BiliQML: A supervised machine-learning model to quantify biliary forms from digitized whole-slide liver histopathological images.

The progress of research focused on cholangiocytes and the biliary tree during development and following injury is hindered by limited available quantitative methodologies. Current techniques include two-dimensional standard histological cell-counting approaches, which are rapidly performed error-prone and lack architectural context; or three-dimensional analysis of the biliary tree in opacified livers, which introduce technical issues along with minimal quantitation. The present study aims to fill these quantitative gaps with a supervised machine learning model (BiliQML) able to quantify biliary forms in the liver of anti-Keratin 19 antibody-stained whole slide images. Training utilized 5,019 researcher-labeled biliary forms, which following feature selection, and algorithm optimization, generated an F-score of 0.87. Application of BiliQML on seven separate cholangiopathy models; genetic (Afp-CRE;Pkd1l1null/Fl, Alb-CRE;Rbp-jkfl/fl, Albumin-CRE; ROSANICD), surgical (bile duct ligation), toxicological (3,5-diethoxycarbonyl-1,4-dihydrocollidine), and therapeutic (Cyp2c70-/- with ileal bile acid transporter inhibition), allowed for a means to validate the capabilities, and utility of this platform. The results from BiliQML quantification revealed biological and pathological differences across these seven diverse models indicate a highly sensitive, robust, and scalable methodology for the quantification of distinct biliary forms. BiliQML is the first comprehensive machine-learning platform for biliary form analysis, adding much needed morphologic context to standard immunofluorescence - based histology, and provides clinical and basic-science researchers a novel tool for the characterization of cholangiopathies.

Active enterohepatic cycling is not required for the choleretic actions of 24-norUrsodeoxycholic acid in mice.

The pronounced choleretic properties of 24-norUrsodeoxycholic acid (norUDCA) to induce bicarbonate-rich bile secretion have been attributed to its ability to undergo cholehepatic shunting. The goal of this study was to identify the mechanisms underlying the choleretic actions of norUDCA and the role of the bile acid transporters. Here, we show that the apical sodium-dependent bile acid transporter (ASBT), organic solute transporter-α (OSTα), and organic anion transporting polypeptide 1a/1b (OATP1a/1b) transporters are dispensable for the norUDCA stimulation of bile flow and biliary bicarbonate secretion. Chloride channels in biliary epithelial cells provide the driving force for biliary secretion. In mouse large cholangiocytes, norUDCA potently stimulated chloride currents that were blocked by siRNA silencing and pharmacological inhibition of calcium-activated chloride channel transmembrane member 16A (TMEM16A) but unaffected by ASBT inhibition. In agreement, blocking intestinal bile acid reabsorption by coadministration of an ASBT inhibitor or bile acid sequestrant did not impact norUDCA stimulation of bile flow in WT mice. The results indicate that these major bile acid transporters are not directly involved in the absorption, cholehepatic shunting, or choleretic actions of norUDCA. Additionally, the findings support further investigation of the therapeutic synergy between norUDCA and ASBT inhibitors or bile acid sequestrants for cholestatic liver disease.

Ileal bile acid transporter inhibition in Cyp2c70 KO mice ameliorates cholestatic liver injury.

Cyp2c70 is the liver enzyme in rodents responsible for synthesis of the primary 6-hydroxylated muricholate bile acid (BA) species. Cyp2c70 KO mice are devoid of protective, hydrophilic muricholic acids, leading to a more human-like BA composition and subsequent cholestatic liver injury. Pharmacological inhibition of the ileal BA transporter (IBAT) has been shown to be therapeutic in cholestatic models. Here, we aimed to determine if IBAT inhibition with SC-435 is protective in Cyp2c70 KO mice. As compared to WT mice, we found male and female Cyp2c70 KO mice exhibited increased levels of serum liver injury markers, and our evaluation of liver histology revealed increased hepatic inflammation, macrophage infiltration, and biliary cell proliferation. We demonstrate serum and histologic markers of liver damage were markedly reduced with SC-435 treatment. Additionally, we show hepatic gene expression in pathways related to immune cell activation and inflammation were significantly upregulated in Cyp2c70 KO mice and reduced to levels indistinguishable from WT with IBAT inhibition. In Cyp2c70 KO mice, the liver BA content was significantly increased, enriched in chenodeoxycholic acid, and more hydrophobic, exhibiting a hydrophobicity index value and red blood cell lysis properties similar to human liver BAs. Furthermore, we determined IBAT inhibition reduced the total hepatic BA levels but did not affect overall hydrophobicity of the liver BAs. These findings suggest that there may be a threshold in the liver for pathological accretion of hydrophobic BAs and reducing hepatic BA accumulation can be sufficient to alleviate liver injury, independent of BA pool hydrophobicity.

Metabolic effects of the schizophrenia-associated 3q29 deletion.

