- Reviewing EditorPeter TontonozUniversity of California, Los Angeles, United States of America
- Senior EditorCarlos IsalesAugusta University, United States of America
Reviewer #1 (Public Review):
This manuscript explores how biliary epithelial cells respond to excess dietary lipids, an important area of research given the increasing prevalence of NAFLD. The authors utilize in vivo models complemented with cultured organoid systems. Interesting, E2F transcription factors appear important for BEC glycolytic activation and proliferation.
Much of the work utilizes the BEC-organoid model, which is complicated by the fact that liver cell organoid models often fail to maintain exclusive cell identity in culture. The method used by the authors (Broutier et al., 2016) can lead to organoids with a mixture of ductal and hepatocyte markers. It would be helpful for the authors to further demonstrate the cholangiocyte identity of the organoid cells.
The authors suggest that BECs form lipid droplets in vivo by detecting BODIPY immunofluorescence of liver cryosections. While confocal microscopy would ensure that the BODIPY fluorescence signal is within the same plane as the cell of interest, the authors use a virtual slide microscope that cannot exclude fluorescence from a different focal plane. The conclusion that BECs accumulate lipids does not seem to be fully supported by this analysis.
Several mouse experiments rely heavily on rare BEC proliferation events with the median proliferation event per bile duct being 0-1 cell. While the proliferative effect appears consistent across experiments, a more quantitative approach, such as performing Epcam+ BEC FACS and flow cytometry-based cell cycle analyses, would be helpful.
Finally, it is not yet clear how relevant the findings in this study are to ductular reaction, which is a non-specific histopathologic indicator of liver injury in the context of severe liver disease. In NAFLD, the ductular reaction is uncommon in benign steatosis, and if seen at all, occurs in the setting of substantial liver inflammation and fibrosis (Gadd et al., Hepatology 2014). The authors use a dietary model containing 60 kcal% fat, which causes adipose lipid accumulation as well as subsequent liver lipid accumulation. This diet does not cause overt inflammation or fibrosis that would represent experimental NASH, which typically requires the addition of cholesterol in dietary lipid NASH models (Farrell et al., Hepatology, 2019). While the E2F-driven proliferation may be important for physiologic bile duct function in the setting of obesity, the claim that E2Fs mediate DR initiation would require an additional pathophysiologic model or human data to demonstrate relevance. The authors could clarify this point in their discussion.
Reviewer #2 (Public Review):
The manuscript by Yildiz et al investigates the early response of BECs to high fatty acid treatment. To achieve this, they employ organoids derived from primary isolated BECs and treat them with a FA mix followed by viability studies and analysis of selected lipid metabolism genes, which are upregulated indicating an adjustment to lipid overload. Both organoids with lipid overload and BECs in mice exposed to a HFD show increased BEC proliferation, indicating BEC activation as seen in DR. Applying bulk RNA-sequencing analysis to sorted BECs from HFD mice identified four E2F transcription factors and target genes as upregulated. Functional analysis of knock-out mice showed a clear requirement for E2F1 in mediating HFD induced BEC proliferation. Given the known function of E2Fs the authors performed cell respiration and transcriptome analysis of organoids challenged with FA treatment and found a shift of BECs towards a glycolytic metabolism.
The study is overall well-constructed, including appropriate analysis. Likewise, the manuscript is written clearly and supported by high-quality figures. My major point is the lack of classification of the progression of DR, since the authors investigate the early stages of DR associated with lipid overload reminiscent of stages preceding late NAFLD fibrosis. How are early stages distinguished from later stages in this study? Molecularly and/or morphologically? While the presented data are very suggestive, a more substantial description would support the findings and resulting claims.