Ferret Lung Transplantation Models Differential Lymphoid Aggregate Morphology Between Restrictive and Obstructive Forms of Chronic Lung Allograft Dysfunction

Abstract

Background. Long-term survival after lung transplantation remains limited by chronic lung allograft dysfunction (CLAD). CLAD has 2 histologic phenotypes, namely obliterative bronchiolitis (OB) and restrictive alveolar fibroelastosis (AFE), which have distinct clinical presentations, pathologies, and outcomes. Understanding of OB versus AFE pathogenesis would improve with better animal models. Methods. We utilized a ferret orthotopic single-lung transplantation model to characterize allograft fibrosis as a histologic measure of CLAD. Native lobes and “No CLAD” allografts lacking aberrant histology were used as controls. We used morphometric analysis to evaluate the size and abundance of B-cell aggregates and tertiary lymphoid organs (TLOs) and their cell composition. Quantitative RNA expression of 47 target genes was performed simultaneously using a custom QuantiGene Plex Assay. Results. Ferret lung allografts develop the full spectrum of human CLAD histology including OB and AFE subtypes. While both OB and AFE allografts developed TLOs, TLO size and number were greater with AFE histology. More activated germinal center cells marked by B-cell lymphoma 6 Transcription Repressor, (B-cell lymphoma 6) expression and fewer cells expressing forkhead box P3 correlated with AFE, congruent with greater diffuse immunoglobulin, plasma cell abundance, and complement 4d staining. Furthermore, forkhead box P3 RNA induction was significant in OB allografts specifically. RNA expression changes were seen in native lobes of animals with AFE but not OB when compared with No CLAD native lobes. Conclusions. The orthotopic ferret single-lung transplant model provides unique opportunities to better understand factors that dispose allografts to OB versus AFE. This will help develop potential immunomodulatory therapies and antifibrotic approaches for lung transplant patients.

Krt14 and Krt15 differentially regulate regenerative properties and differentiation potential of airway basal cells

“…We show that Krt14 and Krt15 expression in surface airway BCs functionally impacts proliferative potential and differentiation. Since Krt14 is upregulated in the SAE after injury, it likely demarcates transit-amplifying cells (8,(10)(11)(12)(13)(14). Transit-amplifying cells have a limited capacity to replicate and are primed to differentiate after a limited number of divisions (29).…”

“…Our group and others observed that Krt14 + cell abundance increases on the airway surface in multiple lung pathologies, such as in idiopathic pulmonary fibrosis, BO occurring after lung transplantation, restrictive allograft syndrome (RAS) after lung transplantation, and in injury models like SO 2 and Cl 2 gas exposure, and injuries caused by naphthalene and polidocanol exposure ( 8 , 10 – 14 ). It is unclear, however, whether Krt14 appearance in the SAE reflects migration and expansion of glandular progenitors that express Krt14 at baseline or a change within surface airway BCs to upregulate Krt14 expression.…”

“…Upregulation of Krt14 expression in the SAE has been reported in multiple lung pathologies and injury models (8,(10)(11)(12)(13)(14). However, considering that keratins are obligate heterodimers and Krt5 (type II) is expressed at homeostasis, it remains unclear which other type I keratin serves as a heterodimerization partner to Krt5 at homeostasis in the absence of Krt14.…”

“…Krt15 expression in basal cells of uninjured airways has been suggested by scRNA-seq, while Krt14 expression is infrequent on the surface of homeostatic airways, but is abundant in submucosal glands (SMG) -a major secretory compartment and a niche for reserve airway stem cells (8,9). Our group and others observed that Krt14+ cell abundance increases on the airway surface in multiple lung pathologies, such as in Idiopathic Pulmonary Fibrosis (IPF), BO following lung transplantation, Restrictive Allograft Syndrome (RAS) following lung transplantation and in injury models like SO2 and Cl2 gas exposure, naphthalene and polidocanol injuries (8,(10)(11)(12)(13)(14). It is, however, unclear whether Krt14 appearance in the SAE reflects migration and expansion of glandular progenitors that express Krt14 at baseline or a change within surface airway basal cells to upregulate Krt14 expression.…”

The Ferret Era of Experimental Lung Transplantation Is Upon Us

“…Based on the novel work by Lynch et al 15 at the University of Iowa, it becomes clear that gaining an understanding of the underlying immunologic mechanisms of CLAD in the ferrets can lay the groundwork for improving both the treatment and knowledge of this disease. Nevertheless, challenges still exist in making this model the "mainstream workhorse" in our field.…”