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Hypoxia inducible factor (HIF)-1 coordinates induction of Toll-like receptors TLR2 and TLR6 during hypoxia.

Kuhlicke J, Frick JS, Morote-Garcia JC, Rosenberger P, Eltzschig HK - PLoS ONE (2007)

Bottom Line: During acute infection and inflammation, dramatic shifts in tissue metabolism are typical, thereby resulting in profound tissue hypoxia.While transcript levels of other TLRs remained unchanged, we found a robust induction of TLR2 (2.36+/-0.7-fold; P<0.05) and TLR6 (3.46+/-1.56-fold; P<0.05).Studies using loss and gain of function of HIF-1 confirmed a critical role of HIF-1alpha in coordinating TLR2 and TLR6 induction.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology and Intensive Care Medicine, Tübingen University Hospital, Tübingen, Germany.

ABSTRACT

Background: During acute infection and inflammation, dramatic shifts in tissue metabolism are typical, thereby resulting in profound tissue hypoxia. Therefore, we pursued the hypothesis, that tissue hypoxia may influence innate immune responses by transcriptional modulation of Toll-like receptor (TLRs) expression and function.

Methodology/principal findings: We gained first insight from transcriptional profiling of murine dendritic cells exposed to hypoxia (2% oxygen for 24 h). While transcript levels of other TLRs remained unchanged, we found a robust induction of TLR2 (2.36+/-0.7-fold; P<0.05) and TLR6 (3.46+/-1.56-fold; P<0.05). Additional studies in different cells types and cell-lines including human dendritic cells, monocytic cells (MM6), endothelia (HMEC-1) or intestinal epithelia (Caco-2) confirmed TLR2 and TLR6 induction of transcript, protein and function during hypoxia. Furthermore, analysis of the putative TLR2 and TLR6 promoters revealed previously unrecognized binding sites for HIF-1, which were shown by chromatin immunoprecipitation to bind the pivotal hypoxia-regulating transcription factor HIF-1alpha. Studies using loss and gain of function of HIF-1 confirmed a critical role of HIF-1alpha in coordinating TLR2 and TLR6 induction. Moreover, studies of murine hypoxia (8% oxygen over 6 h) showed TLR2 and TLR 6 induction in mucosal organs in vivo. In contrast, hypoxia induction of TLR2 and TLR6 was abolished in conditional HIF-1alpha mutant mice.

Conclusions/significance: Taking together, these studies reveal coordinated induction of TLR2 and TLR6 during hypoxia and suggest tissue hypoxia in transcriptional adaptation of innate immune responses during acute infection or inflammation.

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TLR2 transcript, protein and function during hypoxia.
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pone-0001364-g002: TLR2 transcript, protein and function during hypoxia.

Mentions: Based on the above observation of coordinated induction of TLR2 and TLR6 with hypoxia, we next pursued additional details of TLR2 induction with hypoxia using different cellular models. Therefore, we exposed human dendritic cells (hDCs), monocytic cells (MM6), endothelia (HMEC-1) and epithelia (Caco-2) over indicated time periods to hypoxia (2% oxygen), isolated RNA and performed real-time RT-PCR to determine TLR2 transcript levels. Consistent with our studies of murine dendritic cells, we found a robust induction of TLR2 transcript in all examined cells (Figure 2 A, B). For example, real-time RT-PCR analysis revealed prominent induction of TLR2 mRNA expression in hDCs (3.012±0.3002, p<0.01) and MM6 cells (1.812±0.4875, p<0.05) after 24h exposure to hypoxia. Similarly, TLR2 transcript was significantly elevated after 12 h of hypoxia exposure of HMEC-1 (2.170±0.2944, p<0.05) or Caco-2 cells (1.543±0.1009, p<0.05). We next pursued induction of TLR2 protein levels by Western blot analysis. For this purpose, HMEC-1 were grown to full confluency and exposed to hypoxia (0, 24, 48 and 72 h; pO2 20 torr). Indeed, we found increases of TLR2 protein in HMEC-1 after different time points of hypoxia exposure (Fig. 2C). As next step, we studied functional consequences of TLR2 induction by hypoxia. Previous studies had shown specific activation of TLR2 by N-Palmitoyl-bis(palmitoyloxy)-propyl-cysteinyl-seryl-Lys4 (P3C) leading to TLR2-signaling dependent activation of NF-κB and the production of pro-inflammatory cytokine IL6 [18]. For that purpose we stimulated normoxic or post-hypoxic HMEC-1 cells (48 h at 2% oxygen) with the TLR2 agonist P3C and measured IL6 secretion into the supernatant. These studies revealed that TLR2-dependent release of IL6 was dramatically enhanced in post-hypoxic HMEC-1 (Fig. 2D, p<0.01 with 100 ng/ml P3C). Taken together these studies confirm that TLR2 transcript, protein and function are induced by hypoxia.


