The role of hypoxia and inflammatory preconditioning for the function of monocytes and macrophages as well as for the interaction of monocytes with the endothelium

In der vorliegenden Arbeit wurde die Bedeutung des Transkriptionsfaktorkomplexes Hypoxie-induzierbarer Faktor (HIF)-1α für das Verhalten monozytärer Zellen unter Hypoxie sowie nach inflammatorischer Präkonditionierung untersucht. Darüber hinaus wurde der Einfluss akuter, schwerer Hypoxie auf die Wechselwirkung zwischen Monozyten und Endothelzellen charakterisiert. HIF-1α-defiziente Monozyten und Makrophagen weisen nicht nur eine verminderte Vitalität unter Hypoxie auf, sondern zeigen eine deutlich eingeschränkte Funktion, auf einen inflammatorischen Stimulus hin eine angemessene Immunantwort zu generieren. So weisen sie beispielsweise eine geringere Infiltration in inflammatorisches Gewebe auf, die durch eine verminderte Adhäsion, Migration, Chemotaxis und Invasion in extrazelluläre Matrix bedingt ist. In dieser Arbeit konnte gezeigt werden, dass die Etablierung einer Endotoxintoleranz (Toleranzentwicklung bezüglich bakterieller Stimuli, die durch Vorinkubation mit geringen Endotoxinmengen erzielt wird) in vitro und in vivo zu einer verminderten HIF-1α Protein- und mRNA-Expression führt. Als Ursache dafür konnte die Induktion des alternativen NF-κB-Signalwegs während der Toleranzentwicklung bestimmt werden. Endotoxintolerante Monozyten und Makrophagen wiesen eine verminderte Vitalität unter hypoxischen Bedingungen sowie eine verminderte Invasion in extrazelluläre Matrix auf und verhielten sich somit sehr ähnlich zu HIF-1α-defizienten Zellen. Insgesamt führten die erzielten Ergebnisse zu der These, dass mittels Endotoxintoleranz in diesen Zellen ein funktionaler HIF-1α Knock-down erzielt werden kann. Bezüglich der Auswirkungen schwerer Hypoxie auf das Adhäsionsverhalten von Monozyten an Endothelzellen konnte eine deutliche Induktion der monozytären Adhäsion festgestellt werden. Diese erfolgte durch hypoxische - jedoch nicht HIF-1α-abhängige - Induktion des monozytären Adhäsionsmoleküls ICAM-1, das im vorliegenden Fall mit endothelialem ICAM-1 Homodimere ausbilden könnte. Eine Steigerung der Expression dieses Moleküls bedarf einer de novo Synthese, die in den Monozyten durch die hypoxische Aktivierung des klassischen NF-κB-Signalwegs induziert wird. Auch die langfristige Inkubation der Zellen mit sehr geringen LPS-Dosen führte zu einer vermehrten Expression des monozytären ICAM-1, was eine bis heute völlig unbekannte Bedeutung dieses Moleküls innerhalb der Leukozyten-Adhäsionskaskade nahe legt. Eine Ergänzung dieses Modells zur monozytären Adhäsion um die ICAM-1-ICAM-1-Wechselwirkung erscheint deshalb sinnvoll

The hypoxia-regulated ectonucleotidase CD73 is a host determinant of HIV latency

Deciphering the mechanisms underlying viral persistence is critical to achieving a cure for human immunodeficiency virus (HIV) infection. Here, we implement a systems approach to discover molecular signatures of HIV latently infected CD4+ T cells, identifying the immunosuppressive, adenosine-producing ectonucleotidase CD73 as a key surface marker of latent cells. Hypoxic conditioning, reflecting the lymphoid tissue microenvironment, increases the frequency of CD73+ CD4+ T cells and promotes HIV latency. Transcriptomic profiles of CD73+ CD4+ T cells favor viral quiescence, immune evasion, and cell survival. CD73+ CD4+ T cells are capable of harboring a functional HIV reservoir and reinitiating productive infection ex vivo. CD73 or adenosine receptor blockade facilitates latent HIV reactivation in vitro, mechanistically linking adenosine signaling to viral quiescence. Finally, tissue imaging of lymph nodes from HIV-infected individuals on antiretroviral therapy reveals spatial association between CD73 expression and HIV persistence in vivo. Our findings warrant development of HIV-cure strategies targeting the hypoxia-CD73-adenosine axis

