Primary Graft Dysfunction (PGD) remains a leading cause of early morbidity and mortality in lung transplant (Tx) recipients. The Lung Transplant Outcomes Group (LTOG) has been studying this problem for the past decade and a newly NIH-funded LTOG-related project (1U01HL14535-01), headed by Dr. Jason Christie (UPENN), involves long-term follow- up of adult lung transplant recipients. In preliminary studies, undertaken in collaboration with LTOG, we found that patients who subsequently developed PGD had significantly elevated pre-Tx levels of HDAC6 mRNA in their peripheral blood mononuclear cells. We identified CD14+ monocytes as the main source of upregulated HDAC6 in pre-Tx PGD-prone patients, observed an inflammatory phenotype of those monocytes after stimulation in vitro, and found that HDAC6 targeting significantly decreased cytokine production in an in vivo murine lung model of ischemia/reperfusion injury. Accordingly, we hypothesize that recipient blood monocytes with upregulated HDAC6 expression pre-lung Tx are important drivers of graft injury and development of PGD, and that such elevated HDAC6 expression in monocytes may be of prognostic and therapeutic significance. We propose to test this hypothesis by studying pre-Tx blood samples from patients enrolled in the parent U01 that began June 15, 2019.
Aim 1 : Determine if the phenotype of upregulated HDAC6 in monocytes of patients listed for lung Tx is a risk factor for developing PGD? We will expand upon our preliminary data using a larger sample size to evaluate 1.1) pre-Tx levels of monocyte HDAC6 expression, including mRNA and protein levels, and co-expression of classical markers of monocyte activation; and 1.2) post-Tx events (PGD with grade) in relation to the pre-Tx monocyte phenotype.
Aim 2 : What are the mechanisms by which HDAC6 alters key monocyte characteristics? We will: 2.1) evaluate cytokine expression (qPCR, flow cytometry, ELISA) by monocytes isolated from pre-Tx recipients and healthy donors, in relation to their HDAC6 levels; and 2.2) study how high HDAC6 expression regulates TLR/MyD88 pathway activation.
Aim 3 : Test whether HDAC6 inhibitor (HDAC6i) therapy can reverse or diminish the effects of monocyte dysfunction, including ex vivo and in a murine model of PGD. We will: 3.1) test effects of HDAC6i on monocyte activation, cytokine production and interaction with other immune cells; 3.2) test effects of one or more novel HDAC6i small molecules (monocyte specific HDAC6i, HDAC6 zinc-finger ubiquitin binding domain inhibitor and HDAC6i PROTAC); and 3.3) apply HDAC6i in vivo in a recently described new murine model of PGD. Importantly, our studies will ? delineate pathogenic mechanisms of disease; ? identify mechanisms or factors that influence and/or predict response to treatment; and ? discover or validate biomarkers of disease development and/or progression.
We will determine whether abnormalities in a key population of circulating blood cells called monocytes affects the very early outcomes of lung transplants in patients with severe lung diseases. We will assess whether problems with monocytes can be detected pre-transplant using blood samples already being collected as part of an NIH-funded clinical study of lung transplant recipients. Our studies may identify new diagnostic or therapeutic options to decrease the high morbidity and mortality rates currently associated with clinical lung transplantation.
