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. 2003 Mar 7;112(5):645-57.
doi: 10.1016/s0092-8674(03)00154-5.

HIF-1alpha Is Essential for Myeloid Cell-Mediated Inflammation

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Free PMC article

HIF-1alpha Is Essential for Myeloid Cell-Mediated Inflammation

Thorsten Cramer et al. Cell. .
Free PMC article

Erratum in

  • Cell. 2003 May 2;113(3):419

Abstract

Granulocytes and monocytes/macrophages of the myeloid lineage are the chief cellular agents of innate immunity. Here, we have examined the inflammatory response in mice with conditional knockouts of the hypoxia responsive transcription factor HIF-1alpha, its negative regulator VHL, and a known downstream target, VEGF. We find that activation of HIF-1alpha is essential for myeloid cell infiltration and activation in vivo through a mechanism independent of VEGF. Loss of VHL leads to a large increase in acute inflammatory responses. Our results show that HIF-1alpha is essential for the regulation of glycolytic capacity in myeloid cells: when HIF-1alpha is absent, the cellular ATP pool is drastically reduced. The metabolic defect results in profound impairment of myeloid cell aggregation, motility, invasiveness, and bacterial killing. This role for HIF-1alpha demonstrates its direct regulation of survival and function in the inflammatory microenvironment.

