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

Junhan Zhang, Zongxiang Yuan, Xuanrong Li, Fengyi Wang, Xueqin Wei, Yiwen Kang, Chuye Mo, Junjun Jiang  Correspondence email, View ORCID ProfileHao Liang  Correspondence email, View ORCID ProfileLi Ye  Correspondence email
Junhan Zhang
Guangxi Key Laboratory of AIDS Prevention and Treatment, School of Public Health, Guangxi Medical University, Nanning, China
Roles: Investigation, Methodology, Writing—original draft
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Zongxiang Yuan
Guangxi Key Laboratory of AIDS Prevention and Treatment, School of Public Health, Guangxi Medical University, Nanning, China
Roles: Conceptualization, Funding acquisition, Project administration
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Xuanrong Li
Guangxi Key Laboratory of AIDS Prevention and Treatment, School of Public Health, Guangxi Medical University, Nanning, China
Roles: Methodology
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Fengyi Wang
Guangxi Key Laboratory of AIDS Prevention and Treatment, School of Public Health, Guangxi Medical University, Nanning, China
Roles: Formal analysis
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Xueqin Wei
Guangxi Key Laboratory of AIDS Prevention and Treatment, School of Public Health, Guangxi Medical University, Nanning, China
Roles: Software
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Yiwen Kang
Guangxi Key Laboratory of AIDS Prevention and Treatment, School of Public Health, Guangxi Medical University, Nanning, China
Roles: Validation
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Chuye Mo
Guangxi Key Laboratory of AIDS Prevention and Treatment, School of Public Health, Guangxi Medical University, Nanning, China
Roles: Validation
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Junjun Jiang
Guangxi Key Laboratory of AIDS Prevention and Treatment, School of Public Health, Guangxi Medical University, Nanning, China
Roles: Conceptualization, Funding acquisition
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  • For correspondence: jiangjunjun@gxmu.edu.cn
Hao Liang
Guangxi Key Laboratory of AIDS Prevention and Treatment, School of Public Health, Guangxi Medical University, Nanning, China
Roles: Supervision, Writing—review and editing
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  • For correspondence: lianghao@gxmu.edu.cn
Li Ye
Guangxi Key Laboratory of AIDS Prevention and Treatment, School of Public Health, Guangxi Medical University, Nanning, China
Roles: Data curation, Funding acquisition, Writing—review and editing
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  • ORCID record for Li Ye
  • For correspondence: yeli@gxmu.edu.cn
Published 5 October 2023. DOI: 10.26508/lsa.202302148
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    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).

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    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.
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    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.
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    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).

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    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).

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    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).

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    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).

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    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.
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    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).

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    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.
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    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).

Supplementary Materials

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  • Table S1. Gene set enrichment analysis of the MAPK signaling pathway.

  • Table S2. Primer list.

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Metabolism of M1 Mφ in HIV-1 infection
Junhan Zhang, Zongxiang Yuan, Xuanrong Li, Fengyi Wang, Xueqin Wei, Yiwen Kang, Chuye Mo, Junjun Jiang, Hao Liang, Li Ye
Life Science Alliance Oct 2023, 6 (12) e202302148; DOI: 10.26508/lsa.202302148

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Metabolism of M1 Mφ in HIV-1 infection
Junhan Zhang, Zongxiang Yuan, Xuanrong Li, Fengyi Wang, Xueqin Wei, Yiwen Kang, Chuye Mo, Junjun Jiang, Hao Liang, Li Ye
Life Science Alliance Oct 2023, 6 (12) e202302148; DOI: 10.26508/lsa.202302148
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Volume 6, No. 12
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