Zhe-Xiong Lian

Induction of autoimmune cholangitis in non-obese diabetic (NOD).1101 mice following a chemical xenobiotic immunization

Our laboratory has suggested that loss of tolerance to pyruvate dehydrogenase (PDC‐E2) leads to an anti‐mitochondrial antibody response and autoimmune cholangitis, similar to human primary biliary cirrhosis (PBC). We have suggested that this loss of tolerance can be induced either via chemical xenobiotic immunization or exposure to select bacteria. Our work has also highlighted the importance of genetic susceptibility. Using the non‐obese diabetic (NOD) congenic strain 1101 (hereafter referred to as NOD.1101 mice), which has chromosome 3 regions from B6 introgressed onto a NOD background, we exposed animals to 2‐octynoic acid (2OA) coupled to bovine serum albumin (BSA). 2OA has been demonstrated previously by a quantitative structural activity relationship to react as well as or better than lipoic acid to anti‐mitochondrial antibodies. We demonstrate herein that NOD.1101 mice immunized with 2OA‐BSA, but not with BSA alone, develop high titre anti‐mitochondrial antibodies and histological features, including portal infiltrates enriched in CD8+ cells and liver granulomas, similar to human PBC. We believe this model will allow the rigorous dissection of early immunogenetic cause of biliary damage.

Deletion of interleukin (IL)‐12p35 induces liver fibrosis in dominant‐negative TGFβ receptor type II mice

Mice with a dominant‐negative transforming growth factor β receptor restricted to T cells (dnTGFβRII mice) develop an inflammatory biliary ductular disease that strongly resembles human primary biliary cirrhosis (PBC). Furthermore, deletion of the gene encoding interleukin (IL)‐12p40 resulted in a strain (IL‐12p40−/−dnTGFβRII) with dramatically reduced autoimmune cholangitis. To further investigate the role of the IL‐12 cytokine family in dnTGFβRII autoimmune biliary disease, we deleted the gene encoding the IL‐12p35 subunit from dnTGFβRII mice, resulting in an IL‐12p35−/− dnTGFβRII strain which is deficient in two members of the IL‐12 family, IL‐12 and IL‐35. In contrast to IL‐12p40−/− mice, the IL‐12p35−/−mice developed liver inflammation and bile duct damage with similar severity but delayed onset as the parental dnTGFβRII mice. The p35−/− mice also demonstrated a distinct cytokine profile characterized by a shift from a T‐helper 1 (Th1) to a Th17 response. Strikingly, liver fibrosis was frequently observed in IL‐12p35−/− mice. In conclusion, IL‐12p35−/− dnTGFβRII mice, histologically and immunologically, reflect key features of PBC, providing a useful generic model to understand the immunopathology of human PBC. (HEPATOLOGY 2013;)

Characterization of mesenchymal stem cells isolated from mouse fetal bone marrow.

Mesenchymal stem cells (MSCs) are defined as cells that can differentiate into multiple mesenchymal lineage cells. MSCs have some features (surface molecules and cytokine production, etc.) common to so‐called traditional bone marrow (BM) stromal cells, which have the capacity to support hemopoiesis. In the present study, we isolated murine MSCs (mMSCs) from the fetal BM using an anti‐PA6 monoclonal antibody (mAb) that is specific for bone marrow stromal cells. The mMSCs, called FMS/PA6‐P cells, are adherent, fibroblastic, and extensively expanded and have the ability to differentiate not only into osteoblasts and adipocytes but also into vascular endothelial cells. The FMS/PA6‐P cells produce a broad spectrum of cytokines and growth factors closely related to hemopoiesis and show good hemopoiesis‐supporting capacity both in vivo and in vitro, suggesting that they are a component of the hemopoietic stem cell niche in vivo. Interestingly, although the FMS/PA6‐P cells express a high level of the PA6 molecule, which is reactive with anti‐PA6 mAb, they gradually lose their ability to express this molecule during the course of differentiation into osteoblasts and adipocytes, indicating that the PA6 molecule might serve as a novel marker of mMSCs.

