouse models to determine if elevated sodium promotes NLRP3 activation in myeloid-derived APCs via an ENaC and IsoLG dependent pathway in salt-sensitive hypertension.

METHODS

Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. An expanded Methods section is available in the Supplementary Material.

Human subjects:

We used two different cohorts of human participants for the current studies. In cohort one (demographic information in Table 1, previously published ²⁶), we performed RNA sequencing on human monocytes following treatment with elevated sodium (Na⁺) in vitro.

Table 1.

Clinical Characteristics of Subjects Used in Figure 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, mm Hg	69.0±6.8
HTN, %	9
BMI, kg/m2	28.6±17.5

Mean±SD. BMI indicates body mass index; DBP, diastolic blood pressure; HTN, hypertension; and SBP, systolic blood pressure.

In cohort two, we performed cell hashing and CITE-seq analysis of peripheral blood mononuclear cells (PBMCs) from people rigorously phenotyped for salt sensitivity of blood pressure using an established in-patient protocol of rapid salt loading and depletion described in detail below (demographic information in Table 2).

Table 2.

Clinical Characteristics of Subjects Used in Figure 2

	Study population (n=10)
Age, y	49.25±1.98
Male sex, n (%)	8 (80)
White, n (%)	10 (100)
BMI, kg/m2	33.86±2.88
Screening SBP/DBP, mmHg	137.3±4/88.2±2.9
Baseline urinary Na+ excretion, nmol/d	169.1±17.06
Baseline urinary K+ excretion, nmol/d	67.84±10.69

Mean±SD. BMI indicates body mass index; DBP, diastolic blood pressure; and SBP, systolic blood pressure.

All participants gave written informed consent before enrolling. Studies were approved by the Institutional Review Board of Vanderbilt University Medical Center and performed in accordance with the Declaration of Helsinki.

Animals:

Wild-type SV/129 and C57BL6/J mice were obtained from The Jackson Laboratory. All experiments were performed in male mice at approximately 3 months of age and randomly selected for each treatment group using random numbers generated by the RNAD() function in Microsoft Excel. Given that female mice are relatively resistant to salt-sensitive hypertension ²⁷, only male mice were used in this study. Blood pressure was monitored noninvasively using tail cuff as previously described ^{28, 29}, and using state-of-the-art radiotelemetry³⁰, as previously described. The SV/129 mice were fed a high-salt diet (4% NaCl, Teklad TD.92034) for 4 weeks with or without treatment of MCC950 (1mg/kg, every other day, i.p.) or amiloride (1mg/kg/day in drinking water). In a separate experiment, C57BL6/J mice were implanted with a Deoxycorticosterone acetate (DOCA) salt pellet, underwent unilateral nephrectomy, and then were given 1% NaCl in the drinking water, as previously described ³⁰. DOCA-salt mice were pre-treated with IsoLG scavenger 2-hydroxybenzylamine (2-HOBA, Cayman Chemical, Cat# 932-30-9) (1g/L) was delivered via drinking water³⁰. In some studies, CD11c⁺ cells were isolated from the spleens of C57BL6/J wild-type mice fed a high-salt diet for 7 days. These cells were then adoptively transferred to C57BL6/J NLRP3 knock-out mice that then had received L-NAME (0.5 mg/ml) in drinking water for 2 weeks, a washout period for 2 weeks, and a 3-week 4% HSD. Isolation and adoptive transfer of CD11c⁺ cells were performed as previously described³¹. Mice were euthanized at the end of the experiments by CO₂ inhalation. 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.

Results

Elevated Sodium Increases Expression of NLRP3 Inflammasome Associated Genes in Human Monocytes.

We recently found that human monocytes are activated by exposure to elevated sodium concentrations but not equiosmolar mannitol, and that this is NADPH oxidase dependent. These activated human monocytes were found to induce release of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF- α ²⁶. To investigate whether elevated sodium transport into antigen-presenting cells plays a role in NLRP3 inflammasome activation via NADPH oxidase, we employed RNA sequencing on human monocytes from 11 subjects following ex vivo exposure to either normal (150 mMol/L) or high sodium (190 mMol/L) for 72 hours (Figure 1A). The demographics of these participants have been previously published ²⁶ and are also shown in Table 1. Figure 1B shows a representative heatmap for the expression of genes associated with sodium transport, NADPH oxidase, and NLRP3 inflammasome. We found that exposure to elevated sodium increases the expression of channels and transporters including K⁺ voltage-gated channel K_v 1.3 (KCNA3) and P2X purinoreceptor 7 (P2XR7) and decreased ATPase Na⁺/K⁺ transporter catalytic α-subunit (ATP1A1) (Figure 1C). We found no significant difference in gene expressions of Na⁺/H⁺ exchanger (SLC9A1), Ca²⁺-release activated Ca²⁺ modulator (ORAI1), and Na⁺/Ca²⁺ exchanger (SLC8A1) (Figure 1C) and ENaC subunits α (SCNN1A), β (SCNN1B), δ (SCNN1D), or γ (SCNN1G) (Figure 1D). High sodium exposure increased expression of NADPH oxidase subunit p91^phox (CYBB) (Figure 1E) but not NADPH oxidase 1 (NOX1), NADPH oxidase 4 (NOX4), p47^phox (NCF1), p22^phox (CYBA), or p67^phox (NCF2) (Figure 1E). We found that expression of genes associated with activation of the NLRP3 inflammasome including IL-1β (IL1B), which we have previously published²⁶, IL-18 (IL18), and IL-18 receptor (IL18R1) (Figure 1F), inflammatory caspase caspase-1(CASP1), and the initiator and executioner caspases, caspase 2 (CASP2) (Figure 1H–J) and caspase 7 (CASP7) (Figure 1J) were increased in response to elevated sodium exposure. Upon investigation of other NLR proteins, including NLRP1, NLRC4, and NLRP12, in addition to non-NLR proteins such as AIM2 and IFI16, known to assemble into inflammasome complexes, we found that NLRP1, NLRP12, and AIM2 were all significantly downregulated in response to salt (Figure 1G).