Hypoxia inducible factor (HIF)-1 coordinates induction of Toll-like receptors TLR2 and TLR6 during hypoxia.

Kuhlicke J, Frick JS, Morote-Garcia JC, Rosenberger P, Eltzschig HK - PLoS ONE (2007)

TLR2 transcript, protein and function during hypoxia.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2147045&req=5

pone-0001364-g002: TLR2 transcript, protein and function during hypoxia.
Mentions: Based on the above observation of coordinated induction of TLR2 and TLR6 with hypoxia, we next pursued additional details of TLR2 induction with hypoxia using different cellular models. Therefore, we exposed human dendritic cells (hDCs), monocytic cells (MM6), endothelia (HMEC-1) and epithelia (Caco-2) over indicated time periods to hypoxia (2% oxygen), isolated RNA and performed real-time RT-PCR to determine TLR2 transcript levels. Consistent with our studies of murine dendritic cells, we found a robust induction of TLR2 transcript in all examined cells (Figure 2 A, B). For example, real-time RT-PCR analysis revealed prominent induction of TLR2 mRNA expression in hDCs (3.012±0.3002, p<0.01) and MM6 cells (1.812±0.4875, p<0.05) after 24h exposure to hypoxia. Similarly, TLR2 transcript was significantly elevated after 12 h of hypoxia exposure of HMEC-1 (2.170±0.2944, p<0.05) or Caco-2 cells (1.543±0.1009, p<0.05). We next pursued induction of TLR2 protein levels by Western blot analysis. For this purpose, HMEC-1 were grown to full confluency and exposed to hypoxia (0, 24, 48 and 72 h; pO2 20 torr). Indeed, we found increases of TLR2 protein in HMEC-1 after different time points of hypoxia exposure (Fig. 2C). As next step, we studied functional consequences of TLR2 induction by hypoxia. Previous studies had shown specific activation of TLR2 by N-Palmitoyl-bis(palmitoyloxy)-propyl-cysteinyl-seryl-Lys4 (P3C) leading to TLR2-signaling dependent activation of NF-κB and the production of pro-inflammatory cytokine IL6 [18]. For that purpose we stimulated normoxic or post-hypoxic HMEC-1 cells (48 h at 2% oxygen) with the TLR2 agonist P3C and measured IL6 secretion into the supernatant. These studies revealed that TLR2-dependent release of IL6 was dramatically enhanced in post-hypoxic HMEC-1 (Fig. 2D, p<0.01 with 100 ng/ml P3C). Taken together these studies confirm that TLR2 transcript, protein and function are induced by hypoxia.

Bottom Line: During acute infection and inflammation, dramatic shifts in tissue metabolism are typical, thereby resulting in profound tissue hypoxia.While transcript levels of other TLRs remained unchanged, we found a robust induction of TLR2 (2.36+/-0.7-fold; P<0.05) and TLR6 (3.46+/-1.56-fold; P<0.05).Studies using loss and gain of function of HIF-1 confirmed a critical role of HIF-1alpha in coordinating TLR2 and TLR6 induction.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology and Intensive Care Medicine, Tübingen University Hospital, Tübingen, Germany.

ABSTRACT

Background: During acute infection and inflammation, dramatic shifts in tissue metabolism are typical, thereby resulting in profound tissue hypoxia. Therefore, we pursued the hypothesis, that tissue hypoxia may influence innate immune responses by transcriptional modulation of Toll-like receptor (TLRs) expression and function.

Methodology/principal findings: We gained first insight from transcriptional profiling of murine dendritic cells exposed to hypoxia (2% oxygen for 24 h). While transcript levels of other TLRs remained unchanged, we found a robust induction of TLR2 (2.36+/-0.7-fold; P<0.05) and TLR6 (3.46+/-1.56-fold; P<0.05). Additional studies in different cells types and cell-lines including human dendritic cells, monocytic cells (MM6), endothelia (HMEC-1) or intestinal epithelia (Caco-2) confirmed TLR2 and TLR6 induction of transcript, protein and function during hypoxia. Furthermore, analysis of the putative TLR2 and TLR6 promoters revealed previously unrecognized binding sites for HIF-1, which were shown by chromatin immunoprecipitation to bind the pivotal hypoxia-regulating transcription factor HIF-1alpha. Studies using loss and gain of function of HIF-1 confirmed a critical role of HIF-1alpha in coordinating TLR2 and TLR6 induction. Moreover, studies of murine hypoxia (8% oxygen over 6 h) showed TLR2 and TLR 6 induction in mucosal organs in vivo. In contrast, hypoxia induction of TLR2 and TLR6 was abolished in conditional HIF-1alpha mutant mice.

Conclusions/significance: Taking together, these studies reveal coordinated induction of TLR2 and TLR6 during hypoxia and suggest tissue hypoxia in transcriptional adaptation of innate immune responses during acute infection or inflammation.

Show MeSH
Related in: MedlinePlus