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Many aspects of brain development, function, and repair depend on the interaction of neurons with brain immune cells, the microglia. By combining CLEM and SPIM microscopy, a recent study has challenged the current view that microglia can "eat" entire synapses, highlighting the incredible complexity of neuronal-microglial interactions in vivo.

In the liver, the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) regulates bile secretion and other functions at the apical membrane of biliary epithelial cells (i.e., cholangiocytes). CF-related liver disease is a major cause of death in patients with CF. CFTR dysfunction affects innate immune pathways, generating a para-inflammatory status in the liver and other epithelia. This study investigates the mechanisms linking CFTR to toll-like receptor 4 activity. We found that CFTR is associated with a multiprotein complex at the apical membrane of normal mouse cholangiocytes, with proteins that negatively control Rous sarcoma oncogene cellular homolog (Src) activity. In CFTR-defective cholangiocytes, Src tyrosine kinase self-activates and phosphorylates toll-like receptor 4, resulting in activation of nuclear factor kappa-light-chain-enhancer of activated B cells and increased proinflammatory cytokine production in response to endotoxins. This Src/nuclear factor kappa-light-chain-enhancer of activated B cells-dependent inflammatory process attracts inflammatory cells but also generates changes in the apical junctional complex and loss of epithelial barrier function. Inhibition of Src decreased the inflammatory response of CF cholangiocytes to lipopolysaccharide, rescued the junctional defect in vitro, and significantly attenuated endotoxin-induced biliary damage and inflammation in vivo (Cftr knockout mice). Conclusion: These findings reveal a novel function of CFTR as a regulator of toll-like receptor 4 responses and cell polarity in biliary epithelial cells; this mechanism is pathogenetic, as shown by the protective effects of Src inhibition in vivo, and may be a novel therapeutic target in CF-related liver disease and other inflammatory cholangiopathies. (Hepatology 2016;64:2118-2134).

Phagocytic immune cells such as microglia can engulf and process pathogens and dying cells with high efficiency while still maintaining their dynamic behavior and morphology. Effective intracellular processing of ingested cells is likely to be crucial for microglial function, but the underlying cellular mechanisms are poorly understood. Using both living fish embryos and mammalian macrophages, we show that processing depends on the shrinkage and packaging of phagosomes into a unique cellular compartment, the gastrosome, with distinct molecular and ultra-structural characteristics. Loss of the transporter Slc37a2 blocks phagosomal shrinkage, resulting in the expansion of the gastrosome and the dramatic bloating of the cell. This, in turn, affects the ability of microglia to phagocytose and migrate toward brain injuries. Thus, this work identifies a conserved crucial step in the phagocytic pathway of immune cells and provides a potential entry point for manipulating their behavior in development and disease.

Introduction: In polycystic liver diseases, defective PC2 leads to elevated production of cAMP, PKA-dependent activation of the ERK1/2 pathway, HIF1 -mediated VEGF production and stimulation of cyst growthand progression. Activation of the ERK/HIF1 /VEGF pathway in cholangiocytes is fundamental during repair from biliary damage. In this study we hypothesized that PC-2 levels can be modulated during biliary damage/repair. Results: PC2 protein expression was significantly reduced in livers from mice undergoing cholangiocyte damage (Mdr2-/--KO, BDL, treatment with dehydrocollidine–DDC). However, PC-2-gene expression was not reduced,suggesting that the decrease in PC2 was due to increaseddegradation. To understand if factors involved in biliary damage influence PC2 protein expression, mouse cholangiocytes were treated with pro-inflammatory cytokines, nitric oxide (NO) donors and ER stressors. Expression of PC2 protein was again significantly reduced, but not its gene expression. Noteworthy, downregulation of PC-2 leads to increased ERK1/2 phosphorylation, HIF1 transcription activity and secretion of VEGF. Expression of Herp and NEK, proteins that promote PC2 degradation via the proteasome complex was also increased. Pre-treatment with proteasome inhibitor restored the expression of PC2 in cells treated with cytokines but not in cells treated with NO donors or with ER stressors. In the latter conditions, PC2 degradation was inhibited by inhibition of PI3K, that blocks autophagosome andby blockade of lysosomes, suggesting a role for autophagy. Finally, treatment ofDDC-treatedmicewith the proteasome inhibitor bortezomib, significantly reduced the extent of the liver damage and restored PC2 expression. Conclusion: PC2 is modulated post-translationally by proinflammatory cytokines, ER-stressors and NO-donors and is reduced in mice with biliary damage. Down regulation of PC2 protein expression in cholangiocytes increases the ERK1/2/HIF1 /VEGFsecretion, thereby playing a pivotal role in the regulation of cholangiocyte response to biliary damage.Treatments able to restore PC2 expression may represent a new therapeutic approach in biliary diseases.

Polycystin-2 (PC2 or TRPPC2), a member of the transient receptor potential channel family, is a nonselective calcium channel. Mutations in PC2 are associated with polycystic liver diseases. PC2-defective cholangiocytes show increased production of cyclic adenosine monophosphate, protein kinase A–dependent activation of the extracellular signal–regulated kinase 1/2 (ERK1/2) pathway, hypoxia-inducible factor 1α (HIF-1α)–mediated vascular endothelial growth factor (VEGF) production, and stimulation of cyst growth and progression. Activation of the ERK/HIF-1α/VEGF pathway in cholangiocytes plays a key role during repair from biliary damage. We hypothesized that PC2 levels are modulated during biliary damage/repair, resulting in activation of the ERK/HIF-1α/VEGF pathway. PC2 protein expression, but not its gene expression, was significantly reduced in mouse livers with biliary damage (Mdr2–/– knockout, bile duct ligation, 3,5-diethoxycarbonyl-1,4-dihydrocollidine treatment). Treatment of cholangiocytes with proinflammatory cytokines, nitric oxide donors, and endoplasmic reticulum stressors increased ERK1/2 phosphorylation, HIF-1α transcriptional activity, secretion of VEGF, and VEGF receptor type 2 phosphorylation and down-regulated PC2 protein expression without affecting PC2 gene expression. Expression of homocysteine-responsive endoplasmic reticulum–resident ubiquitin-like domain member 1 protein and NEK, ubiquitin-like proteins that promote proteosomal PC2 degradation, was increased. Pretreatment with the proteasome inhibitor MG-132 restored the expression of PC2 in cells treated with cytokines but not in cells treated with nitric oxide donors or with endoplasmic reticulum stressors. In these conditions, PC2 degradation was instead inhibited by interfering with the autophagy pathway. Treatment of 3,5-diethoxycarbonyl-1,4-dihydrocollidine mice and of Mdr2–/– mice with the proteasome inhibitor bortezomib restored PC2 expression and significantly reduced the ductular reaction, fibrosis, and phosphorylated ERK1/2. Conclusion: In response to biliary damage, PC2 expression is modulated posttranslationally by the proteasome or the autophagy pathway, and PC2 down-regulation is associated with activation of ERK1/2 and an increase of HIF-1α-mediated VEGF secretion; treatments able to restore PC2 expression and to reduce ductular reaction and fibrosis may represent a new therapeutic approach in biliary diseases. (Hepatology 2015)