Figure 1. RNAseq data exhibiting the effect of high sodium concentrations on human monocytes.

A) Human monocytes from 11 subjects (Table 1) were isolated from total blood using magnetic separation and cultured in either normal salt (150 mM NaCl) or high salt (190 mM NaCl) for 72-h. B) Heat map of RNA transcripts illustrating gene expression differences in response to normal salt (NS) and high-salt (HS) treatment of select classes of genes in RPKM (reads per kilobase per million mapped reads). Bright pink, bright blue, and white represent highest, lowest, and median read values, respectively. Rows represent individual genes, and columns represent individual samples. C) RPKM for select ion channels and transporters. D) RPKM for ENaC subunits α, β, δ, and γ. E) RPKM for NADPH oxidase-related genes. F) RPKM for NLRP3 inflammasome associated genes and activation markers. G) RPKM for other members of the inflammasome family. H-I) RPKM for initiator, executioner, and inflammatory caspases. n=11, paired analyses were performed (NS vs HS) and adjusted P-values (q) are shown in C-J, false discovery rate (FDR < 0.05).

Changes in Monocyte Inflammasome Expression Mirror Changes in Blood Pressure Following Salt Depletion Among People with Salt-Sensitivity of Blood Pressure (SSBP).

To determine whether the changes we observed in monocyte inflammasome gene expression after salt exposure mirror changes in blood pressure following salt depletion among people with SSBP, we carried out the an established in-patient protocol on 10 participants using the difference in blood pressure from salt-loading to salt-depletion to diagnose salt-sensitivity, having withdrawn anti-hypertensive drugs for two weeks before the experiments to wash out any drug effect (Figure 2A). The demographics of these participants are shown in Table 2. PBMCs were isolated from blood collected at baseline, after salt-loading, and following salt-depletion.

Figure 2. Cite-Seq analysis of PBMCs of patients with SSBP.

A) Experimental design of the inpatient protocol. B) Representative figure showing change in systolic blood pressure (SBP) of a salt-resistant and salt-sensitive subject after salt-loading and then salt washout phase of the inpatient protocol. C) Experimental design of human PBMCs CITE-Seq Analysis. D) UMAP representation of multiple immune cell type clusters using antibody-derived tags identifying different immune cell types. E) UMAP depiction of monocyte and dendritic cell clustering after salt-loading and salt-depletion. F) Heatmap hierarchical clustering for protein data (left) and RNA data (right). G) UMAP representation of monocyte clusters from salt-sensitive subject and changes in gene expression within these clusters between salt-loading and salt-depletion. H) NLRP3 gene expression change in monocyte cluster 3 between salt-loading and salt-depletion in representative salt-resistant and salt-sensitive subjects. I) Correlation between change in mean arterial pressure (MAP) after salt-depletion and IL-1β gene expression in monocyte clusters. H was analyzed using an unpaired Mann-Whitney test. I was analyzed using linear regression and significance computed using 2-tailed Pearson test.

