That cholestatic conditions are accompanied by an enhanced susceptibility to bacterial infection in human and animal models is a known phenomenon. This correlates with the observation that bile acids have suppressive effects on cells of innate and adaptive immunity. The present study provides evidence that in human macrophages, bile acids inhibit the LPS-induced expression of proinflammatory cytokines without affecting the expression of the anti-inflammatory cytokine IL-10. This results in a macrophage phenotype that is characterized by an increased IL-10/IL-12 ratio. Correspondingly, bile acids suppress basal phagocytic activity of human macrophages. These effects of bile acids can be mimicked by cAMP, which is presumably induced TGR5-dependently. The data provided further suggest that in primary human macrophages, modulation of the macrophage response toward LPS by bile acids involves activation of CREB, disturbed nuclear translocation of NF-κB, and PKA-dependent enhancement of LPS-induced cFos expression. The increase in cFos expression is paralleled by an enhanced formation of a protein complex comprising cFos and the p65 subunit of NF-κB. In summary, the data provided suggest that in human macrophages, bile acids induce an anti-inflammatory phenotype characterized by an increased IL-10/IL-12 ratio via activation of PKA and thereby, prevent their activation as classically activated macrophages. This bile acid-induced modulation of macrophage function may also be responsible for the experimentally and clinically observed anti-inflammatory and immunosuppressive effects of bile acids.

Cholestasis is caused by autoimmune reactions, drug-induced hepatotoxicity, viral infections of the liver and the obstruction of bile ducts by tumours or gallstones. Cholestatic conditions are associated with impaired innate and adaptive immunity, including alterations of the cellular functions of monocytes, macrophages, NK cells and T-cells. Bile acids act as signalling molecules, affecting lipopolysaccharide (LPS)-induced cytokine expression in primary human macrophages. The present manuscript investigates the impact of bile acids, such as taurolithocholic acid (TLC), on the transcriptome of human macrophages in the presence or absence of LPS. While TLC itself has almost no effect on gene expression under control conditions, this compound modulates the expression of 202 out of 865 transcripts in the presence of LPS. Interestingly, pathway analysis revealed that TLC specifically supressed the expression of genes involved in mediating pro-inflammatory effects, phagocytosis, interactions with pathogens and autophagy as well as the recruitment of immune cells, such as NK cells, neutrophils and T cells. These data indicate a broad influence of bile acids on inflammatory responses and immune functions in macrophages. These findings may contribute to the clinical observation that patients with cholestasis present a lack of response to bacterial or viral infections.

horseradish peroxidase; HSC, hepatic stellate cells; IL6, interleukin-6; IMDM, Iscove's Modified Dulbecco's Medium; ITGA5, integrin α 5 ; ITGB1, integrin β 1 ; ITS, insulin-selenitetransferrin; LAMA2, laminin α2; LAMA4, laminin α4; LAMA5, laminin α5; LAMB1, laminin β1; LAMB2, laminin β2; LAMC1, laminin γ1; LN-211, laminin-211; LN-521, laminin-521; LRAT, lecithin-retinol acyltransferase; MMP13, metalloproteinase 13; NGF, nerve growth factor; NID, nidogen; PBS, phosphate buffered saline; PCA, principal component analysis; PEG, polyethylene glycol; PPARγ, peroxisome proliferator-activated receptor γ; qPCR, quantitative polymerase chain reaction; SASP, senescence-associated secretory phenotype; SEC, sinusoidal endothelial cells; SEM, standard error of the mean; SPARCL1, secreted protein acidic and rich in cysteines-like 1; TGFb, transforming growth factor β; TLN, talin; TNFR, tumor necrosis factor receptor; VCL, vinculin; α-SMA, α-smooth muscle actin. AbstractHepatic blood flow and sinusoidal endothelial fenestration decrease during aging.Consequently, fluid mechanical forces are reduced in the space of Disse where hepatic stellate cells (HSC) have their niche. We provide evidence that integrin α 5 /β 1 is an important mechanosensor in HSC involved in shear stress-induced release of hepatocyte growth factor (HGF), an essential inductor of liver regeneration which is impaired during aging. The expression of the integrin subunits α 5 and β 1 decreases in liver and HSC from aged rats. CRISPR/Cas9-mediated integrin α 5 and β 1 knockouts in isolated HSC lead to lowered HGF release and impaired cellular adhesion. Fluid mechanical forces increase integrin α 5 and laminin gene expression whereas integrin β 1 remains unaffected. In the aged liver, laminin β2 and γ1 protein chains as components of laminin-521 are lowered. The integrin α 5 knockout in HSC reduces laminin expression via mechanosensory mechanisms. Culture of HSC on nanostructured surfaces functionalized with laminin-521 enhances Hgf expression in HSC, demonstrating that these ECM proteins are critically involved in HSC function. During aging, HSC acquire a senescence-associated secretory phenotype and lower their growth factor expression essential for tissue repair. Our findings suggest that impaired mechanosensing via integrin α 5 /β 1 in HSC contributes to age-related reduction of ECM and HGF release that could affect liver regeneration. K E Y W O R D S aging, hepatic stellate cells, integrins, laminins, mechanobiology S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section.

Impaired proinsulin-to-insulin processing in pancreatic β-cells is a key defective step in both type 1 diabetes and type 2 diabetes (T2D) (refs. 1,2), but the mechanisms involved remain to be defined. Altered metabolism of sphingolipids (SLs) has been linked to development of obesity, type 1 diabetes and T2D (refs. 3–8); nonetheless, the role of specific SL species in β-cell function and demise is unclear. Here we define the lipid signature of T2D-associated β-cell failure, including an imbalance of specific very-long-chain SLs and long-chain SLs. β-cell-specific ablation of CerS2, the enzyme necessary for generation of very-long-chain SLs, selectively reduces insulin content, impairs insulin secretion and disturbs systemic glucose tolerance in multiple complementary models. In contrast, ablation of long-chain-SL-synthesizing enzymes has no effect on insulin content. By quantitatively defining the SL–protein interactome, we reveal that CerS2 ablation affects SL binding to several endoplasmic reticulum–Golgi transport proteins, including Tmed2, which we define as an endogenous regulator of the essential proinsulin processing enzyme Pcsk1. Our study uncovers roles for specific SL subtypes and SL-binding proteins in β-cell function and T2D-associated β-cell failure.

Liver diseases represent a significant global health burden, necessitating the development of reliable biomarkers for early detection, prognosis, and therapeutic monitoring. Extracellular vesicles (EVs) have emerged as promising candidates for liver disease biomarkers due to their unique cargo composition, stability, and accessibility in various biological fluids. In this study, we present an optimized workflow for the identification of EVs-based biomarkers in liver disease, encompassing EVs isolation, characterization, cargo analysis, and biomarker validation. Here we show that the levels of microRNAs miR-10a, miR-21, miR-142-3p, miR-150, and miR-223 were different among EVs isolated from patients with nonalcoholic fatty liver disease and autoimmune hepatitis. In addition, IL2, IL8, and interferon-gamma were found to be increased in EVs isolated from patients with cholangiocarcinoma compared with healthy controls. By implementing this optimized workflow, researchers and clinicians can improve the identification and utilization of EVs-based biomarkers, ultimately enhancing liver disease diagnosis, prognosis, and personalized treatment strategies.