A myeloid hypoxia-inducible factor 1α-Krüppel-like factor 2 pathway regulates gram-positive endotoxin-mediated sepsis

Abstract

Although gram-positive infections account for the majority of cases of sepsis, the molecular mechanisms underlying their effects remains poorly understood. We investigated how cell wall components of gram-positive bacteria contribute to the development of sepsis. Experimental observations derived from cultured primary macrophages and the cell line indicate that gram-positive bacterial endotoxins induce hypoxia-inducible factor 1α (HIF-1α) mRNA and protein expression. Inoculation of live or heat-inactivated gram-positive bacteria with macrophages induced HIF-1 transcriptional activity in macrophages. Concordant with these results, myeloid deficiency of HIF-1α attenuated gram-positive bacterial endotoxin-induced cellular motility and proinflammatory gene expression in macrophages. Conversely, gram-positive bacteria and their endotoxins reduced expression of the myeloid anti-inflammatory transcription factor Krüppel-like transcription factor 2 (KLF2). Sustained expression of KLF2 reduced and deficiency of KLF2 enhanced gram-positive endotoxins induced HIF-1α mRNA and protein expression in macrophages. More importantly, KLF2 attenuated gram-positive endotoxins induced cellular motility and proinflammatory gene expression in myeloid cells. Consistent with these results, mice deficient in myeloid HIF-1α were protected from gram-positive endotoxin-induced sepsis mortality and clinical symptomatology. By contrast, myeloid KLF2-deficient mice were susceptible to gram-positive sepsis induced mortality and clinical symptoms. Collectively, these observations identify HIF-1α and KLF2 as critical regulators of gram-positive endotoxin-mediated sepsis.

Figures

FIGURE 1.

Gram-positive bacteria and endotoxins induce HIF-1α transcriptional activity by modulating HIF-1α expression. A, RAW264.7 cells transfected with the HRE-luciferase plasmid were either incubated with live (5 m.o.i.) or heat-inactivated E. coli and S. aureus separately. B, RAW264.7 cells were treated with teichoic acid (4 μg/ml), lipoteichoic acid (4 μg/ml), and LPS (100 ng/ml) for 12 h. HRE-luciferase activity was measured and indicated as relative fold changes over control. C, wild-type mouse primary peritoneal macrophages were exposed with live (5 m.o.i.) or heat-inactivated E. coli and S. aureus for 6 h. D, wild-type mouse peritoneal macrophages were induced with LPS (100 ng/ml), teichoic acid (4 μg/ml), and lipoteichoic acid (4 μg/ml) for 6 h. Total RNA was isolated, and HIF-1α mRNA expression was analyzed by quantitative PCR and normalized to 36B4. E, wild-type mouse primary peritoneal macrophages were incubated with live (5 m.o.i.) heat-inactivated E. coli and S. aureus for 6 h. F, wild-type mouse peritoneal macrophages were induced with LPS (100 ng/ml), teichoic acid (4 μg/ml), and lipoteichoic acid (4 μg/ml) for 6 h. Cell lysates were analyzed for HIF-1α protein expression by immunoblot analysis. MDA-MB-231 cells exposed to 6 h of hypoxia were used as a positive control, and actin was used as a loading control. Combined data of three experiments are shown in each case. Data represent mean ± S.D. *, p < 0.05 versus the indicated control.

FIGURE 2.

Kinetics of teichoic acid- and lipoteichoic acid-induced HIF-1α expression. Wild-type mouse peritoneal macrophages were stimulated with 0–10 μg/ml teichoic acid (A) for 0–24 h (B). HIF-1α mRNA expression was analyzed by quantitative PCR and normalized to 36B4 (A and B, upper panels). HIF-1α protein levels were analyzed by immunoblot analysis (lower panels). Wild-type mouse primary peritoneal macrophages were stimulated with 0–8 μg/ml lipoteichoic acid (C) for 0–24 h (D). HIF-1α mRNA expression was analyzed by quantitative PCR and normalized to 36B4 (C and D, upper panels). HIF-1α protein levels are analyzed by immunoblot (lower panels). MDA-MB-231 cells exposed to hypoxia were used as a positive control, and actin was used as a loading control. Combined data of three experiments is shown in each case. Data represent mean ± S.D.

FIGURE 3.

