In the liver, CFTR regulates bile secretion and other functions at the apical membrane of biliary epithelial cells (i.e cholangiocytes). CF-related liver disease (CFLD) 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 TLR4 activity. We found that CFTR is associated in a multi-protein complex at the apical membrane of normal mouse cholangiocytes, with proteins that negatively control Src activity. In CFTR-defective cholangiocytes, Src tyrosine kinase self-activates and phosphorylates TLR4, resulting in activation of NF-κB, and increased pro-inflammatory cytokines production in response to endotoxins. This Src/NF-κB-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 LPS, rescued the junctional defect in-vitro and significantly attenuated endotoxin-induced biliary damage and inflammation in vivo (Cftr-KO mice). Conclusion Our findings reveal a novel function of CFTR as regulator of TLR4 responses and cell polarity in biliary epithelial cells. This mechanism is pathogenetic, as shown by the protective effects of Src inhibition in vivo and maybe a novel therapeutic target in CFLD and other inflammatory cholangiopathies.

Cystic fibrosis-associated liver disease (CFLD) is a chronic cholangiopathy that negatively affects the quality of life of cystic fibrosis patients. In addition to reducing biliary chloride and bicarbonate secretion, up-regulation of TLR4/NF-kB-dependent immune mechanisms plays a major role in the pathogenesis of CFLD, and may represent a therapeutic target. Nuclear receptors (NRs) are transcription factors that regulate several intracellular functions. Some NRs, including peroxisome proliferator-activated receptor-γ (PPAR-γ), may counter-regulate inflammation in a tissue-specific manner. In this study, we explored the anti-inflammatory effect of PPAR-γ stimulation in vivo in Cftr-KO mice exposed to DSS, and in vitro in primary cholangiocytes isolated from wild type and from Cftr-KO mice exposed to LPS. We found that in CFTR-defective biliary epithelium, expression of PPAR-γ is increased, but does not result in increased receptor activity because the availability of bioactive ligands is reduced. Exogenous administration of synthetic agonists of PPAR-γ (pioglitazone and rosiglitazone) upregulates PPAR-γ-dependent genes, while inhibiting the activation of NF-kB and the secretion of proinflammatory cytokines (LIX, MCP-1, MIP-2, G-CSF, KC) in response to LPS. PPAR-γ agonists modulate NF-kB-dependent inflammation by upregulating IkBα, a negative regulator of NF-kB. Stimulation of PPAR-γ in vivo (rosiglitazone) significantly attenuates biliary damage and inflammation in Cftr-KO mice exposed to a DSS-induced portal endotoxemia. Conclusion These studies unravel a novel function of PPAR-γ in controlling biliary epithelium inflammation and suggest that impaired activation of PPAR-γ contributes to the chronic inflammatory state of CFTR-defective cholangiocytes.

Graphical Abstract Highlights d Neuronal processing by microglia depends on Slc37a2mediated phagosomal shrinkage d Phagosomes fuse with the gastrosome, a unique compartment in the phagocytic pathway d Loss of Slc37a2 blocks phagosomal shrinkage, resulting in gastrosomal expansion d Gastrosomal expansion affects microglia phagocytosis and migration toward injuries SUMMARYPhagocytic 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.

