Hypoxia-inducible factor 2alpha regulates macrophage function in mouse models of acute and tumor inflammation - PubMed (original) (raw)

. 2010 Aug;120(8):2699-714.

doi: 10.1172/JCI39506. Epub 2010 Jul 19.

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Hypoxia-inducible factor 2alpha regulates macrophage function in mouse models of acute and tumor inflammation

Hongxia Z Imtiyaz et al. J Clin Invest. 2010 Aug.

Abstract

Hypoxia-inducible factor 1alpha (HIF-1alpha) and HIF-2alpha display unique and sometimes opposing activities in regulating cellular energy homeostasis, cell fate decisions, and oncogenesis. Macrophages exposed to hypoxia accumulate both HIF-1alpha and HIF-2alpha, and overexpression of HIF-2alpha in tumor-associated macrophages (TAMs) is specifically correlated with high-grade human tumors and poor prognosis. However, the precise role of HIF-2alpha during macrophage-mediated inflammatory responses remains unclear. To fully characterize cellular hypoxic adaptations, distinct functions of HIF-1alpha versus HIF-2alpha must be elucidated. We demonstrate here that mice lacking HIF-2alpha in myeloid cells (Hif2aDelta/Delta mice) are resistant to lipopolysaccharide-induced endotoxemia and display a marked inability to mount inflammatory responses to cutaneous and peritoneal irritants. Furthermore, HIF-2alpha directly regulated proinflammatory cytokine/chemokine expression in macrophages activated in vitro. Hif2aDelta/Delta mice displayed reduced TAM infiltration in independent murine hepatocellular and colitis-associated colon carcinoma models, and this was associated with reduced tumor cell proliferation and progression. Notably, HIF-2alpha modulated macrophage migration by regulating the expression of the cytokine receptor M-CSFR and the chemokine receptor CXCR4, without altering intracellular ATP levels. Collectively, our data identify HIF-2alpha as an important regulator of innate immunity, suggesting it may be a useful therapeutic target for treating inflammatory disorders and cancer.

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Figures

Figure 1

Figure 1. Myeloid-specific ablation of HIF-2α using LysM-Cre.

(A) Breeding scheme used to obtain myeloid-specific HIF-2α–deficient (Hif2aΔ/Δ, i.e., Hif2a2L/1L, LysM-Cre) and control (Hif2aΔ/+, i.e., Hif2a2L/+, LysM-Cre) mice. 2L, 2loxP allele; 1L, 1loxP or deleted allele. Frequency of each genotype is shown. (B) Southern blot showing deletion of the 2loxP allele in macrophages (M). Tail tissue (T) was used as a control. (C) Western blots confirming the absence of HIF-2α protein under hypoxia in Hif-2αΔ/Δ BMDMs. (D) HIF target expression validating loss of HIF-2α function in HIF-2α–deficient BMDMs. Data represent fold induction of mRNA expression compared with the control BMDMs. Representative data from 4 independent experiments are shown (**P < 0.01). (E) Cytokine/chemokine expression in normoxic and hypoxic BMDMs was evaluated by QRT-PCR. Representative data from at least 3 independent experiments are shown (*P < 0.05, **P < 0.01). Δ/+, control genotype, Hif2aΔ/+; Δ/Δ, mutant genotype, Hif2aΔ/Δ; N, normoxia (21% O2); H, hypoxia (0.5% O2); n.s., nonspecific band.

Figure 2

Figure 2. HIF-2α is critical for cytokine production and cardiac function in LPS-induced endotoxemia.

(A) Control (Δ/+) and mutant (Δ/Δ) mice were challenged with LPS (15 mg/kg body weight) via i.p. injection; serum was obtained 4 hours following the challenge; and cytokine levels were determined using ELISA. Representative data from 5 mice per group are shown (*P < 0.05, **P < 0.01). (B) Hif2aΔ/Δ animals maintained body temperature better than controls 4 hours following LPS treatment (Hif2aΔ/+, n = 3; Hif2aΔ/Δ, n = 6) (*P < 0.05). (C) Echocardiography showed better LV function in mutant mice 4 hours following LPS treatment (Hif2aΔ/+, n = 3; Hif2aΔ/Δ, n = 6) (*P < 0.05). (D) Mutant mice were pretreated with either anti–IL-10 antibody or isotype control IgG, and their LV function was examined 4 hours following LPS induction of endotoxemia (n = 3) (*P < 0.05). (E) A Kaplan-Meier curve representing survival of animals challenged with LPS (Hif2aΔ/+, n = 7; Hif2aΔ/Δ, n = 6; log-rank statistic = 12.50, P = 0.0004).