The 1.6 Mb 3q29 deletion is associated with developmental and psychiatric phenotypes, including a 40-fold increased risk for schizophrenia. Reduced birth weight and a high prevalence of feeding disorders in patients suggest underlying metabolic dysregulation. We investigated 3q29 deletion-induced metabolic changes using our previously generated heterozygous B6.Del16+/Bdh1-Tfrc mouse model. Animals were provided either standard chow (STD) or high-fat diet (HFD). Growth curves were performed on HFD mice to assess weight change (n = 30-50/group). Indirect calorimetry and untargeted metabolomics were performed on STD and HFD mice to evaluate metabolic phenotypes (n = 8-14/group). A behavioral battery was performed on STD and HFD mice to assess behavior change after the HFD challenge (n = 5-13/group). We found that B6.Del16+/Bdh1-Tfrc animals preferentially use dietary lipids as an energy source. Untargeted metabolomics of liver tissue showed a strong sex-dependent effect of the 3q29 deletion on fat metabolism. A HFD partially rescued the 3q29 deletion-associated weight deficit in females, but not males. Untargeted metabolomics of liver tissue after HFD revealed persistent fat metabolism alterations in females. The HFD did not affect B6.Del16+/Bdh1-Tfrc behavioral phenotypes, suggesting that 3q29 deletion-associated metabolic and behavioral outcomes are uncoupled. Our data suggest that dietary interventions to improve weight phenotypes in 3q29 deletion syndrome patients are unlikely to exacerbate behavioral manifestations. Our study also highlights the importance of assessing sex in metabolic studies and suggests that mechanisms underlying 3q29 deletion-associated metabolic phenotypes are sex-specific.

Assessment of the Abbott BinaxNOW SARS-CoV-2 rapid antigen test against viral variants of concern.

As the emergence of SARS-CoV-2 variants brings the global pandemic to new levels, the performance of current rapid antigen tests against variants of concern and interest (VOC/I) is of significant public health concern. Here, we report assessment of the Abbot BinaxNOW COVID-19 Antigen Self-Test. Using genetically sequenced remnant clinical samples collected from individuals positive for SARS-CoV-2, we assessed the performance of BinaxNOW against the variants that currently pose public health threats. We measured the limit of detection of BinaxNOW against various VOC/I in a blinded manner. BinaxNOW successfully detected the Omicron (B.1.1.529), Mu (B.1.621), Delta (B.1.617.2), Lambda (C.37), Gamma (P.1), Alpha (B.1.1.7), Beta (B.1.351), Eta (B.1.525), and P.2 variants and at low viral concentrations. BinaxNOW also detected the Omicron variant in individual remnant clinical samples. Overall, these data indicate that this inexpensive and simple-to-use, FDA-authorized and broadly distributed rapid test can reliably detect Omicron, Delta, and other VOC/I.

Organic Solute Transporter α-ß Protects Ileal Enterocytes From Bile Acid-Induced Injury.

BACKGROUND & AIMS: Ileal bile acid absorption is mediated by uptake via the apical sodium-dependent bile acid transporter (ASBT), and export via the basolateral heteromeric organic solute transporter α-ß (OSTα-OSTß). In this study, we investigated the cytotoxic effects of enterocyte bile acid stasis in Ostα-/- mice, including the temporal relationship between intestinal injury and initiation of the enterohepatic circulation of bile acids. METHODS: Ileal tissue morphometry, histology, markers of cell proliferation, gene, and protein expression were analyzed in male and female wild-type and Ostα-/- mice at postnatal days 5, 10, 15, 20, and 30. Ostα-/-Asbt-/- mice were generated and analyzed. Bile acid activation of intestinal Nrf2-activated pathways was investigated in Drosophila. RESULTS: As early as day 5, Ostα-/- mice showed significantly increased ileal weight per length, decreased villus height, and increased epithelial cell proliferation. This correlated with premature expression of the Asbt and induction of bile acid-activated farnesoid X receptor target genes in neonatal Ostα-/- mice. Expression of reduced nicotinamide adenine dinucleotide phosphate oxidase-1 and Nrf2-anti-oxidant responsive genes were increased significantly in neonatal Ostα-/- mice at these postnatal time points. Bile acids also activated Nrf2 in Drosophila enterocytes and enterocyte-specific knockdown of Nrf2 increased sensitivity of flies to bile acid-induced toxicity. Inactivation of the Asbt prevented the changes in ileal morphology and induction of anti-oxidant response genes in Ostα-/- mice. CONCLUSIONS: Early in postnatal development, loss of Ostα leads to bile acid accumulation, oxidative stress, and a restitution response in ileum. In addition to its essential role in maintaining bile acid homeostasis, Ostα-Ostß functions to protect the ileal epithelium against bile acid-induced injury. NCBI Gene Expression Omnibus: GSE99579.

CD163 interacts with TWEAK to regulate tissue regeneration after ischaemic injury.

