Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Receptor Deficiency Promotes the Ductular Reaction, Macrophage Accumulation, and Hepatic Fibrosis in the Abcb4-/- Mouse.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL; TNFSF10) receptor (TR) is a pro-apoptotic receptor whose contribution to chronic cholestatic liver disease is unclear. Herein, we examined TRAIL receptor signaling in a mouse model of cholestatic liver injury. TRAIL receptor-deficient (Tnsf10 or Tr-/-) mice were crossbred with ATP binding cassette subfamily B member 4-deficient (Abcb4-/-, alias Mdr2-/-) mice. Male and female wild-type, Tr-/-, Mdr2-/-, and Tr-/-Mdr2-/- mice were assessed for liver injury, fibrosis, and ductular reactive (DR) cells. Macrophage subsets were examined by high-dimensional mass cytometry (time-of-flight mass cytometry). Mdr2-/- and Tr-/-Mdr2-/- mice had elevated liver weights and serum alanine transferase values. However, fibrosis was primarily periductular in Mdr2-/- mice, compared with extensive bridging fibrosis in Tr-/-Mdr2-/- mice. DR cell population was greatly expanded in the Tr-/-Mdr2-/- versus Mdr2-/- mice. The expanded DR cell population in Tr-/-Mdr2-/- mice was due to decreased cell loss by apoptosis and not enhanced proliferation. As assessed by time-of-flight mass cytometry, total macrophages were more abundant in Tr-/-Mdr2-/- versus Mdr2-/- mice, suggesting the DR cell population promotes macrophage-associated hepatic inflammation. Inhibition of monocyte-derived recruited macrophages using the CCR2/CCR5 antagonist cenicriviroc in the Mdr2-/- mice resulted in further expansion of the DR cell population. In conclusion, genetic deletion of TRAIL receptor increased the DR cell population, macrophage accumulation, and hepatic fibrosis in the Mdr2-/- model of cholestasis.

Targeted Apoptosis of Ductular Reactive Cells Reduces Hepatic Fibrosis in a Mouse Model of Cholestasis.

BACKGROUND AND AIMS: In cholestatic liver diseases, ductular reactive (DR) cells extend into the hepatic parenchyma and promote inflammation and fibrosis. We have previously observed that multidrug-resistant 2 (Mdr2-/- ) double knockout (DKO) mice lacking tumor necrosis factor-related apoptosis-inducing ligand receptor (Tr-/- ) display a more extensive ductular reaction and hepatic fibrosis compared to Mdr2-/- mice. This observation suggests that the magnitude of the DR-cell population may be regulated by apoptosis. APPROACH AND RESULTS: To examine this concept, we cultured epithelial cell adhesion molecule-positive reactive cholangioids (ERCs) obtained from wild-type (WT), Tr-/- , Mdr2-/- and DKO mice. Single-cell transcriptomics and immunostaining of both WT and DKO ERCs confirmed their DR-cell phenotype. Moreover, DKO ERCs displayed a unique translational cluster with expression of chemokines, indicating a reactive state. Incubation with the myeloid cell leukemia 1 (MCL1) inhibitor S63845, a proapoptotic BH3-mimetic therapy, significantly decreased DKO and Mdr2-/- ERC viability compared to WT. Intravenous administration of S63845 significantly reduced the DR-cell population and markers of inflammation and liver fibrosis in Mdr2-/- and DKO mice. Furthermore, DKO mice treated with S63845 displayed a significant decrease in hepatic B lymphocytes compared to untreated mice as assessed by high-definition mass cytometry by time-of-flight. Coculture of bone marrow-derived macrophages with ERCs from DKO mouse livers up-regulated expression of the B cell-directed chemokine (C-C motif) ligand 5. Finally, DR cells were noted to be primed for apoptosis with Bcl-2 homologous antagonist/killer activation in vitro and in vivo in primary sclerosing cholangitis liver specimens. CONCLUSIONS: DR cells appear to play a key role in recruiting immune cells to the liver to actively create an inflammatory and profibrogenic microenvironment. Pharmacologic targeting of MCL1 in a mouse model of chronic cholestasis reduces DR-cell and B-cell populations and hepatic fibrosis.

Activated cholangiocytes release macrophage-polarizing extracellular vesicles bearing the DAMP S100A11.

In mouse models of biliary tract diseases, macrophages are recruited to the periductal milieu and promote injury and cholestasis. Although cell necrosis with release of biomolecules termed damage-associated molecular patterns (DAMPs) promotes recruitment and activation of macrophages, necrosis was not observed in these studies. Because extracellular vesicles (EVs) are important in cell-to-cell communication, we postulated that activated cholangiocytes may release EVs containing DAMPs as cargo. Both the human (NHC) and mouse cholangiocyte (603B) cell lines display constitutive activation with mRNA expression of chemokines. Proteomic analysis revealed that EVs from both cell lines contained the DAMP S100A11, a ligand for the receptor for advanced glycation end products (RAGE). Bone marrow-derived macrophages (BMDM) incubated with EVs derived from the mouse 603B cell line increased mRNA expression of proinflammatory cytokines. Genetic or pharmacologic inhibition of RAGE reduced BMDM expression of proinflammatory cytokines treated with EVs. RAGE signaling resulted in activation of the canonical NF-κB pathway, and consistently, proinflammatory cytokine expression was blunted by the IKKα/ß inhibitor TPCA-1 in BMDM incubated with EVs. We also demonstrated that primary mouse cholangiocyte-derived organoids express chemokines indicating cholangiocyte activation, release EVs containing S100A11, and stimulate proinflammatory cytokine expression in BMDM by a RAGE-dependent pathway. In conclusion, these observations identify a non-cell death mechanism for cellular release of DAMPs by activated cholangiocytes, namely by releasing DAMPs as EV cargo. These data also suggest RAGE inhibitors may be salutary in macrophage-associated inflammatory diseases of the bile ducts.

