TRPA1; a potential therapeutic target in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a common disease with a bleak prognosis. Half of all patients diagnosed with IPF will die within 3-4 years. A scarring process occurs in the lungs, eventually replacing normal healthy lung tissue with dense scar tissue, which prevents oxygen passing into the blood and the normal lung expansion associated with breathing. As a result patients develop persistent cough, become extremely breathless, and finally die as a result of lung failure. The cause of IPF is unknown. Only two treatments are available, but these are not very effective, and produce frequent and unpleasant side effects.

Considerable evidence implicates cells known as myofibroblasts in the generation of lung scar tissue in IPF. These cells are important in normal wound healing and provide structure to healthy organs including lungs, but in disease they behave abnormally. It is myofibroblasts that produce the dense tissue that fills the lungs. In addition they move to abnormal locations and multiply, leading to dense collections of myofibroblasts and scar tissue that distort normal lung structure. All cells contain proteins called ion channels that conduct ions such as calcium into the cell. We have identified an ion channel called TRPA1 in human lung myofibroblasts, and believe that this is a key signalling molecule through which myofibroblasts generate scar tissue. We will test this hypothesis in this proposal, and if we are correct, we expect that blocking TRPA1 will slow or halt the progress of IPF. Drugs that block TRPA1 are already being studied in other diseases, and to-date, have been well tolerated by patients. We therefore expect that the results from our study will provide a strong rationale for the assessment of TRPA1 blockers in clinical trials for patients with IPF.

General protocols: Primary human lung myofibroblasts (HLMFs) are grown from the lung parenchyma of patients with IPF and healthy non-fibrotic control (NFC) tissue removed at lung resection.

AIM 1: The expression of TRPA1 in HLMFs and human lung parenchymal tissue from non-fibrotic control (NFC) and IPF donors
TRPA1 expression will be examined in NFC- and IPF-derived HLMFs both at rest and following activation with TGFb1 and H2O2 activation. We will examine mRNA expression (qRT-PCR), protein expression (western blotting), functional expression (patch clamp electrophysiology), tissue expression (immunohistochemistry).

AIM 2: The functional role of TRPA1 in HLMF pro-fibrotic activity
We will study the effect of TRPA1 blockers/RNA silencing on HLMF function proliferation, wound healing, soluble collagen secretion, aSMA expression/stress fibre formation, and contractility in collagen gels.

AIM 3: The mechanistic interactions between ROS, TGFb1, and TRPA1 signalling in HLMFs
We will use ratiometric Ca2+ imaging and fluorescent probes to detect intracellular Ca2+ and ROS in HLMFs activated with H202 and TGFb1 +/- TRPA1 antagonists/siRNA. Inhibitors of ROS and NOX4 will be used to investigate ROS-dependency. We will also assess TGFb1-dependent pro-fibrotic signalling (SMAD2/3 phosphorylation and nuclear translocation, phosphorylation of P38 MAPK/JNK) in the presence of ROS inhibitors and TRPA1 antagonists/siRNA.

AIM 4: The contribution of TRPA1 to TGFb1-dependent fibrogenesis in a human lung parenchymal tissue ex-vivo model of fibrosis
We will study TRPA1 antagonists, anti-oxidants and NOX4 inhibitors in our human lung parenchymal model of lung fibrogenesis, using qRT-PCR profiling arrays and immunohistochemistry. This human lung parenchymal assay avoids the important tissue-dependent heterogeneity applicable to TRPA1 pharmacology, and offers the opportunity to benchmark the potential clinical effectiveness of TRPA1 antagonists against pirfenidone.

Who will benefit from this research?

There will be numerous beneficiaries of this research including both respiratory academia and the wider scientific community, the pharmaceutical industry, patients and healthcare systems including the NHS.

How will they benefit from this research?

For academic beneficiaries please see "Academic Beneficiaries" section.

The research proposed will be of great interest to the pharmaceutical industry, and will likely provide a strong rationale for studying TRPA1 antagonists as a novel treatment for IPF. We have worked closely with the pharmaceutical industry over the last decade, both with respect to understanding the basic science driving chronic lung disease, and with the development and phenotype-specific targeting of novel therapies. We are therefore well-placed to ensure that the outcomes of this research are considered for translation to therapy where appropriate.

Longer term, it is anticipated that the development of new therapies derived from this research will improve the quality of life and health for patients and reduce the financial burden of respiratory disease on healthcare systems including the NHS. Because TRPA1 antagonists are already in phase 2 clinical trials for other diseases, and appear well-tolerated, there is the potential for the rapid progression to clinical trials in IPF. A realistic time-frame for start of phase 2 clinical trials in IPF is 3 years from the onset of the study. For implementation in clinical practice, 7-10 years from the onset of the study is feasible.