Bone marrow connexin-43 expression is critical for hematopoietic regeneration after chemotherapy

Contact between bone marrow (BM) hematopoietic stem cells (HSC) and osteoblast/stromal (OS) cells has been shown to be crucial in the regulation of hematopoiesis. However, very little is known about the regulatory mechanisms of direct cell-to-cell communication in the hematopoietic microenvironment. Gap junction channels (connexons) are formed by polypeptides (connexins) arranged in hexamers and represent the best described intercellular communication system. Connexin-43 (Cx43) is expressed by BM OS cells and has been associated with the cadherin/beta -catenin signaling pathway, recently reported as relevant in the OS/HSC interaction at the stem cell niche. Here, we employed an inducible gene-targeted murine approach to study the role of Cx43 in HSC proliferation and differentiation in vivo. Mx-Cre/Cx43+/+ and Mx-Cre/Cx43flox/flox littermates have been analyzed after gene deletion induced in vivo by the interferon-inducer poly (I)-poly (C), generating control (Cx43+) and Cx43-deficient (Cx43-/-) mice. After one week, Cx43+ and Cx43-/- mice were treated with 5-fluorouracil (5-FU). Cx43 expression in Cx43-/- BM was markedly reduced (> 90%) as analyzed on day +14 post-5-FU treatment. Cx43 deficiency did not induce a significant change in peripheral blood counts before 5-FU treatment, but the hematopoiesis recovery after 5-FU treatment was severely impaired as demonstrated by absence of recovery of peripheral blood counts, including profound neutropenia, anemia with reticulocytopenia, thrombocytopenia and a 5- to 8-fold decrease of cellularity and hematopoietic progenitor content (granulomacrophagic colony-forming-units (CFU-GM-), erythroid burst forming units (BFU-E) and mixed colony forming units (CFU-mix-) in BM and spleen on day +14 post-5-FU treatment. However, the femoral content of Lin-/c-kit+/Sca1+ cells in Cx43-/- BM was maintained when compared to Cx43+ BM. Short-term competitive repopulation ability of Cx43-/- BM cells was diminished as compared to Cx43+ mice, specifically for myeloid and B lymphoid cells, but showed spared long-term competitive repopulation ability with roughly normal hematopoietic differentiation. These data suggest that hematopoietic regeneration after cycle-specific chemotherapy is blocked in Cx43-deficient mice at the long-term HSC repopulating level. Cx43 expression within the BM appears to be crucial in the development of an efficient response to hematopoietic stress.     

Telomere dysfunction induces environmental alterations limiting hematopoietic stem cell function and engraftment

Cell-intrinsic checkpoints limit the proliferative capacity of primary cells in response to telomere dysfunction. It is not known, however, whether telomere dysfunction contributes to cell-extrinsic alterations that impair stem cell function and organ homeostasis. Here we show that telomere dysfunction provokes defects of the hematopoietic environment that impair B lymphopoiesis but increase myeloid proliferation in aging telomerase knockout (Terc(-/-)) mice. Moreover, the dysfunctional environment limited the engraftment of transplanted wild-type hematopoietic stem cells (HSCs). Dysfunction of the hematopoietic environment was age dependent and correlated with progressive telomere shortening in bone marrow stromal cells. Telomere dysfunction impaired mesenchymal progenitor cell function, reduced the capacity of bone marrow stromal cells to maintain functional HSCs, and increased the expression of various cytokines, including granulocyte colony-stimulating factor (G-CSF), in the plasma of aging mice. Administration of G-CSF to wild-type mice mimicked some of the defects seen in aging Terc(-/-) mice, including impairment of B lymphopoiesis and HSC engraftment. Conversely, inhibition of G-CSF improved HSC engraftment in aged Terc(-/-) mice. Taken together, these results show that telomere dysfunction induces alterations of the environment that can have implications for organismal aging and cell transplantation therapies.     