Longitudinal analysis of invariant natural killer T cell activation reveals a cMAF-associated transcriptional state of NKT10 cells.

Innate T cells, including CD1d-restricted invariant natural killer T (iNKT) cells, are characterized by their rapid activation in response to non-peptide antigens, such as lipids. While the transcriptional profiles of naive, effector, and memory adaptive T cells have been well studied, less is known about the transcriptional regulation of different iNKT cell activation states. Here, using single-cell RNA-sequencing, we performed longitudinal profiling of activated murine iNKT cells, generating a transcriptomic atlas of iNKT cell activation states. We found that transcriptional signatures of activation are highly conserved among heterogeneous iNKT cell populations, including NKT1, NKT2, and NKT17 subsets, and human iNKT cells. Strikingly, we found that regulatory iNKT cells, such as adipose iNKT cells, undergo blunted activation and display constitutive enrichment of memory-like cMAF+ and KLRG1+ populations. Moreover, we identify a conserved cMAF-associated transcriptional network among NKT10 cells, providing novel insights into the biology of regulatory and antigen-experienced iNKT cells.

Mast cells, macrophages, and crown-like structures distinguish subcutaneous from visceral fat in mice.

Obesity is accompanied by adipocyte death and accumulation of macrophages and mast cells in expanding adipose tissues. Considering the differences in biological behavior of fat found in different anatomical locations, we explored the distribution of mast cells, solitary macrophages, and crown-like structures (CLS), the surrogates for dead adipocytes, in subcutaneous and abdominal visceral fat of lean and diet-induced obese C57BL/6 mice. In fat depots of lean mice, mast cells were far less prevalent than solitary macrophages. Subcutaneous fat contained more mast cells, but fewer solitary macrophages and CLS, than visceral fat. Whereas no significant change in mast cell density of subcutaneous fat was observed, obesity was accompanied by a substantial increase in mast cells in visceral fat. CLS became prevalent in visceral fat of obese mice, and the distribution paralleled mast cells. Adipose tissue mast cells contained and released preformed TNF-α, the cytokine implicated in the pathogenesis of obesity-linked insulin resistance. In summary, subcutaneous fat differed from visceral fat by immune cell composition and a lower prevalence of CLS both in lean and obese mice. The increase in mast cells in visceral fat of obese mice suggests their role in the pathogenesis of obesity and insulin resistance.

Selected Patients with Unresectable Perihilar Cholangiocarcinoma (pCCA) Derive Long-Term Benefit from Liver Transplantation.

Selected patients with unresectable perihilar cholangiocarcinoma (pCCA) derive long-term benefits from liver transplantation. Between 1993-2019, our group at Mayo Clinic performed 237 transplants for pCCA. With this experience, we note that two distinct patient populations comprise this group of pCCA patients: those with underlying primary sclerosing cholangitis (PSC) and those without identifiable risk factors termed sporadic or de novo pCCA. Long-term survival after transplant is better in PSC patients (74% five-year survival) than in those with de novo pCCA (58% five-year survival). Herein, we review the likely clinical factors contributing to the divergence in outcomes for these two patient populations. We also offer our insights on how further advances may improve patient selection and survival, focusing on the de novo pCCA patient population.

Real World Experience of Drug Induced Liver Injury in Patients Undergoing Chemotherapy.

BACKGROUND & AIM: To better understand the clinical significance of drug induced liver injury (DILI) during chemotherapy, we examined the epidemiology, incidence, and treatment effects of DILI in patients undergoing chemotherapy for genitourinary malignancies over a two-year period. METHODS: We conducted a retrospective review of 284 patients who underwent chemotherapy for prostate, bladder, testicular and renal cell carcinomas over a two year period. Those with abnormal or absent liver test (LT) results prior to chemotherapy initiation were excluded. Post chemotherapy LT results were defined as DILI if ALT>3× ULN and/or total bilirubin (TB)>2× ULN, in the absence of other more likely causes of elevated LT. RESULTS: The cumulative incidence of DILI in the total study population was 6.1% (17/284), and in the population who had appropriate LT performed it increased to 18.9% (17/90). Chemotherapeutic agents were determined to be the cause of DILI in 82% (14/17) of patients, and the treatment plans were changed in 59% (10/17) of patients. CONCLUSION: In this real world study, the cumulative incidence of DILI was higher than commonly reported in clinical trials, and the majority of affected patients had to have their cancer treatment altered or interrupted.