The role of CD11c+ hepatic dendritic cells in the induction of innate immune responses

The role of the liver in the initiation and maintenance of tolerance is a critical immune function that involves multiple lineages of immune cells. Included within these populations are liver dendritic cells (DCs). Although there has been significant work on the phenotypic and functional roles of splenic and bone marrow dendritic cells, as well as their subsets, comparable studies in liver have often been difficult. To address this issue we have isolated, from C57BL/6 mice, relatively pure populations of DCs and compared phenotype and function to the data from spleen using flow cytometry, cell sorter assisted purification and culture, morphology by cytospin and May–Giemsa staining, cell cycle progression, antigen uptake, cytokine production and allo‐activation potential. natural killer (NK)1·1–CD11c+ liver DC subsets (conventional DCs, T cell receptor (TcR)β–NK1·1–CD11c+B220– and plasmacytoid DCs, TcRβ–NK1·1–CD11c+B220+) efficiently endocytose dextran and produce significant levels of tumour necrosis factor (TNF)‐α, interleukin (IL)‐6 and IL‐12 p40 in response to Toll‐like receptor (TLR) ligands, with responses higher than splenic DCs. There is also a differential capability of hepatic DCs to respond to innate signals. Indeed, CD11c+ hepatic DCs have a greater capacity to respond to innate stimulation but are less capable of inducing CpG activated‐allogeneic T cells. These data suggest that hepatic dendritic cells function as a critical bridge between innate and adaptive immunity and are capable of inducing stronger innate responses with a lower capacity for allo‐stimulation than splenic dendritic cells. These properties of liver dendritic cells contribute to their unique role in the induction of tolerance.

Intra-Bone Marrow Injection of Donor Bone Marrow Cells Suspended in Collagen Gel Retains Injected Cells in Bone Marrow, Resulting in Rapid Hemopoietic Recovery in Mice

We have recently developed an innovative bone marrow transplantation (BMT) method, intra‐bone marrow (IBM)‐BMT, in which donor bone marrow cells (BMCs) are injected directly into the recipient bone marrow (BM), resulting in the rapid recovery of donor hemopoiesis and permitting a reduction in radiation doses as a pretreatment for BMT. However, even with this IBM injection, some of the injected BMCs were found to enter into circulation. Therefore, we attempted to modify the method to allow the efficient retention of injected BMCs in the donor BM. The BMCs of enhanced green fluorescent protein transgenic mice (C57BL/6 background) were suspended in collagen gel (CG) or phosphate‐buffered saline (PBS), and these cells were then injected into the BM of irradiated C57BL/6 mice. The numbers of retained donor cells in the injected BM, the day 12 colony‐forming units of spleen (CFU‐S) counts, and the reconstitution of donor cells after IBM‐BMT were compared between the CG and PBS groups. The number of transplanted cells detected in the injected BM in the CG group was significantly higher than that in the PBS group. We next carried out CFU‐S assays. The spleens of mice in the CG group showed heavier spleen weight and considerably higher CFU‐S counts than in the PBS group. Excellent reconstitution of donor hemopoietic cells in the CG group was observed in the long term (>100 days). These results suggest that the IBM injection of BMCs suspended in CG is superior to the injection of BMCs suspended in PBS.

Increased Frequency of Pre- Pro B Cells in the Bone Marrow of New Zealand Black (NZB) Mice: Implications for a Developmental Block in B Cell Differentiation

Reductions in populations of both Pre-B cell (Hardy fractions D) and Pro-B cells (Hardy fractions B–C) have been described in association with murine lupus. Recent studies of B cell populations, based on evaluation of B cell differentiation markers, now allow the enumeration and enrichment of other stage specific precursor cells. In this study we report detailed analysis of the ontogeny of B cell lineage subsets in New Zealand black (NZB) and control strains of mice. Our data suggest that B cell development in NZB mice is partially arrested at the fraction A Pre–Pro B cell stage. This arrest at the Pre-Pro B cell stage is secondary to prolonged lifespan and greater resistance to spontaneous apoptosis. In addition, expression of the gene encoding the critical B cell development transcription factor BSAP is reduced in the Pre–Pro B cell stage in NZB mice. This impairment may influence subsequent B cell development to later stages, and thereby accounts for the down-regulation of the B cell receptor component Igα (mb-1). Furthermore, levels of expression of the Rug2, λ5 and Igβ (B29) genes are also reduced in Pre–Pro B cells of NZB mice. The decreased frequency of precursor B cells in the Pre–Pro B cell population occurs at the most primitive stage of B cell differentiation.

