Influence of interferon & Toll like receptors on endothelial hormones: relevance to pneumonitis & pulmonary hypertension

When viruses infect our bodies, our immune system is activated and cells make a hormone called interferon. Interferon is also used as an important drug therapy to treat serious conditions such as hepatitis C and multiple sclerosis. Viruses and interferon drugs can cause severe inflammation in the lungs of some people, which can make them critically ill, and/or cause a chronic life threatening condition called pulmonary hypertension. Pulmonary hypertension is rare, but has no cure and so it is vital that we continue to research it. We have new results suggesting that viruses and interferon drugs can strongly activate human cells, and, if this happened in the body, it could result in lung inflammation or even pulmonary hypertension. One pathway we have found to be important is called endothelin-1. Our aim is to complete our study using human cells so that we can provide vital information that could lead to a clinical study where hormones such as endothelin-1 could be measured in people infected by viruses or using interferon drugs. We think that this is important because we might be able to identify and treat those people who would otherwise go on to get very serious effects in the lungs.

Pulmonary vascular inflammation can occur with viral infection or interferon (IFN) therapy, which is used to treat hepatitis C and multiple sclerosis. Pulmonary hypertension (PH) is linked to infection with HIV and human herpes virus-8 and respiratory viruses are implicated in pneumonitis and acute lung injury. Endothelin-1 (ET-1) is a potent vasoconstrictor and mitogen, which is associated with pulmonary vascular inflammation and, indelibly, with PH. Our pilot data shows that ET-1 is released from human pulmonary vascular smooth muscle cells when stimulated by the virally induced type I and type II IFNs. We have also shown in pilot experiments that human pulmonary vascular cells release ET-1 when activated with viral toll like receptor (TLR)8 ligands, but not when stimulated with bacterial TLR4 ligands. Our main aim is therefore to understand how IFN signalling regulates ET-1 and other vasoactive genes such as prostacyclin and nitric oxide (NO) in pulmonary vascular cells from patients with and without PH. Our other aims will be to determine the effect of viral mimics and of infection of cells with prototype viruses, using protocols firmly established in Professor Johnston‘s group, on IFN release, signalling and vasoactive gene expression in human pulmonary vascular cells.

We will use cell culture and cell based assays. Cultures of human pulmonary artery smooth muscle cells and lung microvascular endothelial cells will be maintained according to protocols established over many years in Professor Mitchell‘s group. Bone morphogenetic protein receptor type 2 (BMPR2) ‘mutant‘ vascular smooth muscle cells from patients with PH will be cultured according to protocols of Professor Nicholas Morrell who is a collaborator. Cell cultures will be characterised by morphology and expression of CD31 (endothelial cells) and ?-actin (smooth muscle cells). ET-1 and prostaglandin release will be measured using ELISA. Cell proliferation will be measured by counting cells, measuring DNA levels and, if required, FACS analysis of proliferation/apoptosis markers. Gene expression will be measured using quantitative PCR. Protein expression will be measured using western blotting and imaged in cells using en face confocal microscopy.

This study has scientific and medical importance. Data obtained will lead to a better understanding of viral and IFN-induced vascular inflammation. If our hypothesis is correct this project will provide the essential pre-clinical data required for a clinical study in which patients at risk of serious life-threatening reactions to virus or IFN therapy may be identified by measuring levels of vasoactive hormones such as ET-1.