Gram-positive, endotoxin-induced macrophage cell motility, bactericidal function, and inflammatory gene expression are HIF-1α-dependent. A and B, primary peritoneal macrophages from control (LysMCre/Cre) and HIF-1αΔ/Δ mice were stimulated with 4 μg/ml teichoic or lipoteichoic acid and subjected to a migration or invasion assay. The number of cells migrated (A) or invaded (B) in unstimulated wells across the membrane were assigned as 100%, and fold changes over this are indicated. C, primary peritoneal macrophages from control (LysMCre/Cre) and HIF-1αΔ/Δ mice were inoculated with S. aureus, and intracellular bacterial killing was analyzed by antibiotic protection assay. D–H, primary peritoneal macrophages and neutrophils from control (LysMCre/Cre) and HIF-1αΔ/Δ mice were stimulated with 4 μg/ml teichoic or lipoteichoic acid for 6 h. Indicated target genes were analyzed by quantitative PCR and normalized to 36B4. Combined data of three experiments are shown in each case. Data represent mean ± S.D. *, p < 0.05 versus indicated control.

FIGURE 4.

KLF2 modulate Gram-positive endotoxins induced HIF-1α expression. Wild-type mouse peritoneal macrophages were induced with heat-inactivated S. aureus, teichoic acid, and lipoteichoic acid for 6 h. A, KLF2 expression was analyzed by quantitative PCR and normalized to 36B4. B, KLF2 protein levels were analyzed by immunoblot analysis. Actin was used as a loading control. C, RAW264.7 cells were cotransfected with a HRE-luciferase reporter plasmid and a pcDNA3 or KLF2 overexpression plasmid. These cells were stimulated with 4 μg/ml teichoic acid or lipoteichoic acid for 12 h. HRE-luciferase activity was measured and indicated as relative fold changes over control. D–G, RAW264.7 cells infected with Ad-GFP/Ad-KLF2 (D and F) or primary peritoneal macrophages from control (LysMCre/Cre) and KLF2Δ/Δ mice (E and G) were induced with 4 μg/ml teichoic acid or lipoteichoic acid for 6 h. HIF-1α mRNA and protein levels were analyzed by quantitative PCR and normalized to 36B4. HIF-1α protein expression was analyzed by immunoblot analysis. Combined data of three experiments are shown in each case. Data represent mean ± S.D. *, p < 0.05 versus indicated control.

FIGURE 5.

KLF2 suppresses Gram-positive, bacterial endotoxin-induced macrophage cell motility, bactericidal function, and inflammatory gene expression. A and B, RAW264.7 cells infected with Ad-GFP/Ad-KLF2 or primary peritoneal macrophages derived from control (LysMCre/Cre) and KLF2Δ/Δ mice were stimulated with 4 μg/ml teichoic or lipoteichoic acid and subjected to a migration assay. The number of cells migrated in unstimulated wells across the membrane was assigned as 100%, and fold changes over this are indicated. C, primary peritoneal macrophages from control (LysMCre/Cre) and KLF2Δ/Δ mice were incubated with S. aureus, and the intracellular bacterial-killing ability of these macrophages was analyzed by antibiotic protection assay. D–F, RAW264.7 cells infected with Ad-GFP/Ad-KLF2 or primary peritoneal macrophages from control (LysMCre/Cre) and KLF2Δ/Δ mice were stimulated with 4 μg/ml teichoic or lipoteichoic acid for 8 h. Indicated target gene expression was analyzed by quantitative PCR and normalized to 36B4. Combined data of three experiments are shown in each case. Data represent mean ± S.D. *, p < 0.05 versus the indicated control.

FIGURE 6.

HIF-1α and KLF2 regulate the Gram-positive, endotoxin-mediated sepsis phenotype in vivo. A, age- and sex-matched control (LysMCre/Cre), HIF-1αΔ/Δ, and KLF2Δ/Δ mice were challenged with lipoteichoic acid (3 mg/kg) supplemented with peptidoglycan by intraperitoneal injection. These mice were observed for 96 h for survival. B–D, age- and sex-matched control (LysMCre/Cre), HIF-1αΔ/Δ, and KLF2Δ/Δ mice were challenged with lipoteichoic acid (3 mg/kg) containing peptidoglycan by intraperitoneal injection and were monitored for changes in core body temperature (rectal probe), systolic blood pressure (tail cuff blood pressure monitor), and shock index (shock index = heart rate/systolic blood pressure). n = 10 mice per group in each experiment (A–D). Data represent mean ± S.D. *, p < 0.05 versus the indicated control.