Developing advanced cell culture models to study COVID19-associated pulmonary fibrosis

Patients with severe COVID-19-associated pneumonia are at risk to develop pulmonary fibrosis. To study the underlying mechanisms, we aim to develop advanced cell culture models that reliably reflect COVID-19-related profibrotic microenvironment. To identify key cellular players, we performed pilot immunohistochemistry analysis on lung tissue from COVID-19 patients with fibrosis collected during autopsy. Results revealed diffuse alveolar damage with macrophage infiltration, and myofibroblast accumulation with enriched collagen deposition surrounding the damaged alveoli. To mimic SARS-CoV-2 infection in alveoli, we infected human primary type II alveolar epithelial cells (AEC2) and found enhanced signaling of profibrotic cytokine transforming growth factor beta (TGFbeta) in some donors. To recreate the early fibrotic niche, an alveolar-macrophage-fibroblast (AMF) tri-culture model was established. After infecting AEC2 with SARS-CoV-2 in this AMF model, gene expression analysis provided evidence for fibroblast-to-myofibroblast transition. Furthermore, we found that overexpression of SARS-CoV-2 papain-like protease (PLpro) can promote TGFbeta signaling in HEK293T and A549 cells. After infecting AEC2 with SARS-CoV-2 PLpro lentivirus in the AMF model, we found signs of epithelial-to-mesenchymal transition and fibroblast-to myofibroblast transition. In future studies, we will use a detailed analysis of COVID-19-associated lung fibrosis with other types of lung fibrosis, to further refine COVID-19-related fibrosis models, including lung-on-chip models.

Rationale for evaluation of PLN-74809 treatment in participants with primary sclerosing cholangitis in Phase 2a study INTEGRIS-PSC

Background and aims: Transforming growth factor beta (TGF-beta) signalling is a key driver of liver fibrosis. In primary sclerosing cholangitis (PSC), integrins over-expressed on injured cholangiocytes (alpha-v/beta-6) and myofibroblasts (alpha-v/beta-1) regulate TGFbeta activity. PLN-74809 is an oral, once-daily, dual-selective inhibitor of integrins alpha-v/beta-6 and alpha-v/beta-1 in development for the treatment of PSC and idiopathic pulmonary fibrosis. It has shown favourable tolerability in over 280 healthy participants, reduced TGF-beta signalling and achieved high target engagement in human lungs. Pre-clinical evaluation of antifibrotic activity resulting from dual integrin inhibition was performed to support clinical evaluation. Method: PLN-74809 was administered orally for 6 weeks in BALBc. Mdr2-/- mice with established fibrosis. A tool alpha-v/beta-6 and alpha-v/beta-1 inhibitor compound, PLN-75068, was tested therapeutically in a diet-induced mouse model of biliary fibrosis using 3, 5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC). Hepatic collagen was quantified by hydroxyproline (OHP) and collagen proportionate area (CPA) and TGF-beta signalling by phosphorylated SMAD3 (pSMAD3) levels. An ex vivo study evaluated the effects of 2-day treatment with PLN-74809 on the expression of profibrotic genes, COL1A1 and COL1A2, in precision-cut liver slices (PCLivS) from tissue explants of participants with biliary fibrosis (n = 2 PSC;n = 2 primary biliary cholangitis [PBC]). A review of available blinded safety data from the enrolling Phase 2a study in participants with PSC was performed (NCT04480840). Results: PLN-74809 dose-dependently reduced OHP (up to ∼30%, p < 0.05), CPA (up to ∼50%, p < 0.05) and pSMAD3 (up to ∼40%, p < 0.001) in the BALBc.Mdr2-/- mouse model, as well as COL1A1 and COL1A2 gene expression (up to ∼30%, p = 0.0789) in PCLivS from tissue explants of participants with PSC and PBC. PLN-75068 reduced OHP (up to ∼20%, p < 0.05) in DDC-injured mice in a dose-dependent manner. PLN-74809waswell tolerated in participants with PSC. Most adverse events (AEs)were mild;nonewere severe. The most common AE was mild headache. One participant experienced serious AEs at least 20 days after the last dose of study drug, deemed not related by the investigator. One participant prematurely discontinued due to COVID-19. PLN-74809 pharmacokinetics in participants with PSC were consistent with those of healthy participants. Conclusion: Pharmacological inhibition of integrins alpha-v/beta-6 and alpha-v/beta-1 demonstrated antifibrotic activity in two models of biliary fibrosis and in PCLivS from participants with PSC or PBC. Available safety findings from participants with PSC enrolled in the ongoing Phase 2a INTEGRIS-PSC study, continue to support the favourable tolerability profile of PLN-74809.

TGF-β SIGNALING PATHWAY SHAPES ADAPTIVE IMMUNITY in EARLY SARS-CoV-2 INFECTION

Background: SARS-CoV-2 induces cytokine response dysregulation and immune dysfunction. What remains unclear is how cytokine signaling shapes immune responses during early SARS-CoV-2 infection when adaptive immunity is developing. Our goal is to identify immune pathways that shape the early development of adaptive immune responses in COVID-19 patients. We performed paired single-cell transcriptomic and epigenomic profiling at two time-points of early SARS-CoV-2 infection to determine immune signatures of acute infection and epigenetic drivers that underpin immune response dynamics. Methods: PBMC samples were collected from four moderate to severe COVID-19 patients at two early time-points (n = 3 for Week 1 and n = 3 for Week 2 after symptom onset, including 2 participants having paired blood sampling at both time points) and from two healthy controls (n = 2). Using paired scRNA-Seq and scATAC-Seq, we captured transcriptomic and epigenomic profiles in the same single cells to identify chromatin accessibility changes as a potential mechanism for the surge and decline of immune responses elicited during acute SARS-CoV-2 infection. Using bioinformatic approaches, we identified heterogeneous immune cell populations, modeled cell differentiation trajectories, determined dysregulated immune pathways through gene set enrichment analysis, and connected chromatin co-accessible landscapes. Results: We captured transcriptomic and epigenomic profiles of 43,726 single cells and identified paired transcriptional and epigenetic landscapes in six major immune cell types: CD4+ T cells, CD8+ T cells, B cells, dendritic cells, monocytes, and NK cells. We found that early SARS-CoV-2 infection induced a surge in IL-2, IL-6, IFN-α, IFN-γ, TNF-α, and NF-κB responses at Week 1 that declined at Week 2 in adaptive immune cells (CD4+ T, CD8+ T, and B cells). In contrast, TGF-β responses surged early at Week 1 and continued to increase at Week 2 in these cells. In B cells and plasmablasts, we found early surges of IGHA1 (encoding IgA heavy chain) and SOX4 (an essential transcription factor for B cell development) expressions that correlated with expression of SMAD-dependent TGF-β signaling pathway. Further, we found a notable increase in chromatin accessibility at the SMAD binding regulatory element 150 kb upstream of SOX4 in B cells of infected patients. Conclusion: Our data suggest a significant increase in TGF-β activity that instructs dynamic B cell-associated protective immunity during early SARS-CoV-2 infection.