Using cell hashing and CITE-seq analysis (Figure 2B), we profiled transcriptomes in immune cells using antibody-derived tags (ADTs). Figure 2C shows UMAP clustering of different cell types. We identified different cell types using ADTs including monocyte marker CD14 in the monocyte cluster, MHC class II cell surface receptor HLA-DR and costimulatory molecule marker CD86 in the monocytes and dendritic cell clusters (Figure 2D). We found no significant differences in the clustering of antigen-presenting cells including dendritic cells and monocytes according to salt-loading/depletion (Figure 2E). We then subset all samples by Cell type, focusing on monocytes and performed cluster analysis to find identifying transcripts. Data was then subset by sample and mean expression for these transcripts was extracted. We created heatmaps with hierarchical clustering by Pearson correlation for protein data and RNA (Figure 2F). We found that samples mainly clustered by patient, not by salt-loading/depletion. However, we found that in salt-sensitive patients, monocytes segregate into 7 different clusters, and salt-depletion is associated with a decrease of cluster 3 monocytes defined in part by IL1B, SGK1, and SOCS3 expression, and an increase in cluster 6 monocytes (Figure 2G and Figure S1). Importantly, we found that the NLRP3 inflammasome expression mirrored changes in blood pressure following salt-depletion (Figure 2H), and the change in blood pressure following salt-depletion correlated with the change in IL1B (Figure 2I). These results suggest that the inflammasome and monocyte activation are dynamically regulated by dietary sodium in vivo and can potentially serve as a diagnostic biomarker for salt-sensitivity of blood pressure.

The High Sodium-Induced Increase in IL-1β is NLRP3 Inflammasome Dependent.

There are different sources of IL-1β in cells. We previously found that experimental hypertension stimulates the production of IL-1β and IsoLGs in murine DCs ¹⁴. To determine mechanisms by which inflammasome activation induce the inflammatory response to elevated sodium in salt sensitivity, we cultured total splenocytes from salt-sensitive SV/129 mice for 24 hours in either normal (150 mM) or high salt media (190 mM) with or without pretreatment with pharmacological inhibitors of NLRP3 inflammasome (MCC950, 1μM) or caspase-1 (YVAD, 5μg/mL) (Figure 3A). We performed flow cytometry on single-cell suspensions using a gating strategy to detect total leukocytes (CD45⁺), dendritic cells (CD11c⁺/MHCII⁺), and monocytes (CD11c⁺/MHCII⁺/CD115⁺) using flow minus one (FMO) controls to set the gates (not shown). Compared to normal salt, high salt induced a striking increase in IL-1β and IL-18 production detected using ELISA, which was prevented by co-incubation with MCC950 and YVAD (Figure 3B and 3C). Similarly, we found high salt increased intracellular IL-1β production in DCs (Figure 3D) and monocytes (Figure 3E), which was abolished with inhibition of NLRP3 inflammasome with MCC950 and caspase-1 inhibitor YVAD. However, we found that MCC950 and YVAD did not prevent expression of NLRP3 in either DCs or monocytes (Figure 3D and 3E). Furthermore, salt-induced increase in IsoLG-protein adducts in DCs and monocytes was not prevented by NLRP3 inflammasome inhibition (Figure 3D and 3E). As shown in Figure 3F, high salt increased co-localization and expression of inflammasome components NLRP3, ASC, and FLICA (a probe for active caspase-1) and D11 ScFv (a marker for isoLG-adducted proteins) in isolated monocytes. As expected, such co-localization was abolished after MCC950 and YVAD pretreatment. These results suggest that high sodium-induced production of IL-1β by myeloid antigen-presenting cells is mediated by the NLRP3 inflammasome and may act downstream of IsoLG-adduct formation.

Figure 3. In vitro high salt activates NLRP3 inflammasome in DCs and monocytes.

A) Experimental strategy where splenocytes were isolated and cultured in normal or high-salt media and co-cultured with inflammasome inhibitor, MCC950 and caspase-1 inhibitor, YVAD. B-C) ELISA showing mature IL-1β (B) and IL-18 (C) released into cell culture media treated with normal or high-salt media with inflammasome inhibitor (n=8, expressed as mean ± SEM) Analysis by paired one-way ANOVA with Tukey’s multiple comparisons test were employed for B and C. D-E) Flow cytometry representatives and average data showing intracellular staining for NLRP3, IL-1β, and IsoLG-protein adducts using the single-chain antibody, D11 ScFv in dendritic cells (D) and monocytes (E) (n= 7–8, expressed as mean ± SEM). Analysis by paired one-way ANOVA with Tukey’s multiple comparisons test were employed for D and E. (F) Representative images and average data showing immunofluorescence staining and colocalization with NLRP3 (Texas Red), ASC (AF647), Caspase-1 (FAM-FLICA), D11 (AF555), and hoechst in isolated monocytes (n=4, expressed as mean ± SEM). Analysis by paired Kruskal-Wallis test followed by a Dunn’s multiple comparison test.

Inhibition of the NLRP3 Inflammasome Attenuates Salt-Sensitive Hypertension in Mice.