The endoplasmic reticulum chaperone protein GRP94 is required for maintaining hematopoietic stem cell interactions with the adult bone marrow niche

Hematopoietic stem cell (HSC) homeostasis in the adult bone marrow (BM) is regulated by both intrinsic gene expression products and interactions with extrinsic factors in the HSC niche. GRP94, an endoplasmic reticulum chaperone, has been reported to be essential for the expression of specific integrins and to selectively regulate early T and B lymphopoiesis. In GRP94 deficient BM chimeras, multipotent hematopoietic progenitors persisted and even increased, however, the mechanism is not well understood. Here we employed a conditional knockout (KO) strategy to acutely eliminate GRP94 in the hematopoietic system. We observed an increase in HSCs and granulocyte-monocyte progenitors in the Grp94 KO BM, correlating with an increased number of colony forming units. Cell cycle analysis revealed that a loss of quiescence and an increase in proliferation led to an increase in Grp94 KO HSCs. This expansion of the HSC pool can be attributed to the impaired interaction of HSCs with the niche, evidenced by enhanced HSC mobilization and severely compromised homing and lodging ability of primitive hematopoietic cells. Transplanting wild-type (WT) hematopoietic cells into a GRP94 null microenvironment yielded a normal hematology profile and comparable numbers of HSCs as compared to WT control, suggesting that GRP94 in HSCs, but not niche cells, is required for maintaining HSC homeostasis. Investigating this, we further determined that there was a near complete loss of integrin α4 expression on the cell surface of Grp94 KO HSCs, which showed impaired binding with fibronectin, an extracellular matrix molecule known to play a role in mediating HSC-niche interactions. Furthermore, the Grp94 KO mice displayed altered myeloid and lymphoid differentiation. Collectively, our studies establish GRP94 as a novel cell intrinsic factor required to maintain the interaction of HSCs with their niche, and thus regulate their physiology.     

Hematopoietic cells from mice that are deficient in both Bcrp1/Abcg2 and Mdr1a/1b develop normally but are sensitized to mitoxantrone

Hematopoietic stem cells (HSCs) express Mdr1a/1b and Bcrp1/Abcg2, which are members of the ATP binding cassette transporter family. Mice lacking both Mdr1-type genes (Mdr1a and Mdr1b) or Bcrp1 had normal hematopoietic development, but it has been unclear whether Mdr1a/1b and Bcrp1 play redundant roles in hematopoiesis. We generated a mouse model lacking both Mdr1a/1b and Bcrp1 expression (M-/-B-/-). The M-/-B-/- mice had normal numbers of peripheral blood cells, bone marrow colony-forming cells (CFCs) and colony-forming units-spleen (CFU-S), and demonstrated normal hematopoietic development. There was a near total elimination of side population (SP) cells in the bone marrow of M-/-B-/- mice compared to M+/+B-/- mice, primarily in the subpopulation lacking other HSC markers, which indicated that Mdr1a/1b was responsible for a small portion of SP cells that were mainly mature cells. Hematopoietic progenitor cells from the bone marrow of M-/-B-/- mice were more sensitive to mitoxantrone in vitro compared to either M-/-B+/+ or M+/+B-/- mice, suggesting that Mdr1a/1b and Bcrp1 may provide additive protection to HSCs against genotoxic agents. These studies demonstrate the lack of functional redundancy between these transporters for HSC development and further clarify their contributing role to the SP phenotype in HSCs and to intrinsic drug resistance within hematopoietic progenitor cells.     

Absence of a Red Blood Cell Phenotype in Mice with Hematopoietic Deficiency of SEC23B

Congenital dyserythropoietic anemia type II (CDAII) is an autosomal recessive disease of ineffective erythropoiesis characterized by increased bi/multinucleated erythroid precursors in the bone marrow. CDAII results from mutations in SEC23B. The SEC23 protein is a core component of coat protein complex II-coated vesicles, which transport secretory proteins from the endoplasmic reticulum to the Golgi apparatus. Though the genetic defect underlying CDAII has been identified, the pathophysiology of this disease remains unknown. We previously reported that SEC23B-deficient mice die perinatally, exhibiting massive pancreatic degeneration, with this early mortality limiting evaluation of the adult hematopoietic compartment. We now report that mice with SEC23B deficiency restricted to the hematopoietic compartment survive normally and do not exhibit anemia or other CDAII characteristics. We also demonstrate that SEC23B-deficient hematopoietic stem cells (HSC) do not exhibit a disadvantage at reconstituting hematopoiesis when compared directly to wild-type HSC in a competitive repopulation assay. Secondary bone marrow transplants demonstrated continued equivalence of SEC23B-deficient and WT HSC in their hematopoietic reconstitution potential. The surprising discordance in phenotypes between SEC23B-deficient mice and humans may reflect an evolutionary shift in SEC23 paralog function and/or expression, or a change in a specific COPII cargo critical for erythropoiesis.     

