A mouse model with high clonal barcode diversity for joint lineage, transcriptomic, and epigenomic profiling in single cells.

Cellular lineage histories and their molecular states encode fundamental principles of tissue development and homeostasis. Current lineage-recording mouse models have insufficient barcode diversity and single-cell lineage coverage for profiling tissues composed of millions of cells. Here, we developed DARLIN, an inducible Cas9 barcoding mouse line that utilizes terminal deoxynucleotidyl transferase (TdT) and 30 CRISPR target sites. DARLIN is inducible, generates massive lineage barcodes across tissues, and enables the detection of edited barcodes in ∼70% of profiled single cells. Using DARLIN, we examined fate bias within developing hematopoietic stem cells (HSCs) and revealed unique features of HSC migration. Additionally, we established a protocol for joint transcriptomic and epigenomic single-cell measurements with DARLIN and found that cellular clonal memory is associated with genome-wide DNA methylation rather than gene expression or chromatin accessibility. DARLIN will enable the high-resolution study of lineage relationships and their molecular signatures in diverse tissues and physiological contexts.

The elegant complexity of mammalian ecto-5'-nucleotidase (CD73).

Purinergic signaling is a fundamental mechanism used by all cells to control their internal activities and interact with the environment. A key component of the purinergic system, the enzyme ecto-5'-nucleotidase (CD73) catalyzes the last step in the extracellular metabolism of ATP to form adenosine. Efforts to harness the therapeutic potential of endogenous adenosine in cancer have culminated in the ongoing clinical development of multiple CD73-targeting antibodies and small-molecule inhibitors. However, recent studies are painting an increasingly complex picture of CD73 mRNA and protein regulation and function in cellular homeostasis, physiological adaptation, and disease development. This review discusses the latest conceptual and methodological advances that are helping to unravel the complexity of this important enzyme that was identified nearly 90 years ago.

Sox9EGFP Defines Biliary Epithelial Heterogeneity Downstream of Yap Activity.

BACKGROUND & AIMS: Defining the genetic heterogeneity of intrahepatic biliary epithelial cells (BECs) is challenging, and tools for identifying BEC subpopulations are limited. Here, we characterize the expression of a Sox9EGFP transgene in the liver and demonstrate that green fluorescent protein (GFP) expression levels are associated with distinct cell types. METHODS: Sox9EGFP BAC transgenic mice were assayed by immunofluorescence, flow cytometry, and gene expression profiling to characterize in vivo characteristics of GFP populations. Single BECs from distinct GFP populations were isolated by fluorescence-activated cell sorting, and functional analysis was conducted in organoid forming assays. Intrahepatic ductal epithelium was grown as organoids and treated with a Yes-associated protein (Yap) inhibitor or bile acids to determine upstream regulation of Sox9 in BECs. Sox9EGFP mice were subjected to bile duct ligation, and GFP expression was assessed by immunofluorescence. RESULTS: BECs express low or high levels of GFP, whereas periportal hepatocytes express sublow GFP. Sox9EGFP+ BECs are differentially distributed by duct size and demonstrate distinct gene expression signatures, with enrichment of Cyr61 and Hes1 in GFPhigh BECs. Single Sox9EGFP+ cells form organoids that exhibit heterogeneous survival, growth, and HNF4A activation dependent on culture conditions, suggesting that exogenous signaling impacts BEC heterogeneity. Yap is required to maintain Sox9 expression in biliary organoids, but bile acids are insufficient to induce BEC Yap activity or Sox9 in vivo and in vitro. Sox9EGFP remains restricted to BECs and periportal hepatocytes after bile duct ligation. CONCLUSIONS: Our data demonstrate that Sox9EGFP levels provide readout of Yap activity and delineate BEC heterogeneity, providing a tool for assaying subpopulation-specific cellular function in the liver.

CD73 Maintains Hepatocyte Metabolic Integrity and Mouse Liver Homeostasis in a Sex-Dependent Manner.