To determine if the NLRP3 inflammasome plays a role in salt-sensitive hypertension, we placed salt-sensitive (SV/129) mice on a normal diet (NSD) or high-salt diet (HSD) (4% NaCl) with or without intraperitoneal injections of MCC950 (10 mg/kg every other day) for 4 weeks (Figure 4A). We found that the MCC950 treated mice exhibited less increases in systolic blood pressure in while on a HSD when compared to the untreated HSD fed mice (Figure 4B). We observed no difference in mice treated with MCC950 on a HSD versus a NSD at the end of the 4-week diet period. Additionally, there was no effect of MCC950 treatment on either diets through the course of the experiment. Using flow cytometric analysis, we found that splenic DCs and monocytes of mice on a HSD exhibited increased accumulation of intracellular NLRP3 expression, IL-1β production, and IsoLG-protein adducts. However, mice treated with MCC950 while fed a HSD had significantly less IL-1β production, IsoLG-protein adduct formation, and NLRP3 expression (Figure 4C and 4D). Interestingly, we found a greater effect of both high salt increases and NLRP3 inhibition decreases of IL-1β in monocytes compared to DCs. These results suggest that activation of the NLRP3 inflammasome in DCs and monocytes contributes to salt-sensitive hypertension.

Figure 4. NLRP3 inflammasome inhibition attenuates high salt-induced high blood pressure.

A) Experimental design: mice were given MCC950 (10 mg/kg/day i.p.) treatment with or without high salt diet (HSD) (4% NaCl) for 4 weeks and spleens were isolated and analyzed by flow cytometry. B) Figure showing the change in systolic blood pressure (SBP) following MCC950 with or without HSD (n=9–10, expressed as mean ± SEM). Analysis by paired 2-way ANOVA with Bonferonni repeated measures post hoc test. (C-D) Representative flow plots and group data for intracellular staining of NLRP3, IL-1β, and isoLG in DCs and monocytes, respectively (n= 9–10, expressed as mean ± SEM). Data were analyzed by an unpaired Student t-tests.

Salt-Induced Activation of the NLRP3 Inflammasome in Myeloid Antigen Presenting Cells (APCs) is ENaC Dependent.

We previously found that elevated sodium enters DCs through amiloride-sensitive ENaC and leads to accumulation of IsoLG-protein adducts and IL-1β production resulting in inflammation and hypertension ¹⁴. We hypothesized that elevated sodium activates the NLRP3 inflammasome in myeloid-derived APCs through an ENaC-mediated mechanism and that this activation leads to salt-sensitive hypertension. To determine the role of ENaC in salt-induced activation of the inflammasome, we cultured SV/129 mouse splenocytes in high-salt media with or without pre-treatment with the ENaC inhibitor amiloride (20μM). Flow cytometry analysis of APCs revealed that inhibition of ENaC markedly reduced intracellular NLRP3, IL-1β, and IsoLG-protein adduct formation in both DCs (Figure 5A) and monocytes (Figure 5B). To confirm these results in vivo, we fed salt-sensitive SV/129 mice a HSD with or without amiloride for 4 weeks (Figure 5C). Mice fed a high-salt diet exhibited increased systolic blood pressure, which was attenuated by co-treatment with amiloride (Figure 5D). Additionally, treatment with amiloride prevented the high salt-induced accumulation of NLRP3, IL-1β, and IsoLG-protein adducts in both DCs and monocytes (Figure 5E and 5F). Similar to what we observed in our MCC950 treated mice, monocytes of mice treated with either high salt alone or high salt with amiloride treatment showed greater respective responses in IL-1β production.

Figure 5. Amiloride-inhibitable sodium channel-dependent high salt activation of the NLRP3 inflammasome.

Mouse splenocytes were isolated and cultured for 48 h in normal salt media (150mMol/L NaCl) or high-salt media (190mMol/L NaCl) for 48 h. Representative histograms and group flow cytometric data for in vitro DC (A) and monocyte (B) intracellular staining of NLRP3, IL-1β, and IsoLG-protein adducts (n=7, expressed as mean ± SEM). Analysis by paired one-way ANOVA with a Tukey multiple comparisons post hoc test. C) Experimental design: SV/129 mice were fed a high-salt diet with or without amiloride (1mg/kg/day) in the drinking water for 4 weeks. Spleens were isolated and analyzed by flow cytometry. D) Systolic blood pressure was monitored by tail-cuff (n=8–9, expressed as mean ± SEM). Analysis by paired 2-way ANOVA with Bonferonni repeated measures post hoc test. (E-F) Representative histograms and group flow cytometric data for instracellular staining of NLRP3, IL-1β, and IsoLG-protein adducts in in vivo DC (E) and monocyte (F) from mice treated with HS and HS+Amiloride (n=8–9, expressed as mean ± SEM). Analysis by an unpaired Student t-tests.

Sodium-Induced Activation of NLRP3 Inflammasome in APCs is IsoLG Dependent.