Disseminated prostate cancer cells can instruct hematopoietic stem and progenitor cells to regulate bone phenotype

Prostate cancer metastases and hematopoietic stem cells (HSC) frequently home to the bone marrow, where they compete to occupy the same HSC niche. We have also shown that under conditions of hematopoietic stress, HSCs secrete the bone morphogenetic proteins (BMP)-2 and BMP-6 that drives osteoblastic differentiation from mesenchymal precursors. As it is not known, we examined whether metastatic prostate cancer cells can alter regulation of normal bone formation by HSCs and hematopoietic progenitor cells (HPC). HSC/HPCs isolated from mice bearing nonmetastatic and metastatic tumor cells were isolated and their ability to influence osteoblastic and osteoclastic differentiation was evaluated. When the animals were inoculated with the LNCaP C4-2B cell line, which produces mixed osteoblastic and osteolytic lesions in bone, HPCs, but not HSCs, were able to induced stromal cells to differentiate down an osteoblastic phenotype. Part of the mechanism responsible for this activity was the production of BMP-2. On the other hand, when the animals were implanted with PC3 cells that exhibits predominantly osteolytic lesions in bone, HSCs derived from these animals were capable of directly differentiating into tartrate-resistant acid phosphatase-positive osteoclasts through an interleukin-6-mediated pathway. These studies for the first time identify HSC/HPCs as novel targets for future therapy involved in the bone abnormalities of prostate cancer.     

Dual role of Mpl receptor during the establishment of definitive hematopoiesis

Cytokine signaling pathways are important in promoting hematopoietic stem cell (HSC) self-renewal, proliferation and differentiation. Mpl receptor and its ligand, TPO, have been shown to play an essential role in the early steps of adult hematopoiesis. We previously demonstrated that the cytoplasmic domain of Mpl promotes hematopoietic commitment of embryonic stem cells in vitro, and postulated that Mpl could be important in the establishment of definitive hematopoiesis. To answer this question, we investigated the temporal expression of Mpl during mouse development by in situ hybridization. We found Mpl expression in the HSCs clusters emerging in the AGM region, and in the fetal liver (FL) as early as E10.5. Using Mpl(-/-) mice, the functional relevance of Mpl expression was tested by comparing the hematopoietic progenitor (HP) content, long-term hematopoietic reconstitution (LTR) abilities and HSC content of control and Mpl(-/-) embryos at different times of development. In the AGM, we observed delayed production of HSCs endowed with normal LTR but presenting a self-renewal defect. During FL development, we detected a decrease in HP and HSC potential associated with a defect in amplification and self-renewal/survival of the lin(-) AA4.1(+) Sca1(+) population of HSCs. These results underline the dual role of Mpl in the generation and expansion of HSCs during establishment of definitive hematopoiesis.     

A novel role for PECAM-1 (CD31) in regulating haematopoietic progenitor cell compartmentalization between the peripheral blood and bone marrow

Although the expression of PECAM-1 (CD31) on vascular and haematopoietic cells within the bone marrow microenvironment has been recognized for some time, its physiological role within this niche remains unexplored. In this study we show that PECAM-1 influences steady state hematopoietic stem cell (HSC) progenitor numbers in the peripheral blood but not the bone marrow compartment. PECAM-1(-/-) mice have higher levels of HSC progenitors in the blood compared to their littermate controls. We show that PECAM-1 is required on both progenitors and bone marrow vascular cells in order for efficient transition between the blood and bone marrow to occur. We have identified key roles for PECAM-1 in both the regulation of HSC migration to the chemokine CXCL12, as well as maintaining levels of the matrix degrading enzyme MMP-9 in the bone marrow vascular niche. Using intravital microscopy and adoptive transfer of either wild type (WT) or PECAM-1(-/-) bone marrow precursors, we demonstrate that the increase in HSC progenitors in the blood is due in part to a reduced ability to migrate from blood to the bone marrow vascular niche. These findings suggest a novel role for PECAM-1 as a regulator of resting homeostatic progenitor cell numbers in the blood.     

