The role of the oxygen sensors PHD2 and PHD3 in the response of macrophages to ischemia-induced inflammation

The role of the oxygen sensors PHD2 and PHD3 in the response of macrophages to ischemia-induced inflammation

Angelika Beneke

Doctoral thesis

Date of Examination: 2016-10-24

Date of issue: 2016-12-12

Advisor: Katschinski, Dörthe Prof. Dr.

Referee: Meyer, Thomas Prof. Dr.

Referee: Reichardt, Holger Prof. Dr.

Persistent Address: http://hdl.handle.net/11858/00-1735-0000-002B-7CD2-B

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Abstract

English

Ischemic diseases, caused by the occlusion of a supplying blood vessel, result in a severe lack of oxygen and nutrients within the tissue. The ischemic insult triggers an inflammatory response of the innate immune system. Myeloid cells such as neutrophils and macrophages are recruited to the tissue to clear up debris and necrotic cells. In ischemia, adaptation to low oxygen levels is essential to cell survival. Prolyl-4-hydroxylase domain enzymes (PHD) 1, 2 and 3 regulate the protein-stability of the α-subunit of hypoxia-inducible factor (HIF), which makes them the oxygen-sensors of the cells. In myeloid cells, PHD3 is a potent regulator of apoptosis in neutrophils and macrophages. PHD2 on the other hand affects macrophage metabolism and migration. The aim of this thesis was to further characterize the functions of PHD2 and 3 for macrophages in the context of ischemic diseases. I studied the role of PHD3 in myeloid cells upon induction of hind limb ischemia in a myeloid-specific PHD3-/- mouse. After hind limb ischemia surgery, the infiltration of myeloid cells into the hind limb muscle as well as their clearance was analyzed via flow cytometry. The inflammatory response of PHD3-/- macrophages was blunted 4 and 5 days after surgery compared to wild type. Furthermore, the PHD3-/- macrophage population showed an increased surface expression of the anti-inflammatory marker CD206 4 and 5 days after surgery, indicating a faster initiation of wound healing compared to wild type. Despite the blunted macrophage response, perfusion recovery and angiogenesis were unaltered between PHD3-/- and wild type mice. However, the development of fibrosis was decreased in PHD3-/- mice 4 weeks after surgery, which could have been caused by the attenuated macrophage response. Neither a change in apoptosis nor in migratory behavior was observed in PHD3-/- macrophages which could serve as a possible explanation for the decreased inflammatory response of PHD3-/- macrophages. RNA sequencing of macrophages sorted from the hind limb 5 days after surgery revealed 10 differentially regulated genes. Cyp2s1, an epoxygenase which metabolizes Prostaglandin (PG) G2 and H2 to 12-hydroxyheptadecatrenoic acid (12-HHT), was significantly downregulated in PHD3-/- macrophages. This downregulation could be mimicked in bone marrow-derived macrophages (BMDM) when cultivated in starvation medium in hypoxia (1% O2). Under these conditions, BMDM also displayed a significantly higher secretion of PGE2, which might be explained by the downregulation of Cyp2s1. PGG2 and PGH2 are substrates for 12-HHT and PGE2 synthesis likewise. Lower Cyp2s1 expression could therefore leave more substrates for PGE2 synthesis. PGE2 can induce anti-inflammatory behavior in macrophages and therefore presents a putative explanation for the attenuated macrophage response in PHD3-/- mice. To study the role of PHD2 in macrophages in an ischemia model, I analyzed the effect of myocardial infarction in myeloid-specific PHD2-/- mice. Macrophage but not neutrophil invasion and clearance in the ischemic heart muscle were altered between wild type and PHD2-/- mice. Macrophage infiltration into the ischemic heart was delayed in PHD2-/- macrophages, which was linked to a worse heart function measured by echocardiography. In line with this, previous in vitro studies, conducted by another member of the Institute of Cardiovascular Physiology, found a migration defect in PHD2-/- BMDM as well as a lower phagocytotic capacity, which might account for the in vivo findings. These findings demonstrate non-redundant roles for PHD-isoforms PHD2 and PHD3 in myeloid cells. Myeloid-specific PHD2-deficiency presents a disadvantage in the resolution of ischemia-induced inflammation while myeloid PHD3-deficiency leads to a faster resolution of inflammation with positive effects on wound-healing.

Keywords: Hypoxia; Macrophage; Prolyl-4-hydroxylase domain enzymes