To determine whether activation of the NLRP3 inflammasome plays a role in another model of salt-induced hypertension, we induced DOCA-salt hypertension in C57BL6/J mice which are otherwise salt-resistant. DOCA-salt hypertension was associated with the formation of IsoLG-protein adducts in APCs as we have previously described, and this was prevented by treatment with the IsoLG specific scavenger, 2-HOBA treatment (Ref and Figure 6)¹⁴. To determine if the formation of IsoLGs in this model of hypertension is mediated by activation of the NLRP3 inflammasome, we fed mice with a regular diet, a DOCA-salt, or DOCA-salt plus 2-HOBA. Previously, we have reported that adoptive transfer of high-salt exposed DCs lead to prohypertensive effects in mice, and these effects were abolished when DCs were pretreated with 2-HOBA. ¹⁴. In the present study, we found that co-treatment with 2-HOBA attenuated DOCA-salt-induced increases in NLRP3 expression, IL-1β production, and IsoLG-protein adduct formation in DCs and monocytes (Figure 6A and 6B). Collectively, these data suggest that high-salt induced ENaC activity and subsequent IsoLG production activate NLRP3 inflammasome in myeloid-derived APCs, which contributes to salt-sensitive blood pressure response.

Figure 6. Scavenging IsoLGs Decreased Production of NLRP3 and IL1b in DCs and Monocytes.

A) ELISA to detect release of mature IL-1β. of media from cultured isolated spleenocytes treated in vivo with 0, 10, 30, and 100nM of IsoLG-PE. (n=7–15, expressed as mean ± SEM). Analysis by paired Kruskal-Wallis test followed by a Dunn’s multiple comparison post hoc test. (B-C) Splenocytes were isolated from spleens of sham, DOCA-salt, or DOCA-salt and 2-HOBA treated mice. Flow cytometry representative histograms identifying intracellular markers NLRP3, IL-1β, and isoLG-adducted peptides in DCs (B) and monocytes (C). (n=6–7, expressed as mean ± SEM). Analysis by unpaired one-way ANOVA with a Tukey multiple comparisons post hoc test.

Adoptive Transfer of Wild-type CD11c⁺ Myeloid Cells Restored Sodium-induced Blood Pressure Elevation in NLRP3 Knockout Mice IL-1β Neutralizing Antibody Attenuates Salt-sensitive Hypertension in Mice.

To determine if the expression of the NLRP3 inflammasome is sufficient to induce blood pressure elevation in response to elevated sodium, we induced salt-sensitive hypertension in otherwise salt-resistant C57BL6/J mice using the L-NAME/HS protocol as previously described³². As shown in Figure 7B, we found that deletion of the NLRP3 inflammasome did not affect baseline blood pressure or the hypertensive response to administration of L-NAME. The systolic blood pressure increased to a similar extent in both wild-type controls and NLRP3 knock-out mice and returned to baseline when L-NAME was discontinued. In contrast, the NLRP3 inflammasome knock-out mice were protected from the elevated sodium-induced increase in systolic pressure (Figure 7B top panel), diastolic pressure, and mean arterial pressure (Figure 7B middle panels). Adoptive transfer of wild-type CD11c⁺ APCs into the NLRP3 inflammasome knock-out mice increased the elevated sodium-induced systolic, diastolic, and mean arterial pressure to near wild-type levels. Interestingly, during high salt feeding, the NLRP3 inflammasome knock-out mice exhibited a lower heart rate compared to the wild-type mice, which was not affected by the adoptive transfer of NLRP3 expressing CD11c⁺ cells (Figure 7B, bottom panel). Figure 7C shows flow cytometric confirmation of wild-type CD11c⁺ cell (CD45.2) adoptive transfer into NLRP3 knock-out mice (CD45.1). To address clinical relevance, we used a mouse species specific IL-1β neutralizing antibody in our model of salt-sensitive hypertension by feeding SV/129 mice a 4-week HSD with treatment of either control IgG or IL-1β IgG (10 μg, i.p., twice per week). We observed a significant decrease in night-time SBP of IL-1β IgG treated mice on a HSD at week 4 of our study compared to mice control IgG on a HSD (Figure 7E).

Figure 7: Adoptive transfer of wild-type CD11c+ myeloid cells restores sodium-induced blood pressure elevation in NLRP3 knockout mice and IL-1β neutralizing antibody attenuates salt-sensitive hypertension in mice.

A) Diagram showing adoptive transfer of splenic CD11c+ cells from C57BL6/J wild-type mice fed a high-salt diet into recipient L-NAME/HS treated NLRP3 knock-out (NLRP3KO) mice. Effect of deletion of the NLRP3 inflammasome and CD11c+ cells specific expression of NLRP3 inflammasome on systolic pressure (top panel), diastolic pressure and mean arterial pressure (middle panels) and heart rate (bottom panel) (B) (n=7, expressed as mean ± SEM). C) Confirmation of CD11c+ cell engraftment of wild-type (WT) CD11c+ cells (CD45.2) into NLRP3 knock-out mice (CD45.1). D) Experimental Design: After baseline telemetry readings, SV/129 mice were fed a HSD (4% NaCl) with Control IgG and IL-1β (10 μg, i.p., twice per week) for 4 weeks and blood pressure was measured during the treatment period. E) Effect of HSD and Control IgG or IL-1β IgG on systolic pressure (top panel), diastolic pressure and mean arterial pressure (middle panels), and heart rate (bottom panels) (n=6–7, expressed as mean ± SEM). Data in (B and E) were analyzed by a paired 2-way ANOVA with Bonferonni repeated measures post hoc test.