Galectin-3 modulates phagocytosis-induced stellate cell activation and liver fibrosis in vivo

Hepatic stellate cells (HSC), the key fibrogenic cells of the liver, transdifferentiate into myofibroblasts upon phagocytosis of apoptotic hepatocytes. Galectin-3, a β-galactoside-binding lectin, is a regulator of the phagocytic process. In this study, our aim was to study the mechanism by which extracellular galectin-3 modulates HSC phagocytosis and activation. The role of galectin-3 in engulfment was evaluated by phagocytosis and integrin binding assays in primary HSC. Galectin-3 expression was studied by real-time PCR and enzyme-linked immunosorbent assay, and in vivo studies were done in wild-type and galectin-3(-/-) mice. We found that HSC from galectin-3(-/-) mice displayed decreased phagocytic activity, expression of transforming growth factor-β1, and procollagen α1(I). Recombinant galectin-3 reversed this defect, suggesting that extracellular galectin-3 is required for HSC activation. Galectin-3 facilitated the α(v)β(3) heterodimer-dependent binding, indicating that galectin-3 modulates HSC phagocytosis via cross-linking this integrin and enhancing the tethering of apoptotic cells. Blocking integrin α(v)β(3) resulted in decreased phagocytosis. Galectin-3 expression and release were induced in active HSC engulfing apoptotic cells, and this was mediated by the nuclear factor-κB signaling. The upregulation of galectin-3 in active HSC was further confirmed in vivo in bile duct-ligated (BDL) rats. Galectin-3(-/-) mice displayed significantly decreased fibrosis, with reduced expression of α-smooth muscle actin and procollagen α1(I) following BDL. In summary, extracellular galectin-3 plays a key role in liver fibrosis by mediating HSC phagocytosis, activation, and subsequent autocrine and paracrine signaling by a feedforward mechanism.     

The emergence of hematopoietic stem cells is initiated in the placental vasculature in the absence of circulation

The mouse placenta was unveiled as an important reservoir for hematopoietic stem cells (HSCs), yet the origin of placental HSCs was unknown. By tracking developing HSCs by expression of Runx1-lacZ and CD41, we have found that HSCs emerge in large vessels in the placenta. Analysis of Ncx1(-/-) embryos, which lack a heartbeat, verified that HSC development is initiated in the placental vasculature independent of blood flow. However, fewer CD41+ hematopoietic cells were found in Ncx1(-/-) placentas than in controls, implying that some HSCs/progenitors colonize the placenta via circulation and/or HSC emergence is compromised without blood flow. Importantly, placentas from Ncx1(-/-) embryos possessed equal potential to generate myelo-erythroid and B and T lymphoid cells upon explant culture, verifying intact multilineage hematopoietic potential, characteristic of developing HSCs. These data suggest that, in addition to providing a niche for a large pool of HSCs prior to liver colonization, the placenta is a true site of HSC generation.     

Long-Term Reproducible Expression in Human Fetal Liver Hematopoietic Stem Cells with a UCOE-Based Lentiviral Vector