Endogenous myoglobin expression in mouse models of mammary carcinoma reduces hypoxia and metastasis in PyMT mice

Myoglobin (MB) is expressed in different cancer types and may act as a tumor suppressor in breast cancer. The mechanisms by which basal MB expression level impacts murine mammary tumorigenesis are unclear. We investigated how MB expression in breast cancer influences proliferation, metastasis, tumor hypoxia, and chemotherapy treatment in vivo. We crossed PyMT and WapCreTrp53$^{flox}$ mammary cancer mouse models that differed in tumor grade/type and onset of mammary carcinoma with MB knockout mice. The loss of MB in WapCre;Trp53$^{flox}$ mice did not affect tumor development and progression. On the other hand, loss of MB decreased tumor growth and increased tissue hypoxia as well as the number of lung metastases in PyMT mice. Furthermore, Doxorubicin therapy prevented the stronger metastatic propensity of MB-deficient tumors in PyMT mice. This suggests that, although MB expression predicts improved prognosis in breast cancer patients, MB-deficient tumors may still respond well to first-line therapies. We propose that determining the expression level of MB in malignant breast cancer biopsies will improve tumor stratification, outcome prediction, and personalized therapy in cancer patients

Dendritic Cells under Hypoxia: How Oxygen Shortage Affects the Linkage between Innate and Adaptive Immunity

Dendritic cells (DCs) are considered as one of the main regulators of immune responses. They collect antigens, process them, and present typical antigenic structures to lymphocytes, thereby inducing an adaptive immune response. All these processes take place under conditions of oxygen shortage (hypoxia) which is often not considered in experimental settings. This review highlights how deeply hypoxia modulates human as well as mouse immature and mature dendritic cell functions. It tries to link in vitro results to actual in vivo studies and outlines how hypoxia-mediated shaping of dendritic cells affects the activation of (innate) immunity

Prolyl hydroxylase domain 2 reduction enhances skeletal muscle tissue regeneration after soft tissue trauma in mice

Abstract The transcription factor Hypoxia-inducible factor 1 (HIF-1) plays a pivotal role in tissue regeneration. HIF-1 is negatively controlled by O2-dependent prolyl hydroxylases with a predominant role of prolyl hydroxylase 2 isoform (Phd2). Transgenic mice, hypomorphic for this isoform, accumulate more HIF-1 under normoxic conditions. Using these mice, we investigated the influence of Phd2 and HIF-1 on the regenerative capability of skeletal muscle tissue after myotrauma. Phd2-hypomorphic and wild type mice (on C57Bl/6 background) were grouped with regeneration times from 6 to 168 hours after closed mechanic muscle trauma to the hind limb. Tissue samples were analysed by immuno-staining and real-time PCR. Bone marrow derived macrophages of wild type and Phd2-hypomorphic mice were isolated and analysed via flow cytometry and quantitative real-time PCR. Phd2 reduction led to a higher regenerative capability due to enhanced activation of myogenic factors accompanied by induction of genes responsible for glucose and lactate metabolism in Phd2-hypomorphic mice. Macrophage infiltration into the trauma areas in hypomorphic mice started earlier and was more pronounced compared to wild type mice. Phd2-hypomorphic mice also showed higher numbers of macrophages in areas with sustained trauma 72 hours after myotrauma application. In conclusion, we postulate that the HIF-1 pathway is activated secondary to a Phd2 reduction which may lead to i) higher activation of myogenic factors, ii) increased number of positive stem cell proliferation markers, and iii) accelerated macrophage recruitment to areas of trauma, resulting in faster muscle tissue regeneration after myotrauma. With the current development of prolyl hydroxylase domain inhibitors, our findings point towards a potential clinical benefit after myotrauma