Discussion

Renal Epithelial Sodium Channel (ENaC) has been extensively studied as a major player in the pathogenesis of salt-sensitivity of blood pressure (SSBP), but recent studies have implicated extra-renal ENaC-dependent mechanisms. Specifically, our studies have focused on the role of ENaC in mediating the entry of sodium into antigen-presenting cells (APCs) leading to the formation of isolevuglandin (IsoLG)-protein adducts, which act as neoantigens and lead to inflammation and SSBP. A critical pro-inflammatory cytokine produced by APCs exposed to elevated sodium environments is IL-1β. Data presented in the current study introduce a novel mechanism attributing IL-1β production to the activation of the NLRP3 inflammasome specifically in myeloid APCs in an ENaC-dependent manner leading to SSBP (Figure 8). Our results indicate that APCs upregulate NLRP3 inflammasome-associated genes in high-salt environments and that their expression changes dynamically according to sodium loading/depletion. We further show that salt-induced activation of the NLRP3 inflammasome is mediated by ENaC-dependent IsoLG-protein adduct formation in the APCs. Moreover, expression of the NLRP3 inflammasome specifically in myeloid CD11c⁺ APCs is sufficient to induce sodium-induced blood pressure elevation in otherwise protected NLRP3 knockout mice. Implicating myeloid-specific NLRP3 inflammasome in the pathogenesis of SSBP adds a new dimension to the conceptual understanding of this important and independent cardiovascular risk factor, and provides new therapeutic and feasible diagnostic strategies.

Figure 8. Schematic illustration of ENaC and IsoLG Dependent Activation of NLRP3 Inflammasome and potential role in Salt-Sensitive Hypertension.

ENaC expression and assembly in immune cells are mediated by SGK1. High salt intake leads to elevated extracellular sodium, which enters APCs through ENaC and increases intracellular Ca⁺ via the activity of the sodium-calcium exchanger. Protein kinase-C, which is activated by Ca⁺, phosphorylates p47phox and leads to the assembly and activation of NADPH oxidase. Superoxide and ROS production by NADPH oxidase results in formation of IsoLGs and IsoLG protein adducts. NLRP3 inflammasome can be activated in response to IsoLGs or ROS directly. NLRP3 inflammasome, in turn, activates caspase 1, which cleaves pro-IL-1β into its mature form. Existing evidence show that IsoLG peptide adducts are presented in major histocompatibility complexes (MHCs), promoting T-cell activation and production of pro-inflammatory cytokines including IFN-γ and IL-17A. T cells infiltrate the kidneys and perivascular space and promote renal sodium retention and vascular dysfunction that ultimately mediate salt-sensitive hypertension.

Although studies have implicated IL-1β in endothelial dysfunction and hypertension^{17 33 34} a role for ENaC-dependent activation of the NLRP3 inflammasome in APCs and SSBP has not been proposed. In a secondary analysis of the recent CANTOS (Canakinumab Anti-Inflammatory Thrombosis Outcome Study) trial, canakinumab, a monoclonal antibody targeting IL-1β, was not found to lower incident hypertension, while effectively reducing cardiovascular events ³⁵. While these studies seem to suggest that IL-1β does not play a role in hypertension, they were not designed to assess the effects of IL-1β inhibition in individuals with or prone to SSBP. The mechanisms governing the pathogenesis of SSBP are different from those of blood pressure regulation. Pioneering studies by Dr. Guyton form the central physiologic tenet that hypertension results from impairment whereby kidneys are no longer able to excrete excess sodium and water leading to increased cardiac output and ultimately blood pressure. However, sodium handling by the kidney and cardiac output are the same in salt-sensitive and salt-resistant people³⁶, suggesting that salt-sensitivity is different from blood pressure regulation by the kidney and further research is needed to understand the extrarenal mechanisms contributing to SSBP.

SSBP, a condition characterized by changes in blood pressure that parallel changes in salt intake, is an independent cardiovascular risk factor which is as powerful as hypertension even in people with otherwise normal blood pressure. The importance of SSBP is demonstrated in a study by Weinberger et al. using an inpatient protocol of acute salt loading and depletion. They found that salt-sensitivity in normotensive and hypertensive people was associated with increased mortality risk ³⁷. The worst prognosis was found in hypertensive salt-sensitive subjects and the best for normotensive salt-resistant. Surprisingly, hypertensive salt-resistant and normotensive salt-sensitive people had an intermediate and similar prognosis, suggesting that salt-sensitivity is a risk factor not only as powerful but also independent of blood pressure. A key important target for study in playing a critical role in the pathogenesis of SSBP is inflammation and immune cell activation. Conditions associated with increased inflammation such as obesity and aging are known to have increased incidents of SSBP, assigning a critical pathogenic role to immune dysregulation. Interestingly, blockade of ENaC is particularly effective in these populations while not effective in most forms of hypertension and has been woefully discontinued in most clinical settings ^{38 39}. These findings suggest that targeting ENaC may help close the gap in health disparities of cardiovascular disease among populations where SSBP is most prevalent.