Figure 3

Figure 3. NO production and activation marker expression are not affected by loss of HIF-2α.

(A) BMDMs were stimulated with various concentrations of LPS plus IFN-γ for 24 hours; NO production was evaluated by measuring nitrate concentration in the culture supernatant using the Griess assay; n = 7. (B) iNOS expression following stimulation with various concentrations of LPS plus IFN-γ under 3% O2 was assayed by Western blotting. No difference was found between the control and HIF-2α–deficient macrophages. (C) Upregulation of cell surface activation markers (MHC class II and CD86) in response to LPS plus IFN-γ was not affected by lack of HIF-2α in macrophages under either normoxia or hypoxia.

Figure 4

Figure 4. Proinflammatory cytokine expression is aberrant in Hif2aΔ/Δ BMDMs responding to low O2 and M1 stimuli.

(A) BMDMs were treated with M1 (5 ng/ml LPS plus 1 ng/ml IFN-γ) stimuli under normoxia or hypoxia for 24 hours, and their expression of cytokines/chemokines (as shown) was evaluated by QRT-PCR. Representative data from at least 3 independent experiments are shown (*P < 0.05, **P < 0.01). (B) BMDMs were activated for 36 hours using LPS (5 ng/ml) plus IFN-γ (1 ng/ml) under normoxia or hypoxia, and culture supernatant was obtained for measurement of secreted cytokine levels via ELISA. Representative data from at least 3 independent experiments are shown (*P < 0.05). Hypoxia: 0.5% O2.

Figure 5

Figure 5. HIF-2α directly regulates cytokine gene expression.

(A) HIF-2α–regulated cytokine expression does not involve NF-κB. BMDMs were treated with M1 (5 ng/ml LPS plus 1 ng/ml IFN-γ) and/or hypoxia (0.5% O2), and DNA binding activity of p65 and RelB to a NF-κB consensus binding site was assessed using TransAM NFκB Family Transcription Factor Assay Kit (Active Motif). Though hypoxia appears to enhance M1-induced p65 binding activity, no significant difference was observed between the control and _Hif2a_-deficient groups (n = 3) (*P < 0.05). (B) U937 cells were differentiated with TPA and activated with M1 (100 ng/ml LPS plus 12 ng/ml IFN-γ) and/or hypoxia (0.5% O2). HIF-2α stabilization under these treatment conditions was examined by Western blotting. Lanes were run on the same gel but were noncontiguous (white lines). (C) IL6 expression in U937 cells under various treatment conditions was determined by QRT-PCR. (***P < 0.001) (D) ChIP was performed on U937 cells to assess binding of HIF-2α protein to potential HRE motifs located at –2.2 kb, –1.7 kb, and –1.6 kb upstream of transcriptional starting site in promoter regions of the IL6 gene. The VEGF gene was used as a positive control. Lanes were run on the same gel but were noncontiguous (white lines).

Figure 6

Figure 6. HIF-2α is required for TPA- and TG-induced inflammation.

(A–G) TPA-induced acute ear inflammation is impaired in myeloid HIF-2α–deficient mice. (A–D) Ear skin was painted with either acetone (control) or TPA; ear tissue was harvested 24 hours later and stained with H&E. Scale bars: 20 μm. (E and F) Ear tissue was stained with Gr-1 antibody to confirm that the majority of infiltrating cells are PMNs. Scale bars: 20 μm. (G) Infiltrating cells (based on H&E staining) and Gr-1+ cells were counted under high-power (×40) fields (n = 3, *P < 0.05). (H) Myeloid HIF-2α–deficient mice displayed reduced total number of TG-elicited peritoneal exudate macro­phages (PEMs) (n = 5, ***P < 0.001). Arrowheads in C, D, E, and F indicate neutrophils.

Figure 7

Figure 7. Decreased TAM infiltration in murine HCC when macrophage HIF-2α is absent.