BACKGROUND & AIMS: Metabolic imbalance and inflammation are common features of chronic liver diseases. Molecular factors controlling these mechanisms represent potential therapeutic targets. CD73 is the major enzyme that dephosphorylates extracellular adenosine monophosphate (AMP) to form the anti-inflammatory adenosine. CD73 is expressed on pericentral hepatocytes, which are important for long-term liver homeostasis. We aimed to determine if CD73 has nonredundant hepatoprotective functions. METHODS: Liver-specific CD73 knockout (CD73-LKO) mice were generated by targeting the Nt5e gene in hepatocytes. The CD73-LKO mice and hepatocytes were characterized using multiple approaches. RESULTS: Deletion of hepatocyte Nt5e resulted in an approximately 70% reduction in total liver CD73 protein (P < .0001). Male and female CD73-LKO mice developed normally during the first 21 weeks without significant liver phenotypes. Between 21 and 42 weeks, the CD73-LKO mice developed spontaneous-onset liver disease, with significant severity in male mice. Middle-aged male CD73-LKO mice showed hepatocyte swelling and ballooning (P < .05), inflammation (P < .01), and variable steatosis. Female CD73-LKO mice had lower serum albumin levels (P < .05) and increased inflammatory genes (P < .01), but did not show the spectrum of histopathologic changes in male mice, potentially owing to compensatory induction of adenosine receptors. Serum analysis and proteomic profiling of hepatocytes from male CD73-LKO mice showed significant metabolic imbalance, with increased blood urea nitrogen (P < .0001) and impairments in major metabolic pathways, including oxidative phosphorylation and AMP-activated protein kinase (AMPK) signaling. There was significant hypophosphorylation of AMPK substrates in CD73-LKO livers (P < .0001), while in isolated hepatocytes treated with AMP, soluble CD73 induced AMPK activation (P < .001). CONCLUSIONS: Hepatocyte CD73 supports long-term metabolic liver homeostasis through AMPK in a sex-dependent manner. These findings have implications for human liver diseases marked by CD73 dysregulation.

Alternative Splicing in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) accounts for the majority of primary liver cancer cases, with more than 850,000 new diagnoses per year globally. Recent trends in the United States have shown that liver cancer mortality has continued to increase in both men and women, while 5-year survival remains below 20%. Understanding key mechanisms that drive chronic liver disease progression to HCC can reveal new therapeutic targets and biomarkers for early detection of HCC. In that regard, many studies have underscored the importance of alternative splicing as a source of novel HCC prognostic markers and disease targets. Alternative splicing of pre-mRNA provides functional diversity to the genome, and endows cells with the ability to rapidly remodel the proteome. Genes that control fundamental processes, such as metabolism, cell proliferation, and apoptosis, are altered globally in HCC by alternative splicing. This review highlights the major splicing factors, RNA binding proteins, transcriptional targets, and signaling pathways that are of key relevance to HCC. We highlight primary research from the past 3-5 years involving functional interrogation of alternative splicing in rodent and human liver, using both large-scale transcriptomic and focused mechanistic approaches. Because this is a rapidly advancing field, we anticipate that it will be transformative for the future of basic liver biology, as well as HCC diagnosis and management.

Tumor-Selective Altered Glycosylation and Functional Attenuation of CD73 in Human Hepatocellular Carcinoma.

CD73, a cell-surface N-linked glycoprotein that produces extracellular adenosine, is a novel target for cancer immunotherapy. Although anti-CD73 antibodies have entered clinical development, CD73 has both protumor and antitumor functions, depending on the target cell and tumor type. The aim of this study was to characterize CD73 regulation in human hepatocellular carcinoma (HCC). We examined CD73 expression, localization, and activity using molecular, biochemical, and cellular analyses on primary HCC surgical specimens, coupled with mechanistic studies in HCC cells. We analyzed CD73 glycan signatures and global alterations in transcripts encoding other N-linked glycoproteins by using mass spectrometry glycomics and RNA sequencing (RNAseq), respectively. CD73 was expressed on tumor hepatocytes where it exhibited abnormal N-linked glycosylation, independent of HCC etiology, tumor stage, or fibrosis presence. Aberrant glycosylation of tumor-associated CD73 resulted in a 3-fold decrease in 5'-nucleotidase activity (P < 0.0001). Biochemically, tumor-associated CD73 was deficient in hybrid and complex glycans specifically on residues N311 and N333 located in the C-terminal catalytic domain. Blocking N311/N333 glycosylation by site-directed mutagenesis produced CD73 with significantly decreased 5'-nucleotidase activity in vitro, similar to the primary tumors. Glycosylation-deficient CD73 partially colocalized with the Golgi structural protein GM130, which was strongly induced in HCC tumors. RNAseq analysis further revealed that N-linked glycoprotein-encoding genes represented the largest category of differentially expressed genes between HCC tumor and adjacent tissue. Conclusion: We provide the first detailed characterization of CD73 glycosylation in normal and tumor tissue, revealing a novel mechanism that leads to the functional suppression of CD73 in human HCC tumor cells. The present findings have translational implications for therapeutic candidate antibodies targeting cell-surface CD73 in solid tumors and small-molecule adenosine receptor agonists that are in clinical development for HCC.