A critical barrier to progress in understanding mechanisms contributing to SSBP is that diagnosis is not feasible in the clinic and is limited to expensive and laborious research protocols. Using the inpatient Weinberger protocol to rigorously phenotype differences in blood pressure from salt-loading to salt-depletion and classify participants as salt-sensitive or salt-resistant, we found that expression of genes related to activation of the NLRP3 inflammasome dynamically changes in a manner that mirrors blood pressure according to salt loading/depletion among the salt-sensitive but not salt-resistant individuals. Taking into consideration the majority of inpatient protocol participants were male and that women tend to be more salt-sensitive than their counterpart, recruitment of more female participants for this study will be paramount in understanding potential sex-differences in NLRP3 inflammasome activation with SSBP. These findings suggest that the NLRP3 inflammasome and its components not only play a role in mediating the blood pressure response to elevated sodium but can also potentially be used as a biomarker to diagnose SSBP. Indeed, our findings that adoptive transfer of NLRP3 inflammasome expressing CD11c⁺ cells was sufficient to induce increased blood pressure in response to elevated sodium in recipient NLRP3 deficient mice confirm that myeloid APC specific NLRP3 inflammasome plays a role in SSBP.

Mechanistically, we found that the NLRP3 inflammasome activation in myeloid APCs is ENaC-dependent. Pharmacological inhibition and genetic deletion of ENaC prevented activation of the NLRP3 inflammasome and salt-induced hypertension. We recently discovered that antigen-presenting dendritic cells and monocytes express ENaC, which has been extensively studied in the kidney in volume and blood pressure regulation. We found that excess sodium enters APCs through ENaC. The increased intracellular sodium is exchanged for calcium via the sodium-calcium exchanger and activates protein kinase C, which phosphorylates NADPH oxidase subunit p47^phox, resulting in the assembly of the enzyme and production of ROS.¹⁴ This leads to lipid peroxidation and formation of IsoLGs which act as neoantigens in hypertension. We found that DCs treated with high-salt are activated and produce inflammatory cytokine IL-1β and induce T cell production of pro-hypertensive cytokines IFN-gamma and IL-17 ¹⁴. Additionally we found exposure of human monocytes to elevated Na⁺ exhibit increased production of IL-1β, IL-6, and IsoLG-adduct formation ²⁶. Our previous studies also indicate that salt-induced increase in IsoLG-protein adduct formation is associated with hypertension ^{30, 40}. In the current study, we found that the increases in IL-1β and blood pressure in response to salt are mediated by the NLRP3 inflammasome activation. We found that the sodium-induced activation of the NLRP3 inflammasome is associated with increased formation of IsoLG-protein adducts in APCs and scavenging of IsoLGs prevents activation of the NLRP3 inflammasome and the associated production of inflammatory cytokine IL-1β. These studies highlight an important role of ENaC and IsoLG-dependent activation of NLRP3 inflammasome as a crucial mediator of salt-sensitive hypertension.

It is unclear how IsoLG-protein adducts interact with the NLRP3 inflammasome. The canonical NLRP3 pathway is activated by cytokines, pathogen-associated molecular patterns (PAMPs), or damage-associated molecular patterns (DAMPs). It is conceivable that IsoLG-protein adducts may act similarly as PAMPs or DAMPs to activate the NLRP3 inflammasome. In addition, we previously found that ENaC-dependent entry of sodium into DCs leads to the production of ROS, which has been found to activate the NLRP3 inflammasome. Interestingly, we found that scavenging IsoLGs does not reduce the production of ROS. This suggests that the ROS-induced activation of the NLRP3-inflammasome is mediated by a downstream product of ROS, such as IsoLGs. Another possibility is that IsoLG-protein adducts indirectly activate the NLRP3 inflammasome via their action as neoantigens leading to the production of pro-inflammatory cytokines, which can, in turn, activate the NLRP3 inflammasome. We found that scavenging IsoLGs with 2-HOBA blocked NLRP3 inflammasome activation in both DCs and monocytes, suggesting that NLRP3 inflammasome activation is a downstream response to IsoLG-adducts formation. However, inhibition of NLRP3 inflammasome decreased IsoLG-production suggesting there may a feedback loop between the inflammasome and production of IsoLG-adducted proteins. Together with our earlier findings, these results point towards a potential relationship between innate immunity, inflammation, and SSBP.

