Immune profiling of murine cardiac leukocytes identifies triggering receptor expressed on myeloid cells 2 as a novel mediator of hypertensive heart failure

Abstract

Aims: Heart failure with preserved ejection fraction (HFpEF) is characterized by diastolic dysfunction, microvascular dysfunction, and myocardial fibrosis with recent evidence implicating the immune system in orchestrating cardiac remodelling.

Methods and results: Here, we show the mouse model of deoxycorticosterone acetate (DOCA)-salt hypertension induces key elements of HFpEF, including diastolic dysfunction, exercise intolerance, and pulmonary congestion in the setting of preserved ejection fraction. A modified single-cell sequencing approach, cellular indexing of transcriptomes and epitopes by sequencing, of cardiac immune cells reveals an altered abundance and transcriptional signature in multiple cell types, most notably cardiac macrophages. The DOCA-salt model results in differential expression of several known and novel genes in cardiac macrophages, including up-regulation of Trem2, which has been recently implicated in obesity and atherosclerosis. The role of Trem2 in hypertensive heart failure, however, is unknown. We found that mice with genetic deletion of Trem2 exhibit increased cardiac hypertrophy, diastolic dysfunction, renal injury, and decreased cardiac capillary density after DOCA-salt treatment compared to wild-type controls. Moreover, Trem2-deficient macrophages have impaired expression of pro-angiogenic gene programmes and increased expression of pro-inflammatory cytokines. Furthermore, we found that plasma levels of soluble TREM2 are elevated in DOCA-salt treated mice and humans with heart failure.

Conclusions: Together, our data provide an atlas of immunological alterations that can lead to improved diagnostic and therapeutic strategies for HFpEF. We provide our dataset in an easy to explore and freely accessible web application making it a useful resource for the community. Finally, our results suggest a novel cardioprotective role for Trem2 in hypertensive heart failure.

Keywords: Heart failure; Hypertension; Inflammation; Macrophage; TREM2.

Graphical Abstract

Figure 1

DOCA-salt model recapitulates key features of HFpEF. (A) Heart weight normalized to tibia length (HW/TL, n = 10–15). (B) Urine albumin to creatinine ratios (n = 6–10). (C) Echocardiographic measures of heart rate (HR), ejection fraction (EF), and fractional shortening (FS) after 3 weeks in sham controls and DOCA-salt treated animals (n = 6–7). (D) Invasive haemodynamic measures of the diastolic relaxation constant Tau (τ) and end-diastolic pressures (EDP, n = 5–8). (E) Exhaustion time during treadmill exercise stress test (n = 7–8). (F) Ratio of lung wet weight to dry weight indicative of pulmonary congestion (n = 7–8). (G) Representative images of Masson’s trichrome staining demonstrating collagen deposition and quantification of positive staining as total left ventricle (LV) fibrosis per total tissue area per section (n = 7). (H) Relative gene expression in left ventricles of sham and DOCA-salt treated mice normalized to Gapdh (n = 5–9). Data are expressed as mean ± SEM and analysed by Mann–Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

CITE-seq of immune cells in healthy and HFpEF hearts reveals broad population changes. (A) Schematic created with Biorender representing hashing and staining of cardiac leucocytes from four sham and four DOCA-salt treated mice. (B) Weighted nearest neighbours uniform manifold approximation and projection (UMAP) plot based on RNA transcriptomes and protein markers displaying clusters of immune cells sequenced from all samples. (C) RNA expression of lineage marking genes supporting cluster identification. (D) Protein expression of antibody panel used in CITE-seq analysis. (E) Log-fold difference of immune cell subsets as percent of CD45 + immune cells using scProp. Clusters significantly different based on a false discovery rate < 0.05 are highlighted in red and denoted by dashed line.

Figure 3

Altered T-cell abundance and transcriptional landscape in HFpEF. (A) UMAP representation of cardiac T-cell subsets. (B) RNA expression of markers differentiating T-cell populations. (C) Log-fold difference of T-cell subsets as a percent of CD3 + cells using scProp. Sub-clusters significantly different based on a false discovery rate < 0.05 are highlighted in red. (D) RNA expression of selected T-cell polarization and activation related genes by cluster. (E) Volcano plot of differentially expressed genes in all T-cell subsets highlighting genes up-regulated in the DOCA-salt treated group (red) vs. genes enriched in the Sham group (blue). Horizontal dotted lines represent adjusted P value of 0.05, and vertical dotted lines represent average log₂-fold change of 0.25. (F) Violin plot of Tmem173 expression split by cluster and experimental condition.

Figure 4

Altered myeloid cell abundance and transcriptional landscape in HFpEF. (A) UMAP representation of myeloid cell subsets along with a UMAP of cardiac macrophages split by experimental condition. (B) RNA expression of markers differentiating myeloid populations. (C) Log-fold difference of myeloid cell subsets as a percent of CD11b + cells using scProp. Sub-clusters significantly different based on a false discovery rate < 0.05 are highlighted in red and denoted by dashed line. (D) Volcano plot of differentially expressed genes in all macrophage subsets highlighting genes up-regulated in the DOCA-salt treated group (red) vs. genes enriched in the Sham group (blue). Horizontal dotted lines represent adjusted P value of 0.05, and vertical dotted lines represent average log₂-fold change of 0.25. (E) Up-regulated pathways in cardiac macrophages from DOCA-salt treated mice. (F) Overlaid expression of selected genes onto UMAP representation of macrophage clusters seen in (A), split by experimental condition.