(A and B) Representative H&E images of liver tissue containing tumor areas. Scale bars: 200 μm. (C–F) HCC samples were stained with the pan-macrophage marker CD68, and positively stained brown cells were identified as infiltrating TAMs. Two representative images per genotype are shown. Scale bars: 50 μm. (G) TAM recruitment in control and myeloid _Hif2a_-deficient mice was determined under high-power (×20) fields and normalized to the units of tumor area (n = 15, **P = 0.0017). (H) Mitotic figures of tumor cells were counted per 10 high-power (×40) fields, and mitotic indices were determined (*P < 0.05). Asterisks in A and B indicate tumor area. Arrowheads in C, D, E, and F indicate CD68+ TAMs.

Figure 8

Figure 8. Loss of TAM HIF-2α leads to reduced tumor burden and progression in murine CAC.

(A and B) Control (Δ/+) and mutant (Δ/Δ) mice were induced to form CAC for 14 weeks, and gross pictures of tumors in the colon and rectum are shown. Arrowheads indicate macroscopic lesions. Scale bars: 3.125 mm. (C and D) Colon sections were stained with H&E; neoplastic lesions are outlined with yellow dashes. Scale bars: 3.125 mm. (E) Top panel shows total number of CAC tumors in both control and mutant cohorts. Bottom panel shows total tumor size (represented as sum of diameters of all tumors). Bars indicate median value of each group. (F) CD68 immunostaining of CAC colons. Left: Distribution of CD68+ macrophages (brown) in normal colon tissue adjacent to tumors. Middle: CD68+ TAM infiltration to the surrounding lamina propria (LP) of hyperplasia. Right: TAM recruitment to LP at stalk area of hyperplasia. Scale bars: 20 μm. (G) CD68 immunostaining of CAC adenomas. Left: Limited TAM infiltration to the center of large lesions. Middle: TAM recruitment to small lesions. Right: TAMs presented in LP at stalk area. Scale bars: 20 μm. (H) Quantification of CD68+ cells. (I) Quantification of tumor stages of CAC. Percentages of hyperplasia and adenoma within control (Δ/+) and mutant (Δ/Δ) groups are shown. (J) Histopathological analysis of mitosis of adenoma tumor cells. Arrowheads in F and G indicate CD68+ TAMs. T, tumor area; S, stalk. *P < 0.05, ***P < 0.001.

Figure 9

Figure 9. Reduced migration and invasion, but normal ATP production in HIF-2α–deficient macrophages.

(A) BMDMs were exposed to normoxia or hypoxia for 16 hours, and their in vitro migration capacity toward the chemoattractant M-CSF was determined by employing a barrier PET membrane (8 μm; BD Biosciences). Representative images are shown. Scale bars: 20 μm. (B) Quantification of migrated macrophages. Representative data are shown from n = 4 mice (*P < 0.05). (C) Invasion was tested as described above for migration, except that the barrier PET membrane was coated with Matrigel basement membrane matrix (BD Biosciences). Representative images are shown. Scale bars: 20 μm. (D) Quantification of macrophages that invaded through the Matrigel. Representative data from n = 3 mice are shown (*P < 0.05, **P < 0.01). (E) BMDMs were exposed to normoxia or hypoxia for 20 hours, and cellular ATP levels were determined using the ApoGlow Assay kit. Representative data from at least 3 independent experiments are shown. (F) Expression of M-CSFR under normoxia and hypoxia was determined by Western blotting. Representative data from 3 independent experiments are shown. Lanes were run on the same gel but were noncontiguous (white lines). (G) Cxcr4 and Fn1 expression in normoxic and hypoxic BMDMs was evaluated by QRT-PCR. Representative data from at least 3 independent experiments are shown (*P < 0.05, **P < 0.01). N, normoxia (21% O2); H, hypoxia (0.5% O2).

Figure 10

Figure 10. Model illustrating the roles of HIF-1α and HIF-2α in macrophages.

HIF-1α exclusively regulates ATP generation and contributes to cytokine production, as well as macrophage function in acute inflammation, migration/invasion, and bacterial killing. HIF-2α also regulates acute inflammation and migration/invasion, but not ATP production in macrophages. Additionally, HIF-2α controls tumor inflammation and appears to play a bigger role in cytokine production than HIF-1α. Further investigation is needed to determine whether HIF-2α is required for macrophage bactericidal activity.

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