Although many studies have used C57BL6/J mice as a model for salt-induced inflammation and hypertension, these mice are not salt-sensitive and do not develop hypertension in response to elevated sodium. Prior studies have linked endothelial dysfunction to SSBP, therefore we induce salt-sensitive hypertension in these mice using L-NAME followed by a HSD. L-NAME inhibits the production of endothelial nitric oxide synthase leading to endothelial dysfunction and leads to the formation of memory T cells, which upon a subsequent hypertensive stimulus such as salt results in hypertension, and C57BL6/J mice treated with L-NAME exhibit salt-sensitivity ⁴¹. We found that the L-NAME and HSD combination effect on systolic blood pressure was indistinguishable between our wild-type and NLRP3 knockout mice. Following a two-week washout period, a HSD-induced blood pressure elevation in the wild-type but not the NLRP3 knockout mice, suggesting that the NLRP3 inflammasome plays a role in mediating SSBP. Interestingly, the adoptive transfer of wild-type CD11c⁺ APCs cells into NLRP3 knockout mice increased their blood pressure to wild-type levels, especially during the active night-time period. To confirm our studies, we also used SV/129 mice that are known to exhibit SSBP and we find that the NLRP3 inflammasome mediated salt-sensitivity in these mice as well.

The use of NLRP3 targeted medications has been explored since the discovery by Gabel and colleagues of the most well-characterized inhibitor of NLRP3 MCC950 (also known as CP-456,773) ⁴². MCC950 was used in phase II clinical trials as a potential treatment for rheumatoid arthritis but was halted due to elevated liver enzyme levels. There were no signs of adverse effects nor biochemical or hematological changes in a preclinical trial of healthy individuals with another NLRP3 inhibiting compound, OLT1177, which is an orally active β-sulfonyl nitrile molecule shown to block NLRP3 and ASC interaction as well as inhibit synthesis of pro-IL-1β ⁴³. Currently, there are four novel NLRP3 inhibitors in clinical trial to study osteoarthritis, heart failure, autoinflammatory disorders, Parkinson’s, and pneumonia (NCT01768975, NCT03595371, NCT03534297, NCT04015076, NCT04338997, NCT04086602, NCT04382053). Preclinical and clinical targeting of NLRP3 activity has already spurred a race towards well-tolerated therapeutic tools towards a treatment of chronic inflammatory diseases.

In conclusion, we have defined a novel pathway whereby the NLRP3 inflammasome formation and activation in APCs contribute to salt-sensitive hypertension through the amiloride-sensitive sodium channel ENaC and IsoLG-adduct formation. The strong relationship between salt sensitivity of blood pressure and poor cardiovascular prognosis identifies salt sensitivity as a major target to decrease cardiovascular mortality. Nevertheless, the precise mechanism of how dietary salt leads to high blood pressure, organ damage, and poor cardiovascular prognosis remains to be elucidated. These data suggest that ENaC and IsoLG-dependent inflammasome activation in APCs may play a major role in the pathogenesis of salt-sensitive hypertension and may be a potential therapeutic target in this population.

Novelty and Significance.

What Is Known?

• Salt-sensitivity of blood pressure puts both normotensive and hypertensive individuals at a higher risk for cardiovascular morbidity and mortality.

• The inflammatory processes and role of immune cells in mitigating salt-sensitive hypertension are still unknown.

What New Information Does This Article Contribute?

• Humans with salt-sensitivity of blood pressure upregulate NLRP3 inflammasome components and IL-1β production in antigen-presenting cells compared to humans who are salt-resistant.

• Activation of NLRP3 inflammasome in myeloid antigen-presenting cells is dependent on the amiloride-sensitive sodium channel ENaC and isolevuglandin-adduct formation and contributes to salt-sensitive hypertension.

• Adoptive transfer of antigen presenting cells from wild-type mice fed a high salt diet increased systolic blood pressure in NLRP3 knockout mice previously protected from salt-sensitive hypertension.

Salt-sensitivity of blood pressure is a major contributor to the development of cardiovascular risks. Production of circulating IL-1β, which is increased in hypertensive patients, has been implicated in the pathogenesis of salt-sensitive hypertension. However, the molecular mechanisms of this inflammatory process remain unknown. In this study, we found that the NLRP3 inflammasome is upregulated and activated in myeloid antigen presenting cells and changes dynamically according to sodium loading/depletion in humans. In response to high-salt environments, the NLRP3 inflammasome is activated in an ENaC-dependent manner leading to increased systolic blood pressure. Salt-induced NLRP3 activation leads to isolevuglandin-protein adduct formation in antigen presenting cells. Adoptive transfer of dendritic cells from high salt-fed wild-type mice into NLRP3-deficient mice restores previously blunted hypertensive responses to elevated sodium. Additionally, treatment with an IL-1β neutralizing antibody attenuated salt-induced hypertension in mice. Thus, we propose a novel mechanism whereby myeloid specific NLRP3 inflammasome activation contributes to salt-sensitive hypertension in an ENaC-dependent manner.