Estimating Drug Efficacy with a Diet-Induced NASH Model in Chimeric Mice with Humanized Livers

Nonalcoholic fatty liver disease/steatohepatitis (NAFLD/NASH) is the most common liver disorder in developed countries. Although many new therapeutics for NASH are present in the drug development pipeline, there are still no approved drugs. One of the reasons that makes NASH drug development challenging is the lack of appropriate animal NASH models that resolve issues arising from inter-species differences between humans and rodents. In the present study, we developed a choline-deficient, L-amino-acid-defined, high-fat-diet (CDAHFD)-induced human NASH model using human liver chimeric mice. We demonstrated human hepatocyte injury by an elevation of plasma human alanine aminotransferase 1 in mice fed CDAHFD. Histological analysis showed that CDAHFD feeding induced similar histological changes to human NASH patients, including ballooning, inflammation, apoptosis, regeneration of human hepatocytes, and pericellular and perisinusoidal fibrosis. The chimeric mice fed CDAHFD were treated with a peroxisome-proliferator-activated receptor α/δ agonist, Elafibranor. Elafibranor ameliorated steatosis, ballooning of hepatocytes, and preserved fibrosis progression. We developed a novel humanized NASH model that can elucidate pathophysiological mechanisms and predict therapeutic efficacy in human NASH. This model will be useful in exploring new drugs and biomarkers in the early stages of human NASH.

IL-1 Signaling Promotes Clonal Expansion and Progression of Bone Marrow Fibrosis in JAK2V617F-Induced Myeloproliferative Neoplasm

Myeloproliferative neoplasms (MPN) are a group of clonal hematopoietic stem cell derived myeloid malignancies characterized by aberrant production of myeloid, erythroid or megakaryocytic lineage cells. JAK2V617F is the most common somatic driver mutation associated with MPN. Interestingly, JAK2V617F mutation can also be detected in healthy individuals with clonal hematopoiesis of indeterminate potential (CHIP) who do not exhibit overt changes in blood counts. This suggests that other factors might be involved in association with JAK2 mutation in clonal expansion and initiation/progression of MPN. Chronic inflammation is frequently associated with MPN. Interleukin 1 (IL-1) is a major regulator of inflammation. IL-1 consists of two related cytokines IL-1α and IL-1β. Both IL-1α and IL-1β bind to the IL-1 receptor 1 (IL-1R1) to initiate downstream signaling. Although elevated expression of IL-1α and IL-1β has been observed in MPN, their role in the pathogenesis of MPN has remained elusive. In this study, we investigated the role of IL-1 signaling in JAK2V617F-induced MPN using a Jak2V617F knock-in mouse model. We observed elevated levels of IL-1α and IL-1β in mice expressing heterozygous (Jak2 VF/+) and homozygous Jak2V617F (Jak2 VF/VF) compared with WT control animals. Notably, IL-1α and IL-1β expression was significantly higher in Jak2 VF/VF mice exhibiting extensive bone marrow (BM) fibrosis compared with Jak2 VF/+ mice exhibiting polycythemia vera (PV), consistent with elevated levels of IL-1 in patients with myelofibrosis (MF). Since both IL-1α and IL-1β levels were elevated in Jak2 VF/VF mice exhibiting MF, we utilized conditional IL-1R1 knockout (IL-1R1cKO) and Jak2 VF/VF mice to assess the role of IL-1 signaling in the initiation/progression of MF. As expected, Jak2 VF/VF mice exhibited a significant increase in WBC, neutrophil and platelet counts compared to WT control mice. Deletion of IL-1R1in Jak2 VF/VF mice (IL-1R1cKO; Jak2 VF/VF) significantly reduced the WBC, neutrophil and platelet counts to almost control levels. Flow cytometric analysis also showed a significant reduction of myeloid (Gr-1 +) and megakaryocytic (CD41 +) precursors in the BM and spleens of IL-1R1cKO; Jak2 VF/VF mice compared to Jak2 VF/VF mice. Moreover, deletion of IL-1R1 significantly reduced hematopoietic stem and progenitor cells (HSPC) in the BM of IL-1R1cKO; Jak2 VF/VF mice compared to Jak2 VF/VF mice. Spleen weight was significantly reduced in IL-1R1cKO; Jak2 VF/VF mice compared with Jak2 VF/VF mice and they were comparable to control WT mice. More importantly, deletion of IL-1R1 markedly reduced BM fibrosis in Jak2 VF/VF mice. These data suggest an important role of IL-1 signaling in the progression of BM fibrosis in Jak2V617F-induced MPN. To test whether IL-1 signaling contributes to clonal expansion of JAK2 mutant HSPC, we performed competitive transplantation assays by mixing Mx1Cre; Jak2 VF/+ and Mx1Cre; IL-1R1 F/F; Jak2 VF/+ mice BM cells with CD45.1 + WT mice BM cells at a ratio of 1:1 and transplanted into lethally irradiated CD45.1 + recipient animals. At 4 weeks after BMT, the recipient animals were injected with pI-pC to induce Jak2V617F expression and IL-1R1 deletion. We observed significantly higher percentages of total CD45.2 + cells as well as CD45.2 + myeloid (Gr-1 +), B- and T-cells in the peripheral blood of chimeric mice receiving Jak2 VF/+ BM compared with chimeric mice receiving IL-1R1cKO; Jak2 VF/+ BM. We also observed significantly reduced percentages of CD45.2 + LSK, LK, Gr-1 + and CD41 + cells in the BM of chimeric recipient animals receiving IL-1R1cKO; Jak2 VF/+ BM compared with Jak2 VF/+ BM. These results suggest a role of IL-1 signaling in clonal expansion of Jak2V617F mutant HSPC. Additionally, we tested the effects of blocking IL-1R1 using an anti-IL-1R1 antibody in the homozygous Jak2V617F knock-in mouse model of MF. We observed that anti-IL-1R1 antibody treatment significantly reduced peripheral blood WBC and neutrophil counts and decreased HSPC and myeloid precursors in the BM of Jak2 VF/VF mice. Furthermore, anti-IL-1R1 antibody treatment significantly reduced splenomegaly and markedly reduced BM fibrosis in Jak2 VF/VF mice, suggesting that therapies targeting IL-1R1 could be useful for the treatment of myelofibrosis. Overall, our results suggest that IL-1 signaling contributes to clonal expansion of Jak2V617F mutant HSPC and progression of bone marrow fibrosis in MPN. Disclosures No relevant conflicts of interest to declare.