Figure 5

Cardiac macrophage Trem2 is increased after DOCA-salt treatment. (A) Left ventricle gene expression of Trem2 in Sham and DOCA-salt treated mice. (B) Plasma sTrem2 levels measured by ELISA in sham and DOCA-salt treated mice. (C) RNAscope images of cardiac sections using probes targeted towards Cd68 (macrophage marker) and Trem2 (left). Quantification of median Trem2 signal per high-powered field and median Trem2 signal per cardiac macrophage using CellProfiler (right). A total of five to eight images were analysed per mouse to obtain median values. Scale bar 25 microns. Inset enlarged for visualization. Data are expressed as mean ± SEM and analysed by Mann–Whitney U test. *P < 0.05, **P < 0.01.

Figure 6

Loss of Trem2 exacerbates cardiac hypertrophy and renal injury in DOCA-salt treated mice. (A) Representative H&E staining of Trem2^+/+ and Trem2^−/− mice after DOCA-salt treatment showing concentric hypertrophic response, scale bar 500 microns. (B) Systolic BP as measured by tail cuff plethysmography (n = 11–12 per group). (C) Heart weight (HW) normalized to body weight (BW) of Trem2^+/+ and Trem2^−/− mice after DOCA-salt treatment and in age-matched unmanipulated mice (untreated). Data from three independent cohorts of mice (n = 6–9). (D) Violin plot of cross-sectional area (CSA) of cardiomyocytes after sham or DOCA-salt treatment. Each dot represents an individual measurement, and each violin represents a mouse from two independent cohorts. The median value for each mouse is quantified on the right (n = 3–6). (E) Invasive haemodynamic measures of the diastolic relaxation constant Tau (τ) (n = 3–4). (F) Urine albumin to creatinine ratios after a 16-h overnight urine collection (n = 6–10). Data are expressed as mean ± SEM and analysed by two-way ANOVA with repeated measures and Sidak’s multiple comparison tests (B), two-way ANOVA with Tukey’s post-test (C, D, F), Mann–Whitney U test (E). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 7

Loss of Trem2 alters macrophage gene expression and cytokine production. (A) Relative expression of Lgals1 (galectin-1), Lgals3 (galectin-3), and Spp1 (osteopontin) in Trem2^+/+ and Trem2^−/− BMDMs. Each point is a biological replicate (n = 4). (B) BMDMs were differentiated and stimulated with LPS (M1) or not (M0) for 24 h and then harvested. Relative expression of Il6 and Il10 normalized to Rplp0. Each point is a biological replicate (n = 4). (C) Enrichment analysis of genes down-regulated in Trem2^−/− BMDMs compared to Trem2^+/+. (D) Z-score values of selected genes from bulk RNA-sequencing of Trem2^+/+ (WT) and Trem2^−/− (KO) BMDMs. (E) Relative expression of Vegfa, Mmp12, and Mmp13 normalized to Rplp0 in BMDMs. Points represent biological replicates (n = 4). (F) VEGF-A concentration in supernatants of BMDMs after 48 h of plating as determined by ELISA. Each point is a biological replicate from two independent experiments (n = 6–8). (G) Gene expression in CD11b + fraction of cardiac cells normalized to Gapdh in Trem2^+/+ and Trem2^−/− mice after DOCA-salt treatment (n = 4–5). (H) Left ventricular tissue gene expression normalized to Gapdh in Trem2^+/+ and Trem2^−/− mice after DOCA-salt treatment (n = 7–9). Data are expressed as mean ± SEM and analysed by two-way ANOVA with Tukey’s post-test (B), Student’s t-test (G: Il10), or Mann–Whitney U test (A, E, F, G, H).*P < 0.05, **P < 0.01, ***P < 0.001.

Figure 8

Loss of Trem2 is associated with decreased cardiac capillary density after DOCA-salt treatment. (A) Representative images of immunohistochemistry for CD31 as a measure of capillary density and quantification of CD31 positivity as percent tissue area. A total of six to eight images with myocytes in cross-section were averaged per animal to obtain a single value per mouse (n = 6–7). Scale bar is 50 microns. (B) Representative gating strategy for quantification of vascular endothelial cells (VECs, CD45-CD31 + PDPN-) based on CD31 and PDPN expression with absolute numbers of cells in Trem2^+/+ (black circles) and Trem2^−/− (red squares) male mice after sham of DOCA-salt treatment from two cohorts (n = 4–6). Data are expressed as mean ± SEM and analysed by Student’s t-test. *P < 0.05.

Figure 9

sTREM2 is elevated in human heart failure. Plasma samples were collected from control individuals without heart failure (n = 9) and from individuals with heart failure (n = 11) within 24 h of an admission for acute heart failure exacerbation and an underlying diagnosis of HFpEF. Levels of sTREM2 were measured by ELISA. Note that the high value in the HFpEF group did not meet outlier criteria by ROUT testing and removal does not change the results. Data are expressed as box and whisker plots showing median and range along with individual points and analysed by Mann–Whitney U test. ***P < 0.001.