β‐Glucosylceramide ameliorates liver inflammation in murine autoimmune cholangitis

We have demonstrated spontaneous development of autoimmune cholangitis, similar to human primary biliary cirrhosis, in mice expressing a dominant negative form of the transforming growth factor‐β receptor (dnTGF‐βRII) restricted to T cells. The autoimmune cholangitis appears to be mediated by autoreactive CD8+ T lymphocytes that home to the portal tracts and biliary system. Because the liver pathology is primarily secondary to CD8+ T cells, we have determined herein whether administration of β‐glucosylceramide (GC), a naturally occurring plant glycosphingolipid, alters the natural history of disease in this model. We chose GC because previous work has demonstrated its ability to alter CD8+ T cell responses and to down‐regulate tissue inflammation. Accordingly, dnTGF‐βRII mice were treated with either GC or control for a period of 18 weeks beginning at 6 weeks of age. Importantly, in mice that received GC, there was a significant decrease in the frequency and absolute number of autoreactive liver‐infiltrating CD8+ T cells, accompanied by a significant decrease in activated CD44high CD8+ T cell populations. Further, there was a significant reduction in portal inflammation in GC‐treated mice. Interestingly, there were no changes in anti‐mitochondrial antibodies, CD4+ T cells, CD19+ B cells or natural killer (NK) T cell populations, indicating further that the beneficial effects of GC on liver inflammation were targeted specifically to liver‐infiltrating CD8+ T cells. These data suggest that further work on GC in models of CD8+ T‐mediated inflammation are needed and point to a new therapeutic venue for potentially treating and/or modulating autoimmune disease.

Generation of functionally distinct B lymphocytes from common myeloid progenitors

Current models of adult haematopoiesis propose that haematopoietic stem cells (HSCs) differentiate into common lymphoid (CLP) and common myeloid (CMP) progenitors and establish an early separation between myeloid and lymphoid lineages. Nevertheless, the developmental potential of CMP‐associated B cells suggests the existence of alternate pathways for B lymphopoesis. The aim of this study was to compare the developmental and functional properties of CMP‐ and CLP‐derived B cells. While both populations matured through pro‐B cell and transitional B cell intermediates in the bone marrow and spleen, respectively, following transfer into irradiated mice, mature CMP‐ and CLP‐derived B cells exhibit distinct functional responses. Specifically, CMP‐derived B cells did not respond to mitogenic stimulation to the same degree as their CLP‐derived counterparts and secrete lower levels of IgM and the inflammatory cytokines such as interleukin (IL)‐6 and IL‐10. Together, these data suggest the existence of multiple pathways for generating functionally distinct B cells from bone marrow precursors.

Age‐Related Alterations in the Lymphohematopoietic and B‐Lineage Precursor Populations in NZB Mice

Significant disturbances in B lineage populations of New Zealand Black (NZB) mice have been reported, both with respect to their phenotypes as well as to their function. Notably, there is a profound age‐dependent decrease in B‐cell precursors in this strain of lupus prone mice. In efforts to characterize the impact of this disturbance in disease, we performed an intensive phenotypic and B‐cell population analysis in young and old NZB mice. Our results revealed that there was a significant age‐dependent decrease in B cell precursors at all levels of the B‐cell‐lineage developmental pathway. Analysis of the proliferative capacity of these cell populations showed a comparative decrease in cycling activity in the B‐cell‐lineage populations of old NZB mice. Furthermore, these cell subsets were much more susceptible to spontaneous apoptosis when compared with similar populations from age‐matched BALB/c or young NZB mice. Since the frequency of cells that express the interleukin‐7 receptor (IL‐7R) declines as NZB mice age, we hypothesize that impairment of IL‐7R signal transduction pathways could contribute to severe perturbations of B‐cell function in aged NZB mice.

Elevated C-met in thymic dendritic cells of New Zealand Black mice.

New Zealand Black (NZB) mice are a well-known animal model of human autoimmune disease. Although the mechanism for development of autoimmunity is unclear, NZB mice are well known for severe thymic microarchitecture abnormalities. It is thought that thymic dendritic cells (DC) may play a role in thymic education and contribute to the autoimmune process. To address this issue and, in particular, that qualitative and/or quantitative differences exist in thymic DC, we took advantage of a novel restriction analysis system that allow definition of differences in the expression of tyrosine kinases using highly enriched populations of thymic DC from NZB compared to BALB/c and C57BL/6 mice. The method chosen, restriction analysis of gene expression, allowed the determination of protein tyrosine kinase transcription profiles. We report herein that NZB mice have a significant upregulation of C-met compared to the control strains. The abnormality of the C-met transcription was confined to thymic DC. We believe that its abnormal expression reflects the resistance of thymic cells to apoptosis, which will ultimately lead to defects and/or abnormal signaling by the interaction of thymic DC and thymocytes. Further studies involving such interactions are under way.