Activation of the JNK/COX-2/HIF-1α axis promotes M1 macrophage via glycolytic shift in HIV-1 infection

Abstract

Chronic inflammation is recognized as a major risk factor for the severity of HIV infection. Whether metabolism reprogramming of macrophages caused by HIV-1 is related to chronic inflammatory activation, especially M1 polarization of macrophages, is inconclusive. Here, we show that HIV-1 infection induces M1 polarization and enhanced glycolysis in macrophages. Blockade of glycolysis inhibits M1 polarization of macrophages, indicating that HIV-1-induced M1 polarization is supported by enhanced glycolysis. Moreover, we find that this immunometabolic adaptation is dependent on hypoxia-inducible factor 1α (HIF-1α), a strong inducer of glycolysis. HIF-1α-target genes, including HK2, PDK1, and LDHA, are also involved in this process. Further research discovers that COX-2 regulates HIF-1α-dependent glycolysis. However, the elevated expression of COX-2, enhanced glycolysis, and M1 polarization of macrophages could be reversed by inactivation of JNK in the context of HIV-1 infection. Our study mechanistically elucidates that the JNK/COX-2/HIF-1α axis is activated to strengthen glycolysis, thereby promoting M1 polarization in macrophages in HIV-1 infection, providing a new idea for resolving chronic inflammation in clinical AIDS patients.

Graphical abstract

Figure 1.. HIV-1 infection induces M1 polarization of macrophages.

(A) Morphologic features of monocyte-derived macrophages (MDMs) after HIV-1 infection for 1–3 d. Scale bar, 100 μm. (B, C) Changes in mRNA (B) and protein expressions (C) of M1 and M2 polarization markers in MDMs after HIV-1 infection for 1–3 d. (Statistical analysis was performed using a t test, *P < 0.05, **P < 0.01, and ***P < 0.001.) (D) Representative results of expressions of M1 and M2 polarization markers in MDMs detected by flow cytometry. (Statistical analysis was performed using a t test, *P < 0.05).

Figure S1.. Impacts of HIV-1 on monocyte-derived macrophages are similar to LPS and IFN-γ co-stimulation.

THP-1 cells were seeded in a culture plate with PMA (50 μM) stimulation (48 h) to differentiate macrophages. LPS (1 μg/ml) and IFN-γ (20 mg/ml) were co-used for typical M1 polarization, whereas IL-4 (20 mg/ml) was used in combination with IL-10 (20 mg/ml) for M2 polarization. (A) Immunofluorescence staining of DAPI (representing nucleus) and β-actin (representing cytoskeleton) was used to observe morphological changes in macrophages with different stimuli for 48 h. Scale bar, 150 μm. (B) RT–qPCR analysis showed the mRNA levels of TNF-α, IL-1β, and IL-6 in THP-1 macrophages after 2 d (upper) and 3 d (bottom) of stimulation. (Statistical analysis was performed using a t test, *P < 0.05, **P < 0.01, and ***P < 0.001).

Figure 2.. Metabolism reprogramming affects polarization in HIV-1–infected macrophages.

(A) ATP/ADP ratio (left panel) and relative ATP production from glycolysis and oxidative phosphorylation (right panel) in monocyte-derived macrophages (MDMs) infected or uninfected with HIV-1. (Statistical analysis was performed using a t test, *P < 0.05, **P < 0.01, and ***P < 0.001.) (B) Representative results of glucose uptake analyzed by flow cytometry. (Statistical analysis was performed using a t test, ***P < 0.001.) (C) Lactate concentration in the supernatant of MDMs infected with HIV-1 for 48 h was detected using colorimetry. (Statistical analysis was performed using a t test, ***P < 0.001.) (D, E) Extracellular acidification rate and oxygen consumption rate of MDMs infected with HIV-1 for 48 h. Time-resolved fluorescence was applied and monitored for 120 min. Results were relative to 0 min. (Statistical analysis was performed using a t test, *P < 0.05, **P < 0.01, and ***P < 0.001.) (F) Mitochondrial membrane potential was assessed using the JC-1 assay. A change from red to green fluorescence indicates a decrease in the mitochondrial membrane potential. The red/green fluorescence ratio was calculated for comparison. (Statistical analysis was performed using a t test, **P < 0.01.) Scale bar, 300 μm. (G) Bar plot showed glycolytic index (GI) differences in control and HIV-1–infected MDMs. (H) mRNA expressions of key genes in TCA and oxidative phosphorylation. (Statistical analysis was performed using a t test, ns non-significant, *P < 0.05, **P < 0.01, and ***P < 0.001.) (I, J) MDMs were pretreated with or without 2-DG (10 mM) for 2 h, followed by infection of HIV-1 for 48 h. (I, J) Lactate concentration in the culture supernatant (I), and mRNA expressions of TNF-α, IL-1β, and IL-6 (J) were detected. (Statistical analysis was performed using a t test, *P < 0.05, **P < 0.01, and ***P < 0.001).

