Biliary atresia (BA) is a rapidly progressive perinatal inflammatory disease, resulting in liver failure. Hepatic Ly6CLo non-classical monocytes promote the resolution of perinatal liver inflammation during rhesus rotavirus-mediated (RRV) BA in mice. In this study, we aim to investigate the effects of inflammation on the transcription factor Nr4a1, a known regulator of non-classical monocytes. Nr4a1-GFP reporter mice were injected with PBS for control or RRV within 24 h of delivery to induce perinatal liver inflammation. GFP expression on myeloid immune populations in the liver and bone marrow (BM) was quantified 3 and 14 days after injection using flow cytometry. Statistical significance was determined using a student’s t-test and ANOVA, with a p-value < 0.05 for significance. Our results demonstrate that non-classical monocytes in the neonatal liver exhibit the highest mean fluorescence intensity (MFI) of Nr4a1 (Ly6CLo MFI 6344 vs. neutrophils 3611 p < 0.001; macrophages 2782; p < 0.001; and Ly6CHi classical monocytes 4485; p < 0.0002). During inflammation, hepatic Ly6CLo non-classical monocytes showed a significant increase in Nr4a1 expression intensity from 6344 to 7600 (p = 0.012), while Nr4a1 expression remained unchanged on the other myeloid populations. These findings highlight the potential of using Nr4a1 as a regulator of neonatal hepatic Ly6CLo non-classical monocytes to mitigate perinatal liver inflammation.

Perinatal hepatic inflammation can have devastating consequences. Monocytes play an important role in the initiation and resolution of inflammation, and their diverse functions can be attributed to specific cellular subsets: pro-inflammatory or classical monocytes (Ly6c^Hi) and pro-reparative or non-classical monocytes (Ly6c^Lo). We hypothesized that inherent differences in Ly6c^Hi classical monocytes and Ly6c^Lo non-classical monocytes determine susceptibility to perinatal hepatic inflammation in late gestation fetuses and neonates. We found an anti-inflammatory transcriptional profile expressed by Ly6c^Lo non-classical monocytes, and a physiologic abundance of these cells in the late gestation fetal liver. Unlike neonatal pups, late gestation fetuses proved to be resistant to rhesus rotavirus (RRV) mediated liver inflammation. Furthermore, neonatal pups were rendered resistant to RRV-mediated liver injury when Ly6c^Lo non-classical monocytes were expanded. Pharmacologic inhibition of Ly6c^Lo non-classical monocytes in this setting restored susceptibility to RRV-mediated disease. These data demonstrate that Ly6c^Lo monocytes promote resolution of perinatal liver inflammation in the late gestation fetus, where there is a physiologic expansion of non-classical monocytes, and in the neonatal liver upon experimental expansion of these cells. Therapeutic strategies directed towards enhancing Ly6c^Lo non-classical monocyte function may mitigate the detrimental effects of perinatal liver inflammation.

Pediatric hepatoblastoma is the most common primary liver cancer in infants and children. Studies of hepatoblastoma that focus exclusively on tumor cells demonstrate sparse somatic mutations and a common cell of origin, the hepatoblast, across patients. In contrast to the homogeneity these studies would suggest, hepatoblastoma tumors have a high degree of heterogeneity that can portend poor prognosis. In this study, we use single-cell transcriptomic techniques to analyze resected human pediatric hepatoblastoma specimens, and identify five hepatoblastoma tumor signatures that may account for the tumor heterogeneity observed in this disease. Notably, patient-derived hepatoblastoma spheroid cultures predict differential responses to treatment based on the transcriptomic signature of each tumor, suggesting a path forward for precision oncology for these tumors. In this work, we define hepatoblastoma tumor heterogeneity with single-cell resolution and demonstrate that patient-derived spheroids can be used to evaluate responses to chemotherapy.