Background/Aims Genetic defects in Polycystins -1 or -2 (PC1 or PC2) cause polycystic liver disease associated with ADPKD (PLD-ADPKD). Progressive cyst growth is sustained by a cAMP-dependent Ras/ERK/HIFα pathway leading to increased autocrine/paracrine VEGF-A signalling. In PC2-defective cholangiocytes, store-operated Ca2+ entry (SOCE), intracellular and endoplasmic reticulum [Ca2+]ER levels are reduced, while cAMP production in response to [Ca2+]ER depletion is increased. We hypothesized that in PC2-defective cells, in response to [Ca2+]ER depletion, the Ca2+-inhibitable adenylyl-cyclases AC5 or AC6 are activated by the ER chaperon STIM1 resulting in cAMP/PKA-dependent Ras/ERK/HIFα pathway activation. Methods/Results PC2/AC6 conditional double-KO mice were generated (Pkd2/AC6-KO) and compared to Pkd2-KO mice, however no decrease in liver cyst was found and cellular cAMP generated by [Ca2+]ER depletion decreased only by 12%. Conversely, in PC2-defective cells, inhibition of AC5 with siRNA or SQ22,536 and NKY80 significantly reduced [Ca2+]ER depletion-stimulated cAMP production, and pERK1/2 expression and VEGF-A secretion. AC5 inhibitors significantly reduced also growth of biliary organoids derived from Pkd2-KO and Pkd2/AC6-KO mice. Consistent with these data, in vivo treatment with SQ22,536 significantly reduced liver cystic area and cell proliferation in PC2-defective mice. Confocal imaging and proximity ligation assay demonstrated that in PC2-defective cells, after [Ca2+]ER depletion, STIM1 interacts with AC5 but not with Orai1, the Ca2+ channel that mediates SOCE. Conclusion in PC2-defective cells, in response to [Ca2+]ER depletion, activation of AC5 results in stimulation of cAMP/ERK1–2 signalling, VEGF production and cyst growth. As shown by in vivo experiments this mechanism is of pathophysiological relevance and may represent a novel therapeutic target.

Polycystin-2 (PC2 /TRPP2), a member of the transient receptor potential channels (TRP) family, is a non-selective calcium channel. Mutations in PC2/TRPP2 are associated with Polycystic Liver Diseases. PC2-defective cholangiocytes shows increased production of cAMP, PKA-dependent activation of the ERK1/2 pathway, HIF1α-mediated VEGF production, and stimulation of cyst growth and progression. Activation of the ERK/HIF1α/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/HIF1α/VEGF pathway. Results PC2 protein expression, but not its gene expression, was significantly reduced in mouse livers with biliary damage (Mdr2−/−-KO, bile duct ligation, DDC-treatment). Treatment of colangiocytes with pro-inflammatory cytokines, nitric oxide (NO) donors and ER stressors), increased ERK1/2 phosphorylation, HIF1α transcriptional activity, secretion of VEGF, VEGFR2 phosphorylation and downregulated PC2 protein expression without affecting PC2 gene expression. Expression of Herp and NEK, ubiquitin-like proteins that promote proteosomal PC2 degradation was increased. Pre-treatment with the proteasome inhibitor MG-132 restored the expression of PC2 in cells treated with cytokines but not in cells treated with NO donors or with ER stressors. In these conditions, PC2 degradation was instead inhibited by interfering with the autophagy pathway. Treatment of DDC-mice and of Mdr2−/−-mice with the proteasome inhibitor bortezomib, restored PC2 expression and significantly reduced the ductular reaction, fibrosis and p-ERK1/2. In conclusion, in response to biliary damage, PC2 expression is modulated post-translationally by the proteasome or the autophagy pathways. PC2-dowregulation is associated with activation of ERK1/2 and increase of HIF1α-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.

During brain development, many newborn neurons undergo apoptosis and are engulfed by microglia, the tissue-resident phagocytes of the brain, in a process known as efferocytosis. A hallmark of microglia is their highly branched morphology characterized by the presence of numerous dynamic extensions that these cells use for scanning the brain parenchyma and engulfing unwanted material. The mechanisms driving branch formation and apoptotic cell engulfment in microglia are unclear. By taking a live-imaging approach in zebrafish, we show that while microglia generate multiple microtubule-based branches, they only successfully engulf one apoptotic neuron at a time. Further investigation into the mechanism underlying this sequential engulfment revealed that targeted migration of the centrosome into one branch is predictive of phagosome formation and polarized vesicular trafficking. Moreover, experimentally doubling centrosomal numbers in microglia increases the rate of engulfment and even allows microglia to remove two neurons simultaneously, providing direct supporting evidence for a model where centrosomal migration is a rate-limiting step in branch-mediated efferocytosis. Conversely, light-mediated depolymerization of microtubules causes microglia to lose their typical branched morphology and switch to an alternative mode of engulfment, characterized by directed migration towards target neurons, revealing unexpected plasticity in their phagocytic ability. Finally, building on work focusing on the establishment of the immunological synapse, we identified a conserved signalling pathway underlying centrosomal movement in engulfing microglia.

No abstract

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.