Cell type- and tissue-specific functions of ecto-5'-nucleotidase (CD73).

Ecto-5'-nucleotidase [cluster of differentiation 73 (CD73)] is a ubiquitously expressed glycosylphosphatidylinositol-anchored glycoprotein that converts extracellular adenosine 5'-monophosphate to adenosine. Anti-CD73 inhibitory antibodies are currently undergoing clinical testing for cancer immunotherapy. However, many protective physiological functions of CD73 need to be taken into account for new targeted therapies. This review examines CD73 functions in multiple organ systems and cell types, with a particular focus on novel findings from the last 5 years. Missense loss-of-function mutations in the CD73-encoding gene NT5E cause the rare disease "arterial calcifications due to deficiency of CD73." Aside from direct human disease involvement, cellular and animal model studies have revealed key functions of CD73 in tissue homeostasis and pathology across multiple organ systems. In the context of the central nervous system, CD73 is antinociceptive and protects against inflammatory damage, while also contributing to age-dependent decline in cortical plasticity. CD73 preserves barrier function in multiple tissues, a role that is most evident in the respiratory system, where it inhibits endothelial permeability in an adenosine-dependent manner. CD73 has important cardioprotective functions during myocardial infarction and heart failure. Under ischemia-reperfusion injury conditions, rapid and sustained induction of CD73 confers protection in the liver and kidney. In some cases, the mechanism by which CD73 mediates tissue injury is less clear. For example, CD73 has a promoting role in liver fibrosis but is protective in lung fibrosis. Future studies that integrate CD73 regulation and function at the cellular level with physiological responses will improve its utility as a disease target.

Development of multiplex PCR and multi-color fluorescent in situ hybridization (m-FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease.

BACKGROUND: Several pathogens have been debated to play a role in inflammatory bowel disease (IBD) including Crohn's disease (CD). None of these pathogens have been investigated together in same clinical samples. We developed a multiplex PCR and multi-color fluorescent in situ hybridization (m-FISH) protocols for simultaneous detection of CD-associated pathogens including Mycobacterium avium subspecies paratuberculosis (MAP), Klebsiella pneumoniae, and adherent-invasive Escherichia coli strain LF82. METHODS: The multiplex PCR is based on 1-h DNAzol® extraction protocol modified for rapid extraction of bacterial DNA from culture, blood, and intestinal biopsies. Oligonucleotide primers sequences unique to these pathogens were evaluated individually and in combinations using bioinformatics and experimental approaches. m-FISH was based on fluorescent-tagged oligonucleotides and confocal scanning laser microscopy (CSLM). RESULTS: Following several attempts, the concentration of the oligonucleotide primers and DNA templates and the PCR annealing temperatures were optimized. Multiplex PCR analyses revealed excellent amplification signal in trials where a single primer set and combinations of two and three primers sets were tested against a mixture of DNA from three different bacteria or a mixture of three bacterial cultures mixed in one tube before DNA extraction. Slides with individual and mixtures of bacterial cultures and intestinal tissue sections from IBD patients were tested by m-FISH and the CSLM images verified multiplex PCR results detected on 3% agarose gel. CONCLUSION: We developed a 4-h multiplex PCR protocol, which was validated by m-FISH images, capable of detecting up to four genes from major pathogens associated with CD. The new protocol should serve as an excellent tool to support efforts to study multi-pathogens involved in CD and other autoimmune disease.

Frontline Science: RIP2 promotes house dust mite-induced allergic airway inflammation.

