Search

Adult kidney organoids have been described as strictly tubular epithelia and termed tubuloids. While the cellular origin of tubuloids has remained elusive, here we report that they originate from a distinct CD24+epithelial subpopulation. Long-term-cultured CD24+cell-derived tubuloids represent a functional human kidney tubule. We show that kidney tubuloids can be used to model the most common inherited kidney disease, namely autosomal dominant polycystic kidney disease (ADPKD), reconstituting the phenotypic hallmark of this disease with cyst formation. Single-cell RNA sequencing of CRISPR–Cas9 gene-edited PKD1-and PKD2-knockout tubuloids and human ADPKD and control tissue shows similarities in upregulation of disease-driving genes. Furthermore, in a proof of concept, we demonstrate that tolvaptan, the only approved drug for ADPKD, has a significant effect on cyst size in tubuloids but no effect on a pluripotent stem cell-derived model. Thus, tubuloids are derived from a tubular epithelial subpopulation and represent an advanced system for ADPKD disease modeling.

Lipocalin 2 (LCN2), an acute-phase protein produced during acute liver injury, plays an important role in the innate immune response against bacterial infection via iron scavenging. LCN2 further influences neutrophil development and physiology leading to increased inflammatory responses. We investigated the roles of LCN2 in chronic inflammation and fibrosis, using repeated carbon tetrachloride (CCl4) in mineral-oil injection. Surprisingly, mice treated with the mineral oil vehicle alone showed liver inflammation, evidenced by neutrophil and monocyte-macrophage infiltration. Fluorescence-activated cell sorting (FACS) of isolated liver leukocytes showed significantly high CD45+leukocyte concentrations in CCl4mice, but no difference of Ly6G+neutrophils between mineral oil and CCl4application. Liver CD11b+F4/80+cells counted higher in CCl4mice, but the proportions of Gr1high, an indicator of inflammation, were significantly higher in mineral oil groups. Liver myeloperoxidase (MPO), expressed in neutrophils and monocytes, showed higher levels in wild type mice compared to Lcn2−/−in both mineral-oil and CCl4treated groups. Hepatic and serum LCN2 levels were remarkably higher in the mineral oil-injected wild type group compared to the CCl4. Wild type animals receiving mineral oil showed significantly higher inflammatory cytokine- and chemokine mRNA levels compared to Lcn2−/−mice, with no differences in the CCl4treated groups. RNA sequencing (RNA-Seq) confirmed significant downregulation of gene sets involved in myeloid cell activation and immune responses in Lcn2null mice receiving chronic mineral oil versus wild−type. We observed significant upregulation of gene sets and proteins involved in cell cycle DNA replication, with downregulation of collagen-containing extracellular matrix genes in Lcn2–/–mice receiving CCl4,compared to the wild type. Consequently, the wild type mice developed slightly more liver fibrosis compared to Lcn2−/−mice, evidenced by higher levels of collagen type I in the CCl4groups and no liver fibrosis in mineral oil-treated mice. Our findings indicate that serum and hepatic LCN2 levels correlate with hepatic inflammation rather than fibrosis.

Lipocalin 2 (LCN2), an acute-phase protein produced during acute liver injury, plays an important role in the innate immune response against bacterial infection via iron scavenging. LCN2 further influences neutrophil development and physiology leading to increased inflammatory responses. We investigated the roles of LCN2 in chronic inflammation and fibrosis, using repeated carbon tetrachloride (CCl4) in mineral-oil injection. Surprisingly, mice treated with the mineral oil vehicle alone showed liver inflammation, evidenced by neutrophil and monocyte-macrophage infiltration. Fluorescence-activated cell sorting (FACS) of isolated liver leukocytes showed significantly high CD45+leukocyte concentrations in CCl4mice, but no difference of Ly6G+neutrophils between mineral oil and CCl4application. Liver CD11b+F4/80+cells counted higher in CCl4mice, but the proportions of Gr1high, an indicator of inflammation, were significantly higher in mineral oil groups. Liver myeloperoxidase (MPO), expressed in neutrophils and monocytes, showed higher levels in wild type mice compared to Lcn2−/−in both mineral-oil and CCl4treated groups. Hepatic and serum LCN2 levels were remarkably higher in the mineral oil-injected wild type group compared to the CCl4. Wild type animals receiving mineral oil showed significantly higher inflammatory cytokine- and chemokine mRNA levels compared to Lcn2−/−mice, with no differences in the CCl4treated groups. RNA sequencing (RNA-Seq) confirmed significant downregulation of gene sets involved in myeloid cell activation and immune responses in Lcn2null mice receiving chronic mineral oil versus wild−type. We observed significant upregulation of gene sets and proteins involved in cell cycle DNA replication, with downregulation of collagen-containing extracellular matrix genes in Lcn2–/–mice receiving CCl4,compared to the wild type. Consequently, the wild type mice developed slightly more liver fibrosis compared to Lcn2−/−mice, evidenced by higher levels of collagen type I in the CCl4groups and no liver fibrosis in mineral oil-treated mice. Our findings indicate that serum and hepatic LCN2 levels correlate with hepatic inflammation rather than fibrosis.

