Reprogramming of type 2 alveolar epithelial cells in idiopathic pulmonary fibrosis and regulation by TGFb1. - Idiopathic Pulmonary Fibrosis (IPF) is the most common fibrotic interstitial lung disease among
adults. The cause of IPF is not fully understood, and it is frequently progressive, often leading
to death within several years of diagnosis. In IPF, there is loss of alveolar epithelial cells,
including type 1 cells (AEC1s), which line the alveolar airspace surface, and type 2 (AEC2s), which
secrete surfactant, self-renew, and give rise to AEC1s and development of honeycomb cysts. These
cysts are lined by "bronchiolized" epithelium, so-called because of expression of airway and
secretory cell markers such as p63, KRT5, KRT17, and MUC5B. The origin of the cells lining these
cysts is not understood, but have generally been thought to be the result of migration of airway
epithelial cells (basal and/or club), in a failed attempt at alveolar repair. Recent single-cell
RNA (sc-RNA) sequencing studies have uncovered widespread AEC2 and other epithelial cell
abnormalities in end-stage IPF tissue, such as intermediate/transitional cell states and ectopic
expression of genes associated with airway cells (such as KRT5+ AEC2s). Our lab has recently shown
that AEC2s are capable of reprogramming into KRT5+ basal-cell like cells in in vitro organoid
cultures. These suggest a new hypothesis that the bronchiolized epithelium lining honeycomb cysts
may actually be derived from reprogrammed AEC2s. This study seeks to characterize whether the
epithelial abnormalities present in end-stage IPF tissue are also present earlier in the IPF
disease course and to determine the role of TGFβ1 in regulating the aforementioned reprogramming.
Samples from patients undergoing surgical biopsy for the purpose of clinical diagnosis will be
analyzed by sc-RNA sequencing, to characterize the AEC2 and other epithelial cell populations and
reconstruct estimated lineages, especially surrounding the induction of the basal-cell
differentiation master- regulator Sox2 within AEC2s. These samples will be compared to normal and
end-stage IPF tissue, in order to test the hypothesis that AEC2 reprogramming is an early feature
of IPF. In addition, diagnostic biopsy samples will be obtained from patients who took
epigallocatechin gallate (EGCG) for two weeks prior to biopsy. EGCG is a mesenchyme-specific
inhibitor of TGFβ signaling under study in humans and will therefore allow us to examine the
hypothesis that AEC2 reprogramming abnormalities seen in diagnostic biopsies can be reversed by
TGFβ blockade. Finally, organoid co-cultures and precision-cut lung slices cultures will be used to
examine the contribution of important signaling pathways, such as TGFβ, Wnt, and Notch, in driving
AEC2 reprograming towards a SPC-/KRT5+ basal-cell like state. Knowledge of the mechanisms driving
AEC2 reprograming in IPF may provide fundamental insight into the cause of this disorder and
contribute to the development of targeted therapies for this incurable and frequently fatal
disease. This proposed project will be performed as part of the research phase of Pulmonary &
Critical Care Medicine Fellowship at UCSF and utilizes comprehensive institutional support and
resources, in order to prepare the applicant for an independent research career.