Hematopoietic Stem Cell (HSC) targeted gene transfer is an attractive treatment option for a number of hematopoietic disorders caused by single gene defects. However, extensive methylation of promoter sequences results in silencing of therapeutic gene expression. The choice of an appropriate promoter is therefore crucial for reproducible, stable and long-term transgene expression in clinical gene therapy. Recent studies suggest efficient and stable expression of transgenes from the ubiquitous chromatin opening element (UCOE) derived from the human HNRPA2B1-CBX3 locus can be achieved in murine HSC. Here, we compared the use of HNRPA2B1-CBX3 UCOE (A2UCOE)-mediated transgene regulation to two other frequently used promoters namely EF1α and PGK in human fetal liver-derived HSC (hflHSC). Efficient transduction of hflHSC with a lentiviral vector containing an HNRPA2B1-CBX3 UCOE-eGFP (A2UCOE-eGFP) cassette was achieved at higher levels than that obtained with umbilical cord blood derived HSC (3.1x; p<0.001). While hflHSC were readily transduced with all three test vectors (A2UCOE-eGFP, PGK-eGFP and EF1α-eGFP), only the A2-UCOE construct demonstrated sustained transgene expression in vitro over 24 days (p<0.001). In contrast, within 10 days in culture a rapid decline in transgene expression in both PGK-eGFP and EF1α-eGFP transduced hflHSC was seen. Subsequently, injection of transduced cells into immunodeficient mice (NOD/SCID/Il2rg ^-/-) demonstrated sustained eGFP expression for the A2UCOE-eGFP group up to 10 months post transplantation whereas PGK-eGFP and EF1α-eGFP transduced hflHSC showed a 5.1 and 22.2 fold reduction respectively over the same time period. We conclude that the A2UCOE allows a more efficient and stable expression in hflHSC to be achieved than either the PGK or EF1α promoters and at lower vector copy number per cell.     

Forced expression of HoxB4 enhances hematopoietic differentiation by human embryonic stem cells

HoxB4 has been shown to enhance hematopoietic engraftment by hematopoietic stem cells (HSC) from differentiating mouse embryonic stem cell (mESC) cultures. Here we examined the effect of ectopic expression of HoxB4 in differentiated human embryonic stem cells (hESCs). Stable HoxB4-expressing hESCs were established by lentiviral transduction, and the forced expression of HoxB4 did not affect stem cell features. HoxB4-expressing hESC-derived CD34+ cells generated higher numbers of erythroid and blast-like colonies than controls. The number of CD34+ cells increased but CD45+ and KDR+ cell numbers were not significantly affected. When the hESC derived CD34+ cells were transplanted into NOD/SCID beta 2m-/- mice, the ectopic expression of HoxB4 did not alter their repopulating capacity. Our findings show that overexpression of HoxB4 in differentiating hESCs increases hematopoietic colony formation and hematopoietic cell formation in vitro, but does not affect in vivo repopulation in adult mice hosts.     

The endothelial antigen ESAM monitors hematopoietic stem cell status between quiescence and self-renewal

Whereas most hematopoietic stem cells (HSC) are quiescent in homeostasis, they actively proliferate in response to bone marrow (BM) injury. Signals from the BM microenvironment are thought to promote entry of HSC into the cell cycle. However, it has been cumbersome to assess cycle status of viable HSC and thus explore unique features associated with division. In this study, we show that expression of endothelial cell-selective adhesion molecule (ESAM) can be a powerful indicator of HSC activation. ESAM levels clearly mirrored the shift of HSC between quiescence and activation, and it was prominent in comparison with other HSC-related Ags. ESAM(hi) HSC were actively dividing, but had surprisingly high long-term reconstituting capacity. Immunohistochemical analyses showed that most ESAM(hi) HSC were located near vascular endothelium in the BM after 5-fluorouracil treatment. To determine the importance of ESAM in the process of BM recovery, ESAM knockout mice were treated with 5-fluorouracil and their hematopoietic reconstruction was examined. The ESAM deficiency caused severe and prolonged BM suppression, suggesting that ESAM is functionally indispensable for HSC to re-establish homeostatic hematopoiesis. With respect to intracellular regulators, NF-κB and topoisomerase II levels correlated with the ESAM upregulation. Thus, our data demonstrate that the intensity of ESAM expression is useful to trace activated HSC and to understand molecular events involved in stem cell states.     