Figure S2.. Heptelidic acid inhibits glycolysis and inflammation induced by HIV-1 infection, whereas oligomycin exerts the opposite effect.

Monocyte-derived macrophages were treated with 10 μM heptelidic acid (HA) or 40 μM oligomycin (Omy) for 8 h during the 48 h of HIV-1 infection. (A, B, C, D) Lactate concentration in the culture supernatant (A), and expressions of TNF-α, IL-1β, and IL-6 (B, C, D) were detected. (Statistical analysis was performed using a t test, *P < 0.05, **P < 0.01, and ***P < 0.001 as compared to Control; #P < 0.05, ##P < 0.01, and ###P < 0.001 as compared to HIV-1).

Figure 3.. Activation of HIF-1ɑ impinges upon gene regulation of glucose metabolism.

(A) Immunofluorescence showed HIF-1ɑ protein localization and expression in monocyte-derived macrophages (MDMs). Blue: DAPI; green: HIF-1ɑ. Scale bar, 300 μm. (B, C) MDMs were pretreated with or without LW6 (15 μM) for 2 h, followed by HIV-1 infection for another 48 h. (B, C) RT–qPCR analysis showed the mRNA levels of key genes in the glycolysis (B) and TCA cycle (C). (Statistical analysis was performed using a t test, *P < 0.05, **P < 0.01, and ***P < 0.001.) (D, E) MDMs treated with or without YC-1 (1 μM) and infected with or without HIV-1 were assessed for expressions of key genes in the glycolysis (D) and TCA cycle (E). (Statistical analysis was performed using a t test, *P < 0.05, **P < 0.01, and ***P < 0.001).

Figure 4.. Effect of HIF-1ɑ inhibition on glycolysis, mitochondrial activity, and M1 polarization in monocyte-derived macrophages (MDMs).

MDMs were infected with HIV-1 for 48 h, in the presence or absence of LW6 (15 μM) pretreatment (2 h). (A) Lactate concentration in the culture supernatant of MDMs. (Statistical analysis was performed using a t test, **P < 0.01 and ***P < 0.001.) (B) Mitochondrial membrane potential was assessed by the JC-1 assay. (Statistical analysis was performed using a t test, *P < 0.05.) Scale bar, 100 μm. (C, D) mRNA (C) and protein levels (D) of TNF-α, IL-1β, and IL-6 were detected using RT–qPCR and ELISA, respectively. (Statistical analysis was performed using a t test, *P < 0.05, **P < 0.01, and ***P < 0.001).

Figure 5.. Activation of COX-2 mediates HIF-1ɑ–dependent glucose metabolism reprogramming and M1 polarization in monocyte-derived macrophages (MDMs).

(A, B) mRNA (A) and protein expressions (B) of COX-2 in MDMs after HIV-1 infection for 48 h. β-Actin was used as the normalization control in Western blot analysis. (Statistical analysis was performed using a t test, **P < 0.001.) (C, D, E, F, G, H, I) MDMs were pretreated with meloxicam (50 μM) for 2 h; then, HIV-1 particles were added to incubate for another 48 h. COX-2 expression (C), lactate production (D), key glycolytic gene expression (E), hypoxia-inducible factor 1α expression ((F), scale bar, 300 μm), and M1 polarization (H, I) were measured. (G) Quantification of hypoxia-inducible factor 1α fluorescence intensity. (Statistical analysis was performed using a t test, *P < 0.05, **P < 0.01, and ***P < 0.001).