Macrophages are an essential component of the immune response to ischaemic injury and play an important role in promoting inflammation and its resolution, which is necessary for tissue repair. The type I transmembrane glycoprotein CD163 is exclusively expressed on macrophages, where it acts as a receptor for haemoglobin:haptoglobin complexes. An extracellular portion of CD163 circulates in the blood as a soluble protein, for which no physiological function has so far been described. Here we show that during ischaemia, soluble CD163 functions as a decoy receptor for TWEAK, a secreted pro-inflammatory cytokine of the tumour necrosis factor family, to regulate TWEAK-induced activation of canonical nuclear factor-κB (NF-κB) and Notch signalling necessary for myogenic progenitor cell proliferation. Mice with deletion of CD163 have transiently elevated levels of TWEAK, which stimulate muscle satellite cell proliferation and tissue regeneration in their ischaemic and non-ischaemic limbs. These results reveal a role for soluble CD163 in regulating muscle regeneration after ischaemic injury.

Hepcidin-ferroportin axis controls toll-like receptor 4 dependent macrophage inflammatory responses in human atherosclerotic plaques.

OBJECTIVES: Toll-like Receptor 4 (TLR4) is implicated in modulating inflammatory cytokines though its role in atherosclerosis remains uncertain. We have recently described a non-foam cell macrophage phenotype driven by ingestion of hemoglobin:haptoglobin complexes (HH), via the scavenger receptor CD163, characterized by reduced inflammatory cytokine production. In this study, we examined the role of iron metabolism in modulating TLR4 signaling in these cells. METHODS AND RESULTS: Areas in human atherosclerotic plaque with non-foam cell, CD163 positive macrophages demonstrated reduced expression of tumor necrosis factor alpha (TNF-α) and interferon-beta (INF-ß) compared to foam cells. Human macrophages differentiated in hemoglobin:haptoglobin (HH) complexes expressed the CD163 positive non-foam cell phenotype and demonstrated significantly less TNF-α and INF-ß compared to control macrophages when exposed to oxidized LDL (oxLDL) or lipopolysaccharide (LPS). LPS stimulated expression of TNF-α and INF-ß could be restored in HH macrophages by pretreatment with hepcidin, an endogenous suppressor of ferroportin1 (FPN), or by genetic suppression of FPN in macrophages derived from myeloid specific FPN knockout mice. LPS stimulated control macrophages demonstrated increase in TLR4 trafficking to lipid rafts; this response was suppressed in HH macrophages but was restored upon pretreatment with hepcidin. Using a pharmacologic hepcidin suppressor, we observed a decrease in cytokine expression and TLR4-lipid raft trafficking in LPS-stimulated in a murine macrophage model. CONCLUSION: TLR4 dependent macrophage signaling is controlled via hepcidin-ferroportin1 axis by influencing TLR4-lipid raft interactions. Pharmacologic manipulation of iron metabolism may represent a promising approach to limiting TLR4-mediated inflammatory responses.

Pharmacological suppression of hepcidin increases macrophage cholesterol efflux and reduces foam cell formation and atherosclerosis.

OBJECTIVE: We recently reported that lowering of macrophage free intracellular iron increases expression of cholesterol efflux transporters ABCA1 and ABCG1 by reducing generation of reactive oxygen species. In this study, we explored whether reducing macrophage intracellular iron levels via pharmacological suppression of hepcidin can increase macrophage-specific expression of cholesterol efflux transporters and reduce atherosclerosis. METHODS AND RESULTS: To suppress hepcidin, increase expression of the iron exporter ferroportin, and reduce macrophage intracellular iron, we used a small molecule inhibitor of bone morphogenetic protein (BMP) signaling, LDN 193189 (LDN). LDN (10 mg/kg IP b.i.d.) was administered to mice, and its effects on atherosclerosis, intracellular iron, oxidative stress, lipid efflux, and foam cell formation were measured in plaques and peritoneal macrophages. Long-term LDN administration to apolipoprotein E-/- mice increased ABCA1 immunoreactivity within intraplaque macrophages by 3.7-fold (n=8; P=0.03), reduced Oil Red O-positive lipid area by 50% (n=8; P=0.02), and decreased total plaque area by 43% (n=8; P=0.001). LDN suppressed liver hepcidin transcription and increased macrophage ferroportin, lowering intracellular iron and hydrogen peroxide production. LDN treatment increased macrophage ABCA1 and ABCG1 expression, significantly raised cholesterol efflux to ApoA-1, and decreased foam cell formation. All preceding LDN-induced effects on cholesterol efflux were reversed by exogenous hepcidin administration, suggesting modulation of intracellular iron levels within macrophages as the mechanism by which LDN triggers these effects. CONCLUSIONS: These data suggest that pharmacological manipulation of iron homeostasis may be a promising target to increase macrophage reverse cholesterol transport and limit atherosclerosis.