House dust mites (HDMs) are one of the most significant environmental allergens in the establishment of the so-called "Atopic March." It is known that the immune response to HDM is Th2 dominant, but the innate mechanisms leading to HDM-induced type 2 responses are still not completely understood. A number of innate immune receptors have been implicated in the response to HDM including toll-like receptors, C-type lectin receptors, and protease activated receptors. NOD2 is a member of the NOD-like receptor family, which has been reported to be involved in the establishment of type 2 immunity and in blocking respiratory tolerance. NOD2 mediates its effects through its downstream effector kinase, receptor interacting protein (RIP2). It has not been shown if RIP2 is involved in the innate response to HDM and in the resulting generation of type 2 immunity. Furthermore, the role of RIP2 in modulating allergic airway inflammation has been controversial. In this study, we show that RIP2 is activated in airway epithelial cells in response to HDM and is important for the production of CCL2. Using a murine HDM asthma model, we demonstrate that lung pathology, local airway inflammation, inflammatory cytokines, HDM-specific IgG1 antibody production, and HDM-specific Th2 responses are all reduced in RIP2 knockout mice compared to WT animals. These data illustrate that RIP2 can be activated by a relevant allergic stimulus and that such activation can contribute to allergic airway inflammation. These findings also suggest that RIP2 inhibitors might have some efficacy in down-regulating the inflammatory response in type 2 dominated diseases.

RHB-104 triple antibiotics combination in culture is bactericidal and should be effective for treatment of Crohn's disease associated with Mycobacterium paratuberculosis.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) has been implicated as an etiological agent of Crohn's disease (CD), a debilitating chronic inflammatory bowel disease. Clarithromycin (CLA), clofazimine (CLO), rifabutin (RIF) and other antibiotics have been used individually or in combinations with other drugs to treat mycobacterial diseases including CD. The treatment has varied by regimen, dosage, and duration, resulting in conflicting outcomes and additional suffering to the patients. RHB-104, a drug formula with active ingredients composed of (63.3 %) CLA, (6.7 %) CLO, and (30 %) RIF, has been recently subjected to investigation in an FDA approved Phase III clinical trial to treat patients with moderate to severe CD. In this study, we determined the efficacy of RHB-104 active ingredients against MAP strains isolated from the blood, tissue, and milk of CD patients. Based on fluorescence quenching technology using the Bactec MGIT Para-TB medium, we determined the minimum inhibitory concentration (MIC) of CLA, CLO, RIF individually and in dual and triple combinations against 16 MAP clinical strains and 19 other mycobacteria. RESULTS: The MIC of all drugs against 35 different mycobacteria ranged between 0.25-20 µg/mL. However, the MIC of RHB-104 active ingredients regimen was the lowest at 0.25-10 µg/mL compared to the MIC of the other drugs at 0.5-20 µg/mL. The components of RHB-104 active ingredients at their individual concentrations or in dual combinations were not effective against all microorganisms compared to the triple combinations at MIC level. The MIC of CLA-CLO, CLA-RIF, and CLO-RIF regimens ranged between 0.5-1.25 µg/mL compared to 0.25 µg/mL of bactericidal effect of the triple combination. CONCLUSION: The data clearly demonstrated that lower concentrations of the triple combination of RHB-104 active ingredients provided synergistic anti-MAP growth activity compared to individual or dual combinations of the drugs. Consequently, this is favorable and should lead to tolerable dosage that is desirable for long-term treatment of CD and Mycobacterium avium complex disease.

Domino effect of hypomagnesemia on the innate immunity of Crohn's disease patients.

Digestive diseases play major role in development and complications of other disorders including diabetes. For example, Crohn's disease (CD) is an inflammatory bowel disease associated with Mycobacterium avium subspecies paratuberculosis. The inflammation is a complex process that involves the activity of both innate and adaptive immune responses. CD lesions are primarily due to T cell response, however; innate immune response has a significant role in initiating its pathogenesis. Toll-like receptors and NOD-like receptors promote the activity of nuclear factor (NF)-κB pathway for cytokines production. This results in the production of high levels of tumor necrosis factor-α, interleukin (IL)-1ß and IL-6. Moreover, intestinal inflammation of CD is related to increased activity of NMDA receptors and the release of substance P. Imbalanced magnesium homeostasis in CD is a frequent finding in CD, Diabetes and others. The loss of such a major mineral affects many physiological processes in the body including its role as an immunomodulator. This review aims to (1) describe the significance of hypomagnesemia in the release of pro-inflammatory mediators in CD; (2) demonstrate effects of magnesium on pathways like NF-κB; (3) address the role of hypomagnesemia in the activity of CD; and (4) examine possible future research to establish a standard magnesium supplementation strategy; helping patients with CD or other disorders to maintain a sustained remission.