ObjectiveBone marrow-derived myeloid cells accumulate in the liver as monocytes and macrophages during the progression of obesity-related non-alcoholic fatty liver disease (NAFLD) to steatohepatitis (NASH). Myeloid cells comprise heterogeneous subsets, and dietary overnutrition may affect macrophages in the liver and bone marrow. We therefore aimed at characterising in depth the functional adaptations of myeloid cells in fatty liver.DesignWe employed single-cell RNA sequencing to comprehensively assess the heterogeneity of myeloid cells in the liver and bone marrow during NAFLD, by analysing C57BL/6 mice fed with a high-fat, high-sugar, high-cholesterol ‘Western diet’ for 16 weeks. We also characterised NAFLD-driven functional adaptations of macrophages in vitro and their functional relevance during steatohepatitis in vivo.ResultsSingle-cell RNA sequencing identified distinct myeloid cell clusters in the liver and bone marrow. In both compartments, monocyte-derived populations were largely expanded in NASH-affected mice. Importantly, the liver myeloid compartment adapted a unique inflammatory phenotype during NAFLD progression, exemplarily characterised by downregulated inflammatory calprotectin (S100A8/A9) in macrophage and dendritic cell subsets. This distinctive gene signature was also found in their bone marrow precursors. The NASH myeloid phenotype was principally recapitulated by in vitro exposure of bone marrow-derived macrophages with fatty acids, depended on toll-like receptor 4 signalling and defined a characteristic response pattern to lipopolysaccharide stimulation. This imprinted and stable NASH myeloid immune phenotype functionally determined inflammatory responses following acute liver injury (acetaminophen poisoning) in vivo.ConclusionLiver myeloid leucocytes and their bone marrow precursors adapt a common and functionally relevant inflammatory signature during NAFLD progression.

Group 1 innate lymphoid cells (ILCs) comprise a heterogeneous family of cytotoxic natural killer (NK) cells and ILC1s. We identify a population of “liver-type” ILC1s with transcriptional, phenotypic, and functional features distinct from those of conventional and liver-resident NK cells as well as from other previously described human ILC1 subsets. LT-ILC1s are CD49a+CD94+CD200R1+, express the transcription factor T-BET, and do not express the activating receptor NKp80 or the transcription factor EOMES. Similar to NK cells, liver-type ILC1s produce IFN-γ, TNF-α, and GM-CSF; however, liver-type ILC1s also produce IL-2 and lack perforin and granzyme-B. Liver-type ILC1s are expanded in cirrhotic liver tissues, and they can be produced from blood-derived ILC precursors in vitroin the presence of TGF-β1 and liver sinusoidal endothelial cells. Cells with similar signature and function can also be found in tonsil and intestinal tissues. Collectively, our study identifies and classifies a population of human cross-tissue ILC1s.

Ehninger: Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership; GEMoaB Monoclonals GmbH: Employment, Equity Ownership; Bayer: Research Funding. Brümmendorf:Merck: Consultancy; Janssen: Consultancy; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Takeda: Consultancy. Rollig:Janssen: Research Funding; Bayer: Research Funding. Thiede:Novartis: Honoraria, Research Funding; AgenDix: Other: Ownership.

Introduction:Acute myeloid leukemia (AML) is characterized by heterogeneous cytogenetic and molecular aberrations. Deletions on the long arm of chromosome 9 (del(9q)) are observed in 2% of AML patients. In about 24% of the cases, del(9q) is observed as sole karyotypic abnormality, while in the remaining 76%, it is associated with a t(8;21) translocation or other aberrations. Among all del(9q) AML cases, 36%-50% exhibit an additional t(8;21), whereas 7%-14% of AML cases with t(8;21) show del(9q) as an additional aberration. A commonly deleted region (CDR) of del(9q) was defined and further analysis specified a minimally deleted region (MDR) composed of seven annotated genes (GKAP1, KIF27, C9ORF64, HNRNPK, RMI1, SLC28A3and NTRK2) (Kronke J et al. Blood. 2013). However, the function of these genes and their impact on the pathogenesis of AML remain elusive. A recent study demonstrated that reduced expression of the HNRNPKgene product can contribute to leukemogenesis in AML (Gallardo M, Cancer Cell. 2015). The multifunctional protein hnRNP K interacts with other proteins, DNA and RNA, to modulate gene activity and gene expression on different levels. For example, hnRNP K not only regulates SRCgene transcription, but as well SRCmRNA translation and the activity of c-Src kinase. In the context of AML, hnRNP K was shown to interact with the mRNAs encoding C/EBPa (CEBPA) and p21 (CDKN1A). We analyzed a cohort of 31 del(9q) AML patients in order to further analyze the deleted region and to analyze the impact of HNRNPKdeletion on leukemogenesis.