Signaling through Syk or CARD9 Mediates Species-Specific Anti- Candida Protection in Bone Marrow Chimeric Mice

While C. albicans remains the most clinically significant Candida species, C. parapsilosis is an emerging pathogen with increased affinity to neonates. Syk/CARD9 signaling is crucial in immunity to C. albicans , but its role in in vivo responses to other pathogenic Candida species is largely unexplored.

Strong Replication Interference Between Hepatitis Delta Viruses in Human Liver Chimeric Mice

BackgroundHepatitis D Virus (HDV) is classified into eight genotypes with distinct clinical outcomes. Despite the maintenance of highly conserved functional motifs, it is unknown whether sequence divergence between genotypes, such as HDV-1 and HDV-3, or viral interference mechanisms may affect co-infection in the same host and cell, thus hindering the development of HDV inter-genotypic recombinants. We aimed to investigate virological differences of HDV-1 and HDV-3 and assessed their capacity to infect and replicate within the same liver and human hepatocyte in vivo.MethodsHuman liver chimeric mice were infected with hepatitis B virus (HBV) and with one of the two HDV genotypes or with HDV-1 and HDV-3 simultaneously. In a second set of experiments, HBV-infected mice were first infected with HDV-1 and after 9 weeks with HDV-3, or vice versa. Also two distinct HDV-1 strains were used to infect mice simultaneously and sequentially. Virological parameters were determined by strain-specific qRT-PCR, RNA in situ hybridization and immunofluorescence staining.ResultsHBV/HDV co-infection studies indicated faster spreading kinetics and higher intrahepatic levels of HDV-3 compared to HDV-1. In mice that simultaneously received both HDV strains, HDV-3 became the dominant genotype. Interestingly, antigenomic HDV-1 and HDV-3 RNA were detected within the same liver but hardly within the same cell. Surprisingly, sequential super-infection experiments revealed a clear dominance of the HDV strain that was inoculated first, indicating that HDV-infected cells may acquire resistance to super-infection.ConclusionInfection with two largely divergent HDV genotypes could be established in the same liver, but rarely within the same hepatocyte. Sequential super-infection with distinct HDV genotypes and even with two HDV-1 isolates was strongly impaired, suggesting that virus interference mechanisms hamper productive replication in the same cell and hence recombination events even in a system lacking adaptive immune responses.

Non-conditioned bone marrow chimeric mouse generation using culture-based enrichment of hematopoietic stem and progenitor cells

Bone marrow (BM) chimeric mice are a valuable tool in the field of immunology, with the genetic manipulation of donor cells widely used to study gene function under physiological and pathological settings. To date, however, BM chimera protocols require myeloablative conditioning of recipient mice, which dramatically alters steady-state hematopoiesis. Additionally, most protocols use fluorescence-activated cell sorting (FACS) of hematopoietic stem/progenitor cells (HSPCs) for ex vivo genetic manipulation. Here, we describe our development of cell culture techniques for the enrichment of functional HSPCs from mouse BM without the use of FACS purification. Furthermore, the large number of HSPCs derived from these cultures generate BM chimeric mice without irradiation. These HSPC cultures can also be genetically manipulated by viral transduction, to allow for doxycycline-inducible transgene expression in donor-derived immune cells within non-conditioned immunocompetent recipients. This technique is therefore expected to overcome current limitations in mouse transplantation models.