Distinct metabolic responses to heme in inflammatory human and mouse macrophages - Role of nitric oxide

Abstract

Activation of inflammation is tightly associated with metabolic reprogramming in macrophages. The iron-containing tetrapyrrole heme can induce pro-oxidant and pro-inflammatory effects in murine macrophages, but has been associated with polarization towards an anti-inflammatory phenotype in human macrophages. In the current study, we compared the regulatory responses to heme and the prototypical Toll-like receptor (TLR)4 ligand lipopolysaccharide (LPS) in human and mouse macrophages with a particular focus on alterations of cellular bioenergetics. In human macrophages, bulk RNA-sequencing analysis indicated that heme led to an anti-inflammatory transcriptional profile, whereas LPS induced a classical pro-inflammatory gene response. Co-stimulation of heme with LPS caused opposing regulatory patterns of inflammatory activation and cellular bioenergetics in human and mouse macrophages. Specifically, in LPS-stimulated murine, but not human macrophages, heme led to a marked suppression of oxidative phosphorylation and an up-regulation of glycolysis. The species-specific alterations in cellular bioenergetics and inflammatory responses to heme were critically dependent on the availability of nitric oxide (NO) that is generated in inflammatory mouse, but not human macrophages. Accordingly, studies with an inducible nitric oxide synthase (iNOS) inhibitor in mouse, and a pharmacological NO donor in human macrophages, reveal that NO is responsible for the opposing effects of heme in these cells. Taken together, the current findings indicate that NO is critical for the immunomodulatory role of heme in macrophages.

Keywords: Heme; Inflammation; Lipopolysaccharide; Macrophages; Mitochondrial metabolism; Nitric oxide.

Graphical abstract

Fig. 1

Differential effect of heme and LPS on the gene expression profile of human macrophages. hMDMs were treated with heme (10 μM) and LPS (1 μg/ml) alone for 6 h and the bulk transcriptome was compared (n = 4). (A) Heatmap from differentially expressed genes (DEGs) in heme- vs LPS-treated cells. (B) Gene ontology cellular compartments (GOCC) analysis of differentially expressed genes in heme vs LPS. Gene Set Enrichment Analysis was performed using EnrichR. Significant GOCC database were defined as those having an adjusted p-value smaller than 0.05. (C) Heatmap representation of DEGs. The heatmap was scaled across genes and generated based on R package pheatmap v1.0.12. (D) Volcano plots for the DEGs of heme vs LPS. The x-axis represents the log2-fold change values, and the y-axis represents -log10 adjusted p-values. DESeq2 was used to calculate the normalized reads from raw counts. (E) Boxplots for the top 10 up-regulated and down-regulated DEGs of heme vs LPS. See also Figs. S1 and S2.

Fig. 2

Heme is anti-inflammatory in LPS-stimulated human macrophages. hMDMs were treated with various concentrations of heme and with LPS (1 μg/ml) alone or in combination for 16 h, as indicated. (A) Gene expression of TNF-α, IL-6, COX-2 were measured by real-time RT-PCR and cell culture supernatants were collected for ELISA to detect TNF-α and IL-6. (B) Gene expression of HO-1: values were normalized to the expression of HPRT and the respective ΔΔCT values are shown. (C) hMDMs were stimulated with IFN-γ (10 ng/ml), IL-4 (20 ng/ml), LPS or heme for 24 h. Expression of the M1 marker CD64 and the M2 marker CD206 were analyzed by flow cytometry and the fold induction of mean fluorescence intensity relative to unstimulated control macrophages is shown (n = 3). The values represent mean ± SEM of three independent experiments. Statistical analysis was performed using one-way ANOVA with Tukey's test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Fig. S3.

Fig. 3

Heme is pro-inflammatory in LPS stimulated mouse macrophages. mBMDMs were treated with heme (5 or 10 μM) and LPS (1 μg/ml) for 16 h. (A) Gene expression of TNF-α, IL-6, COX-2 were measured by real-time RT-PCR and culture supernatants were collected for ELISA to detect TNF-α and IL-6. (B) Gene expression of HO-1: values were normalized to the expression of HPRT and the respective ΔΔCT values are shown. The values represent mean ± SEM of three independent experiments. Statistical analysis was performed using one-way ANOVA with Tukey's test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 4

Heme differentially affects bioenergetics in human and mouse macrophages. (A–B) hMDMs and mBMDMs were treated with heme (5 or 10 μM) or LPS (1 μg/ml) alone for 16 h and subjected to a Mito Stress test consisting of the sequential addition of oligomycin (1 mg/ml), FCCP (0.7 μM), and rotenone (1 μM) plus antimycin A (1 μM). The Mito Stress profile and the values of the calculated respiratory parameters are shown as mean ± SEM of three independent experiments. (C) Extracellular acidification rate (ECAR), an index of glycolysis, is shown. (D) ATP levels were assessed in hMDMs and mBMDMs by an ATP assay 16 h after heme or LPS treatment. Statistical analysis was performed using one-way ANOVA with Tukey's test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Con, control; FCCP, carbonyl cyanide p-trifluoro methoxyphenylhydrazone.

Fig. 5

Heme differentially affects mitochondrial bioenergetics in LPS-activated human and mouse macrophages. hMDMs and mBMDMs were treated with heme (5 or 10 μM) or LPS (1 μg/ml) alone for 16 h and subjected to a Mito Stress test after the sequential addition of oligomycin (1 mg/ml), FCCP (0.7 μM), and rotenone (1 μM) plus antimycin A (1 μM). (A) Mito-stress profile in hMDMs and respiratory parameters. (B) Extracellular acidification rate (ECAR), an index of glycolysis, is shown. (C) The Mito-Stress profile in mBMDMs and the values of the calculated respiratory parameters are shown as mean ± SEM of three independent experiments. (D) ECAR: statistical analysis was performed using one-way ANOVA with Tukey's test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Con, control; FCCP, carbonyl cyanide p-trifluoro methoxyphenylhydrazone. See also Fig. S4.

Fig. 6

Inhibition of NO activity reduces pro-inflammatory cytokine production in mouse macrophages. (A) mBMDMs were pre-treated with 1400W for 30 min followed by exposure to heme (10 μM) and LPS (1 μg/ml) alone or in combination for 16 h (B) Nitrite production was measured by the Griess assay. (C) Culture supernatants were collected for ELISA to detect TNF-α and IL-6. (D) Gene expression of TNF-α and IL-6 was measured by real-time RT-PCR. The values were normalized to the expression of HPRT and the respective ΔΔCT values are shown. (E) hMDMs were pre-treated with DETA (1 mM) for 1 h followed by stimulation with heme (10 μM) and LPS (1 μg/ml) for 16 h. Gene expression of TNF-α, IL-6 and COX-2 were measured by real-time RT-PCR. The values were normalized to the expression of HPRT and the respective ΔΔCT values are shown. The values are shown as mean of ±SEM of at least three independent experiments. Statistical analysis was performed using one-way ANOVA with Tukey's test or unpaired t-test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.