A Time- and Compartment-Specific Activation of Lung Macrophages in Hypoxic Pulmonary Hypertension

Abstract

Studies in various animal models suggest an important role for pulmonary macrophages in the pathogenesis of pulmonary hypertension (PH). Yet, the molecular mechanisms characterizing the functional macrophage phenotype relative to time and pulmonary localization and compartmentalization remain largely unknown. In this study, we used a hypoxic murine model of PH in combination with FACS to quantify and isolate lung macrophages from two compartments over time and characterize their programing via RNA sequencing approaches. In response to hypoxia, we found an early increase in macrophage number that was restricted to the interstitial/perivascular compartment, without recruitment of macrophages to the alveolar compartment or changes in the number of resident alveolar macrophages. Principal component analysis demonstrated significant differences in overall gene expression between alveolar and interstitial macrophages (IMs) at baseline and after 4 and 14 d hypoxic exposure. Alveolar macrophages at both day 4 and 14 and IMs at day 4 shared a conserved hypoxia program characterized by mitochondrial dysfunction, proinflammatory gene activation, and mTORC1 signaling, whereas IMs at day 14 demonstrated a unique anti-inflammatory/proreparative programming state. We conclude that the pathogenesis of vascular remodeling in hypoxic PH involves an early compartment-independent activation of lung macrophages toward a conserved hypoxia program, with the development of compartment-specific programs later in the course of the disease. Thus, harnessing time- and compartment-specific differences in lung macrophage polarization needs to be considered in the therapeutic targeting of macrophages in hypoxic PH and potentially other inflammatory lung diseases.

Figure 1

Accumulation of CD68-positive perivascular macrophages (red) is observed at 4 days and 14 days hypoxic (simulated 18,000 feet) exposure in WT mice, compared to sea-level controls. Thin arrows label perivascular macrophages while thick arrows label alveolar macrophages. Cell nuclei are labeled with DAPI (blue), elastic lamellae (green) are visualized via autofluorescence. Scale bar 100 μm. SL, sea level; 4d-Hx, 4 days hypoxia; 14d-Hx, 14 days hypoxia. Representative of 5 mice per group.

Figure 2

FACS quantification of lung macrophages. a,b WT mice were exposed to hypoxia for 4, 14 days or remained at sea level. IM’s and AM’s were quantified using whole lung via flow cytometry. IM’s and AM’s expressed as total number per lung determined by FACS/counting bead method. N= 5 mice per group. Error bars show SEM. * p<0.05. c: Cells were lavaged from sea level mice or after 4 days of hypoxia exposure. Macrophages were quantified using counting beads and FACS or by hemocytometer. N=4–6 mice per group. There were no significant differences between groups. d. Time course of CD64+, CD11b^hi or CD64+, CD11c^hi cells represented as percentage of macrophages in BAL fluid. No significant differences between groups. N=4–5 mice per group. Quant, quantification; BAL, bronchoalveolar lavage; SL, sea level; D, day; WT, wild type; IM, interstitial macrophage; AM, alveolar macrophage.

Figure 3

a. Principle component analysis (PCA) of AM’s and IM’s at baseline and after 4 and 14 days hypoxia exposure. Each dot represents RNAseq data from 3 pooled mice performed in biologic triplicate except for sea level AM’s which were done in biologic replicate. Power analysis demonstrated 99% power for sample size with a p value<0.05. The red line separates sea level samples from hypoxic samples. b. Global analysis of differentially expressed genes between AM and IM populations. Identification of differentially expressed genes was performed in all pairwise comparisons between the cell population (AMs & IMs) and between different time points (0, 4 & 14 day hypoxia) from the RNA-seq analysis (2,672 genes). Hierarchical clustering and heatmap were generated on the set of 2,672 genes and highly expressed genes in specific populations were clustered. Cluster of RNA-seq data segregated in two clusters (sea level and hypoxia) and further segregated in AMs and IMs. SL, sea level; IM, interstitial macrophage; AM, alveolar macrophage; D, day.

Figure 4

VENN diagrams showing up and down regulated genes in alveolar macrophages (AM) and interstitial macrophages (IM) after 4 days (D4) or 14 days (D14) hypoxia as compared to baseline sea level macrophages. Listed are total numbers of differentially regulated genes in each subset as well as the percentages of total up or down regulated genes in each group. Cutoff criteria for differential gene expression: ≥ 2 fold absolute change from sea level baseline, q<0.05.

Figure 5

IPA analysis of top canonical pathways and upstream regulators in alveolar and interstitial macrophages. a. List of all canonical pathways and b. upstream regulators (abbreviated list) with a p<0.05, absolute Z ≥ 2 in AM’s and IM’s after 4 or 14 days hypoxia as compared to baseline sea level macrophages. Colors indicate higher or lower Z scores. c. List of top canonical pathways (abbreviated list) in IM’s and AM’s after 4 or 14 days hypoxia with a p<0.05 sorted by increasing p values (dark purple color). IM, interstitial macrophage; AM, alveolar macrophage; D, day.

Figure 6

Schematic of the major pathways/upstream regulators and their interaction as identified by IPA analysis in AM’s at days 4 and 14 and IM’s at day 4. Red boxes indicate predicted activation, while blue boxes predicted inhibition with an absolute Z score cutoff of 2 and a p<0.05. Red arrows indicate activation of downstream pathway, while blue lines indicate inhibition of downstream pathway. * ERK was not significant in AM’s at days 4 or 14, while MAP4K4, PPRG, SREBF1/2 were not significant in IM’s at day 4.

Figure 7

a. Canonical pathways in IM’s after 4 and 14 days hypoxic exposure as compared to baseline sea level macrophages. “Role of pattern recognition receptors in bacteria and viruses” was the only pathway with an absolute Z score>2, p<0.05 that was unique in IM’s at day 14 as compared to IM’s at day 4. The individual genes in this pathways are listed with the down arrow indicating decreased transcription as compared to baseline sea level macrophages. b. Selected unique upstream regulators in IM’s after 14 days hypoxia with an absolute Z score>2, p<0.05. Table denotes Z score of corresponding regulator in IM’s after 4 days hypoxia. IM, interstitial macrophage; AM, alveolar macrophage; D, day; NS, non-significant.

Figure 8

All canonical pathways and upstream regulators in IM’s after 14 days of hypoxia as compared to IM’s after 4 days hypoxia with an absolute Z score≥ 2, p<0.05. Adjusted p value (q value) is listed in the table for canonical pathways. The filtered data set used for IPA used the cutoff of q <0.2 and an absolute fold change of ≥1.5.