Figures

Figure 1
Figure 1. Efficiency of CRE Recombinase-Mediated Deletion in Myeloid Cells
Genomic DNA was isolated from peritoneal macrophages (A) and neutrophils (B) from HIF-1α-, VEGF-, and VHL-LysM-CRE mice and subjected to real-time PCR with primers spanning the targeted region as well as primers for an undeleted control gene for normalization. Efficiency of deletion was calculated by quantitative PCR. (C) Differential leukocyte counts were performed on samples harvested by retinal bleeding.
Figure 2
Figure 2. Functional Inactivation of Hypoxic Response-Related Genes Significantly Impairs Gene Expression Patterns of Macrophages
(A) Peritoneal macrophages were cultured under normoxic (open bars) or hypoxic (closed bars) conditions for 8 hr, and total RNA was isolated and VEGF mRNA expression determined by real-time PCR. Values were normalized to RT-PCR results for ribosomal RNA. Normoxic WT levels were arbitrarily set as one. VEGF RNA was not analyzed in VEGF mutants due to the expression of a nontranslatable transcript in these cells. (B) Peritoneal macrophages were cultured under normoxic (open bars) or hypoxic (closed bars) conditions for 24 hr, and conditioned supernatant was harvested and VEGF protein analyzed by ELISA. Values were normalized to cell number. (C) Hypoxic induction of gene expression of the glycolytic enzyme phosphoglyceratekinase (PGK) and (D) glucose transporter 1 (Glut-1) was characterized by means of real-time PCR. Normoxic WT levels were arbitrarily set as one and normalization performed as outlined under (A). (E) Macrophages were stimulated with LPS and cultured under normoxic or hypoxic conditions for 8 hr, and conditioned supernatant was assayed for TNF-α protein by ELISA and values normalized to cell number. Statistical analysis was performed using the unpaired Student’s t test, *p < 0.05, **p < 0.01.
Figure 3
Figure 3. Glycolysis and Energy Generation in Myeloid Cells Are Severely Affected by the Loss of HIF-1α
(A) Lactate concentrations in macrophage supernatant were quantified under either control conditions (open bars) or after the addition of LPS (closed bars). Values were normalized to total protein content. (B) Peritoneal macrophages were isolated from WT, HIF-1α-, VEGF-, and VHL-LysM-CRE mice and cultured under ambient conditions for 24 hr. Cell lysates were harvested and intracellular ATP concentrations measured by means of a luciferase-based chemiluminescent assay. Values were normalized to total protein content. (C) Peritoneal neutrophils were harvested and assayed for ATP synthesis. 2-deoxyglucose (500 mg/kg b.w.) was injected i.p. 60 min prior to harvest to block glycolysis in peritoneal exudate cells. (D) Engulfment of viable bacteria was characterized by inoculating macrophages with GFP-expressing GBS for 2 hr. Deconvolution fluorescence microscopy was used for documentation. (E) Bone marrow derived macrophages were inoculated with Group B streptococci (GBS) at a MOI of 2.5 and intracellular killing analyzed by determination of viable colony forming units in the macrophage lysates after washing and antibiotic treatment to remove nonengulfed bacteria. Statistical analysis was performed using the unpaired Student’s t test, **p < 0.01.
Figure 4
Figure 4. HIF-1α Is an Essential Prerequisite for Aggregation, Motility, and Invasion of Macrophages
Macrophages were isolated from the peritoneum and plated at a concentration of 106/ml on growth factor-reduced matrigel. Cells were allowed to aggregate for 24 hr: (A) WT, (B) HIF-1α null macrophages. Invasive capacity of peritoneal macrophages was studied using modified Boyden chambers. Cell culture inserts were coated with matrigel, (C) WT, or (D) HIF-1α null cells added to the upper wells. Macrophages were allowed to invade toward 5% FCS in the lower wells for 24 hr, stained with 0.1% Alcian blue, and photographed. Bound stain was extracted with acetic acid and quantified spectophotometrically (E) WT levels were set arbitrarily as 100. (F) Migration of macrophages was analyzed in modified Boyden chambers without added matrigel. Cells were allowed to migrate for 24 hr and further processed as outlined above. Statistical analysis was performed using the unpaired Student’s t test, **p < 0.01.
Figure 5
Figure 5. Phorbol Ester-Induced Ear Inflammation Is Differentially Regulated by Members of the Hypoxic Response Pathway in Myeloid Cells
Ears of (A) WT, (B) HIF-1α-, (C) VEGF-, and (D) VHL-LysM-CRE mice were painted once with 2.5 µg TPA in acetone carrier solution. 24 hr later, ears were harvested and fixed in 4% PFA. The leukocyte common antigen (CD45) was immunolocalized on paraffin sections and detected with DAB. (E) Ears were treated with TPA as outlined above, and 24 hr later a defined area of 6mm was punched out. Weight was determined as a measurement of edema formation. (F) Tissue was homogenized and myeloperoxidase activity assayed by spectophotometrically monitored conversion of o-dianisidine. (G and H). Total RNA was isolated from ear samples and subjected to RPA analysis using murine chemokine (G) and cytokine (H) probe sets. Statistical analysis was performed using the unpaired Student’s t-test, **p < 0.01.
Figure 6
Figure 6. Loss of HIF-1α in Myeloid Cells Impairs Chronic Cutaneous Inflammation
The back skin of mice was freed of hair and 5% SDS solution applied epicutaneously once daily for a total of 10 days. Macroscopic appearance of skin after (A) 5 and (B) 10 days of treatment. (C and D) Histological analysis of skin after 5 days of SDS application, H&E, magnification 100×. (E and F) Immunolocalization of CD45 in mouse skin after 5 days of 5% SDS treatment. Magnification 200×.
Figure 7
Figure 7. Passively Induced Arthritis Is Dependent on Functional HIF-1α in Myeloid Cells
Animals were injected twice with heterologous serum from KbxN-TCR transgenic mice and monitored over a period of 3 weeks. Macroscopic appearance of hindpaws from (A)WT and (B) HIF-1α null mice 21 days after the initiation of serum injection. Limbs were fixed in 4% PFA and paraffin embedded. Histological analysis of (C) WT and (D) HIF-1α null ankle joints 23 days after arthritis induction. H&E, magnification 100×. (E) Throughout the course of the experiment, mice were blind scored every second day for clinical signs of arthritis as outlined in Experimental Procedures. Statistical analysis was performed using the unpaired Student’s t test, **p < 0.01.

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