Transregional Collaborative Research Center SFB-TR 84 - “Innate Immunity of the Lung: Mechanisms of Pathogen Attack and Host Defence in Pneumonia“

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C6-Subproject

The Angiopoietin-/Tie-2 system in severe pneumonia
(Witzenrath / Weißmann)

 

In pneumonia, lung endothelium importantly contributes to the regulation of the innate immune response upon pathogen-host interaction. Here, endothelial cell activation may provoke hyperpermeability, resulting in life-threatening lung edema. Constitutive ligation of pericyte/smooth muscle cell-derived protective angiopoietin (Ang) -1 to the endothelial receptor tyrosine kinase Tie2 preserves endothelial quiescence and sealing. In contrast, autocrine action of endothelial cell-released Ang-2 as a direct antagonist of Ang-1 locally disturbs the vascular homeostasis and is required, e.g. for blood vessel formation. Our aim is to decipher the mechanistic role of the Ang-1, -2 / Tie2 signaling system in the pulmonary innate immune response and on this basis, to develop new strategies to prevent and treat lung failure in pneumonia.

We detected reduced expression and liberation of Ang-1, whereas Ang-2 was increased following infection, by analysing patient plasma, mice with pneumonia, or endothelial cells in vitro. Analysing ex vivo cultured human lung tissue and endothelial cells by employing antibodies, plasmids and state-of-the-art superresolution microscopy; we revealed that Ang-2 was localized intracellularly in endothelial cells independent from Weibel-Palade-bodies, which is in contrast to some previously reported results.

We detected an important functional role for angiopoietins in pneumonia. Whereas endogenous Ang-2 contributed to increased lung permeability in pneumonia models, exogenous Ang-2 only slightly increased endothelial permeability in vitro, and Ang-2 did not evoke inflammation or vascular permeability in healthy mice. In contrast to Ang-2, Ang-1 improved endothelial barrier integrity in vitro, and Ang-1 therapy of established pneumonia reduced pulmonary inflammation and barrier dysfunction in mice.

We observed strong correlation of Ang-1 and sphingosine-1-phosphate (S1P) levels in plasma of pneumonia patients. Previous reports suggested that Ang-1 may exert its beneficial effects on endothelial barrier function via S1P. However, in sphingosine-kinase-1 (SphK1)-deficient mice with reduced S1P production, we found diminished permeability in pneumonia, whereas exogenous S1P increased permeability in pneumonia. Human macrophages expressed SphK1, increased their S1P production upon stimulation with S. pneumoniae, and were found in inflamed lung areas of human post mortem lungs with pneumonia. S1P and pneumolysin augmented permeability synergistically in a S1P receptor 2 and Rho-kinase dependent manner. Thus, a novel role for S1P in pneumonia was observed.

Microarray studies in combination with cluster and pathway analysis of lungs from Ang-1 treated mice with pneumonia suggested a regulation of junctional proteins and apoptosis-controlling factors.
On the basis of these findings, we will now employ (1) our newly developed endothelium-specific inducible Ang-2 knockout mice, (2) GFP-labeled Tie-2 mice, and (3) the novel synthetic Tie-2 agonist Vasculotide as well as neutralizing antibodies against Ang-2 to unravel the signalling pathways underlying the Ang-1/2 effects in pneumonia.

In particular, we aim (i) to elucidate the exact cellular and subcellular localization and kinetics of Ang-2 and Tie-2 following infection; (ii) to further decipher the impact of Ang-2 on endothelial barrier dysfunction and leukocyte transmigration in pneumonia and to delineate the relative importance of monocytic vs. endothelial Ang-2 in this context; (iii) to clarify the mechanisms of the observed beneficial effects of Ang-1 by focussing on (a) recruitment, migration and activation of leukocytes, (b) endothelial activation, cytoskelettal changes, junctional integrity and apoptosis, and (c) impact of Ang-1 on the transcriptional level as discovered by our current mRNA screening in vivo and miRNA screening in vitro; (iv) to further enhance the preclinical relevance of our findings. We will investigate whether the outstanding therapeutic success of Ang-1 in murine pneumococcal pneumonia can be reproduced in pneumonia due to pathogens other than S. pneumonia. Also, we will expand our observations towards the clinically relevant interplay of pneumonia and ventilator-induced lung injury. (v) to evaluate Angiopoietin plasma levels as possible biomarkers, and correlate Ang-1 and Ang-2 plasma levels in the time course of pneumonia along with comprehensively documented clinical and paraclinical parameters from 200 patients with pneumonia (provided by the PROGRESS consortium).
With these investigations we aim to propose Ang-1 as potential novel therapeutic approach to treat severe pneumonia.