Knockdown of myeloid cell hypoxia-inducible factor-1α ameliorates the acute pathology in DSS-induced colitis

<div><p>Inflammation and hypoxia are hallmarks of inflammatory bowel disease. Low oxygen levels activate hypoxia-inducible factors as central transcriptional regulators of cellular responses to hypoxia, particularly in myeloid cells where hypoxia-inducible factors control immune cell function and survival. Still, the role of myeloid hypoxia-inducible factor-1 during inflammatory bowel disease remains poorly defined. We therefore investigated the role of hypoxia-inducible factor-1 for myeloid cell function and immune response during colitis. Experimental colitis was induced by administration of 2.5% dextran sulfate sodium to mice with a conditional knockout of hypoxia-inducible factor-1α in myeloid cells and their wild type siblings. Murine colon tissue was examined by histologic analysis, immunohistochemistry, and quantitative polymerase chain reaction. Induction of experimental colitis increased levels of hypoxia and accumulation of hypoxia-inducible factor-1α positive cells in colon tissue of both treated groups. Myeloid hypoxia-inducible factor-1α knockout reduced weight loss and disease activity index when compared to wild type mice. Knockout mice displayed less infiltration of macrophages into intestinal mucosa and reduced mRNA expression of markers for dendritic cells and interleukin-17 secreting T helper cells. Expression of inflammatory and anti-inflammatory cytokines also showed a reduced and delayed induction in myeloid hypoxia-inducible factor-1α knockout mice. Our results show a disease promoting role of myeloid hypoxia-inducible factor-1 during intestinal inflammation. This might result from a hypoxia-inducible factor-1 dependent increase in pro-inflammatory interleukin-17 secreting T helper cells in the absence of obvious changes in regulatory T cells. In contrast, knockout mice appear to shift the balance to anti-inflammatory signals and cells resulting in milder intestinal inflammation.</p></div

Colon tissue damage in DSS-induced colitis in mice lacking myeloid HIF-1α.

<p>H&E staining (<b>A</b>) and histology scoring (<b>B</b>) of paraffin-embedded colon sections from wild type (HIF-1α^+f/+f) and knockout (Lyz2-Cre/HIF-1α^+f/+f) mice treated for four to six days (4d-6d) with 2.5% DSS. (<b>C</b>) Real time PCR of Lipocalin-2 in colon RNA samples of wild type (HIF-1α^+f/+f) and knockout (Lyz2-Cre/HIF-1α^+f/+f) mice treated as in (A). Data are representative for experiments with five or six mice/group. Each time point represents the mean value ± SEM. *P < 0.05; **P < 0.01 and ***P < 0.001 compared as indicated. # displays significance to respective control. Original bars, 50 μm.</p

Hypoxia and HIF-1 accumulation after DSS-treatment.

<p>Real time PCR of HIF-1α exon 2 (<b>A</b>) in RNA samples of hypoxic treated BMDM of wild type (HIF-1α^+f/+f) and knockout (Lyz2-Cre/HIF-1α^+f/+f) mice with 14 samples/group. Each bar represents the mean value ± SEM. ***P < 0.001. Immunohistochemical staining of hypoxia with Hypoxyprobe-1 (HP-1) antibody (<b>B</b>) or staining of HIF-1α (<b>C</b>) and HIF-2α (<b>D</b>) in paraffin-embedded colon tissue of wild type (HIF-1α^+f/+f) and knockout (Lyz2-Cre/HIF-1α^+f/+f) mice after treatment with drinking water (C = control) or with 2.5% DSS for six days (6d). Note, that the HIF-1α antibody recognizes both wild type and knockout HIF-1α (lacking exon 2) and therefore detects (non-functional) HIF-1α protein in knockout mouse tissue. Representative images of stained colon slices of experiments with five or six mice/group. Original bars 100 μm.</p