Hypoxia Inducible Factor (HIF)-1 Coordinates Induction of Toll-Like Receptors TLR2 and TLR6 during Hypoxia

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.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.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

The importance of biofilm formation for cultivation of a Micrarchaeon and its interactions with its Thermoplasmatales host

Micrarchaeota is a distinctive lineage assigned to the DPANN archaea, which includes poorly characterised microorganisms with reduced genomes that likely depend on interactions with hosts for growth and survival. Here, we report the enrichment of a stable co-culture of a member of the Micrarchaeota (Ca. Micrarchaeum harzensis) together with its Thermoplasmatales host (Ca. Scheffleriplasma hospitalis), as well as the isolation of the latter. We show that symbiont-host interactions depend on biofilm formation as evidenced by growth experiments, comparative transcriptomic analyses and electron microscopy. In addition, genomic, metabolomic, extracellular polymeric substances and lipid content analyses indicate that the Micrarchaeon symbiont relies on the acquisition of metabolites from its host. Our study of the cell biology and physiology of a Micrarchaeon and its host adds to our limited knowledge of archaeal symbioses

Oxygen-Independent Stabilization of Hypoxia Inducible Factor (HIF)-1 during RSV Infection

BACKGROUND: Hypoxia-inducible factor 1 (HIF)-1alpha is a transcription factor that functions as master regulator of mammalian oxygen homeostasis. In addition, recent studies identified a role for HIF-1alpha as transcriptional regulator during inflammation or infection. Based on studies showing that respiratory syncytial virus (RSV) is among the most potent biological stimuli to induce an inflammatory milieu, we hypothesized a role of HIF-1alpha as transcriptional regulator during infections with RSV. METHODOLOGY, PRINCIPAL FINDINGS: We gained first insight from immunohistocemical studies of RSV-infected human pulmonary epithelia that were stained for HIF-1alpha. These studies revealed that RSV-positive cells also stained for HIF-1alpha, suggesting concomitant HIF-activation during RSV infection. Similarly, Western blot analysis confirmed an approximately 8-fold increase in HIF-1alpha protein 24 h after RSV infection. In contrast, HIF-1alpha activation was abolished utilizing UV-treated RSV. Moreover, HIF-alpha-regulated genes (VEGF, CD73, FN-1, COX-2) were induced with RSV infection of wild-type cells. In contrast, HIF-1alpha dependent gene induction was abolished in pulmonary epithelia following siRNA mediated repression of HIF-1alpha. Measurements of the partial pressure of oxygen in the supernatants of RSV infected epithelia or controls revealed no differences in oxygen content, suggesting that HIF-1alpha activation is not caused by RSV associated hypoxia. Finally, studies of RSV pneumonitis in mice confirmed HIF-alpha-activation in a murine in vivo model. CONCLUSIONS/SIGNIFICANCE: Taking together, these studies suggest hypoxia-independent activation of HIF-1alpha during infection with RSV in vitro and in vivo

Role of hypoxia inducible factor (HIF)-1 in TLR2 and TLR6 expression during hypoxia in vivo.

<p>Real-time RT-PCR analysis of murine epithelial TLR2 and TLR6 mRNA in conditional HIF-1α mutant (HIF-/-) and littermate control (WT) animals subjected to normoxia or hypoxia. Data were calculated relative to ß-actin and are expressed as fold change over normoxia±SEM, where transcript levels of control animals were normalized to 1. *, significant differences from normoxic control animals (p<0.05). Results are derived from 8 animals in each condition.</p

Human and murine Primer pairs as used for real-time RT-PCR.

<p>Human and murine Primer pairs as used for real-time RT-PCR.</p

Influence of hypoxia inducible factor (HIF)-1α on TLR6 expression during hypoxia.

<p><i>A</i>, Map of TLR6 promoter region showing positions of the putative HIF binding sites and the binding site for NFκB relative to the transcription start site (TSS). <i>B</i>, Stable transfected HMEC-1 monolayers containing either HIF-1α siRNA or control-siRNA were exposed to normoxia or hypoxia for indicated time points. Total RNA was isolated, and 1 µg of RNA was transcribed into first strand cDNA. Relative expressional levels of TLR6 transcripts were compared to normoxic controls by real-time RT-PCR. Data were calculated relative to internal control gene (ß-actin), and are expressed as fold change over normoxia±SEM, *, significant differences from normoxia and control cells. Results are derived from three different experiments in each condition. <i>C</i>, Total RNA of normoxic monolayers of either wildtype (WT) or oxygen-stable HIF-1α expressing (HIF^+/+) HMEC-1 cells was isolated and realt-time RT-PCR was performed as described above. *, significant differences from wildtype cells. <i>D</i>, Western blot analysis of TLR6 protein of normoxic HMEC-1 wildtype (WT) and oxygen-stable HIF-1α expressing (HIF+/+) cells. The same blot was probed for ß-actin expression as a control for protein loading. <i>E</i>, HMEC-1 monolayers were treated with 1mM of dimethyloxalylglycine (DMOG) for 24 hours. Afterwards transcript levels of TLR6 where quantified by real-time RT-PCR as described above. *, significant differences from untreated cells. <i>F</i>, ChIP assay was utilized to examine HIF-1α binding to the TLR6 promoter in normoxic and hypoxic HMEC-1 cells. Reaction controls included immunoprecipitations using a nonspecific igG monoclonal antibody (IgG) and PCR performed using HMEC-1 DNA (input). An example of three experiments is shown.</p

TLR6 transcript, protein and function during hypoxia.

<p><i>A</i> and <i>B</i>, Quantification of TLR6 transcripit levels in freshly purified blood DCs, MM6 cells, confluent HMEC-1 monolayers and confluent Caco-2 monolayers. Cells were exposed to normoxia and hypoxia for indicated time points. Total RNA was isolated and TLR6 mRNA levels were determined by real-time RT-PCR. Data were calculated relative to ß-actin and expressed as fold change relative to normoxia±SEM, where transcript levels in normoxic cells were normalized to 1. Results are derived from three different experiments. *, significant differences from normoxic cells (p<0.01). <i>C</i>, Confluent HMEC-1 cells were grown to confluence and exposed to indicated periods of hypoxia. Result depicts a representative TLR6 Western blot from three separate experiments. The same blot was probed for ß-actin expression as a control for protein loading. <i>D</i>, Same amount of HMEC-1 cells were grown to confluence in 24-well plates. Cells were then stimulated with TLR2/6 agonist (FSL-1) at the indicated concentrations and exposed to hypoxia or normoxia for 24 h. After 24 h, generation of IL6 was measured by ELISA in the cell supernatant. Data are mean±SEM from 3 separate replicates. *, significant differences from untreated cells (p<0.05); #, significant differences from normoxia and untreated cells (p<0.05).</p

Influence of hypoxia on TLR mRNA expression in murine dendritic cells.

<p>Isolated murine bone-marrow-derived dendritic cells (BMDCs) were exposed to normoxia or hypoxia for 24 hours. Total RNA was isolated, and quantitative mRNA levels of TLR1-9 and TLR11-13 were assessed by real-time RT-PCR. Data were calculated relative to ß-actin and expressed as fold change relative to normoxia±SEM and transcript levels in normoxic BMDCs were normalized to 1. Results are derived from three different experiments (*p<0.05, significant differences from normoxia).</p

TLR2 transcript, protein and function during hypoxia.

<p>TLR2 transcript, protein and function during hypoxia.</p