Figure 6.. Knockdown of COX-2 represses HIF-1ɑ–dependent glycolytic shift and M1 polarization in THP-1 macrophages.

(A) EGFP fluorescence of THP-1 macrophages transfected with lentivirus. (B, C) COX-2–silencing THP-1 macrophages were verified by RT–qPCR and Western blot. (Statistical analysis was performed using a t test, **P < 0.01.) (D, E, F, G) HIF-1ɑ expression (D), lactate production (E), and mRNA and protein expressions of TNF-α, IL-1β, and IL-6 (F, G) were decreased in COX-2–silencing THP-1 macrophages, as quantified by RT–qPCR and ELISA. (Statistical analysis was performed using a t test, *P < 0.05, **P < 0.01, and ***P < 0.001).

Figure 7.. HIV-1 urges phosphorylation of JNK in macrophages.

THP-1 macrophages and primary monocyte-derived macrophages (MDMs) were infected with or without HIV-1 for 48 h. (A) KEGG enrichment analysis of differentially expressed genes in HIV-1–infected THP-1 macrophages and control macrophages. The size of the bubble positively correlated with the number of enriched genes. The x-axis represents the gene ratio, and the color of the bubble represents the adjusted P-value of enrichment analysis. (B) Gene set enrichment analysis was performed in Control and HIV-1 groups. In this figure, the y-axis represents enrichment score (ES), and on the x-axis are genes (vertical black lines) included in gene sets. The analysis demonstrates that the MAPK signaling pathway is enriched in the HIV-1 group (NES = 1.315, P-value < 0.0001, FDR = 0.301). The detailed information is provided in Table S1. (C) RT–qPCR analysis showed the mRNA levels of JNK, ERK, and p38 in MDMs. (Statistical analysis was performed using a t test, *P < 0.05.) (D, E) Western blot confirmed phosphorylation of JNK is increased in MDMs (D) and THP-1 macrophages (E) upon HIV-1 infection. (Statistical analysis was performed using a t test, *P < 0.05).

Figure 8.. SP600125 reverses HIV-1–induced M1 polarization by inhibiting JNK activation.

Monocyte-derived macrophages (MDMs) were pretreated with or without SP600125 (50 μM) for 2 h, followed by infection of HIV-1 for 48 h. (A) Expressions of JNK and COX-2 were measured by Western blot. Total JNK was used as the normalization control of phosphorylated JNK, and β-actin was used as the normalization control of COX-2. (Statistical analysis was performed using a t test, *P < 0.05, **P < 0.01, and ***P < 0.001.) (B) Western blot analysis of hypoxia-inducible factor 1α, HK1, HK2, and LDHA in MDMs. β-Actin was used as the normalization control. SP600125, meloxicam, LW6, and YC-1 were administered as mentioned above. (Statistical analysis was performed using a t test, *P < 0.05 and **P < 0.01 as compared to Control; #P < 0.05, ##P < 0.01, and ###P < 0.001 as compared to HIV-1.) (C) Lactate concentration in the culture supernatant of MDMs was detected using colorimetry. (Statistical analysis was performed using a t test, *P < 0.05 and ***P < 0.001.) (D, E) MDMs were collected, and the expression of TNF-α, IL-1β, and IL-6 was measured by RT–qPCR (D) and ELISA (E). (Statistical analysis was performed using a t test, *P < 0.05, **P < 0.01, and ***P < 0.001).

Figure S3.. Blockade of glycolysis inhibits HIV-1 replication.

Monocyte-derived macrophages were pretreated with or without 2-DG (10 mM) for 2 h, followed by infection of HIV-1 for 48 h. The content of p24 protein in the supernatant was detected by ELISA. (Statistical analysis was performed using a t test, *P < 0.05, **P < 0.01, and ***P < 0.001).