Granulocyte colony stimulating factor expands hematopoietic stem cells within the central but not endosteal bone marrow region

Granulocyte colony stimulating factor (G-CSF) is clinically well established for the mobilization of hematopoietic stem cells (HSC). Extensive data on the underlying mechanism of G-CSF induced mobilization is available; however, little is known regarding the functional effect of G-CSF on HSC within the bone marrow (BM). In this study we analyzed the proportion and number of murine HSC in the endosteal and central bone marrow regions after 4 days of G-CSF administration. We demonstrate that the number of HSC, defined as CD150(+)CD48(-)LSK cells (LSKSLAM cells), increased within the central BM region in response to G-CSF, but not within the endosteal BM region. In addition the level of CD150 and CD48 expression also increased on cells isolated from both regions. We further showed that G-CSF mobilized proportionally fewer LSKSLAM compared to LSK cells, mobilized LSKSLAM had colony forming potential and the presence of these cells can be used as a measure for mobilization efficiency. Together we provide evidence that HSC in the BM respond differently to G-CSF and this is dependent on their location. These findings will be valuable in developing new agents which specifically mobilize HSC from the endosteal BM region, which we have previously demonstrated to have significantly greater hematopoietic potential compared to their phenotypically identical counterparts located in other regions of the BM.     

Glycogen synthase kinase-3 is an in vivo regulator of hematopoietic stem cell repopulation

The in vivo regulation of hematopoietic stem cell (HSC) function is poorly understood. Here, we show that hematopoietic repopulation can be augmented by administration of a glycogen synthase kinase-3 (GSK-3) inhibitor to recipient mice transplanted with mouse or human HSCs. GSK-3 inhibitor treatment improved neutrophil and megakaryocyte recovery, recipient survival and resulted in enhanced sustained long-term repopulation. The output of primitive Lin(-)c-Kit(+)Sca-1(+) cells and progenitors from HSCs increased upon GSK-3 inhibitor treatment without altering secondary repopulating ability, suggesting that the HSC pool is maintained while overall hematopoietic reconstitution is increased. GSK-3 inhibitors were found to modulate gene targets of Wnt, Hedgehog and Notch pathways in cells comprising the primitive hematopoietic compartment without affecting mature cells. Our study establishes GSK-3 as a specific in vivo modulator of HSC activity, and suggests that administration of GSK-3 inhibitors may provide a clinical means to directly enhance the repopulating capacity of transplanted HSCs.     

Allelic variation and differential expression of the mSIN3A histone deacetylase complex gene Arid4b promote mammary tumor growth and metastasis

Accumulating evidence suggests that breast cancer metastatic progression is modified by germline polymorphism, although specific modifier genes have remained largely undefined. In the current study, we employ the MMTV-PyMT transgenic mouse model and the AKXD panel of recombinant inbred mice to identify AT-rich interactive domain 4B (Arid4b; NM_194262) as a breast cancer progression modifier gene. Ectopic expression of Arid4b promoted primary tumor growth in vivo as well as increased migration and invasion in vitro, and the phenotype was associated with polymorphisms identified between the AKR/J and DBA/2J alleles as predicted by our genetic analyses. Stable shRNA-mediated knockdown of Arid4b caused a significant reduction in pulmonary metastases, validating a role for Arid4b as a metastasis modifier gene. ARID4B physically interacts with the breast cancer metastasis suppressor BRMS1, and we detected differential binding of the Arid4b alleles to histone deacetylase complex members mSIN3A and mSDS3, suggesting that the mechanism of Arid4b action likely involves interactions with chromatin modifying complexes. Downregulation of the conserved Tpx2 gene network, which is comprised of many factors regulating cell cycle and mitotic spindle biology, was observed concomitant with loss of metastatic efficiency in Arid4b knockdown cells. Consistent with our genetic analysis and in vivo experiments in our mouse model system, ARID4B expression was also an independent predictor of distant metastasis-free survival in breast cancer patients with ER+ tumors. These studies support a causative role of ARID4B in metastatic progression of breast cancer.     

Hepatitis B Virus Infection and Immunopathogenesis in a Humanized Mouse Model: Induction of Human-Specific Liver Fibrosis and M2-Like Macrophages

The mechanisms of chronic HBV infection and immunopathogenesis are poorly understood due to a lack of a robust small animal model. Here we report the development of a humanized mouse model with both human immune system and human liver cells by reconstituting the immunodeficient A2/NSG (NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ mice with human HLA-A2 transgene) with human hematopoietic stem cells and liver progenitor cells (A2/NSG-hu HSC/Hep mice). The A2/NSG-hu HSC/Hep mouse supported HBV infection and approximately 75% of HBV infected mice established persistent infection for at least 4 months. We detected human immune responses, albeit impaired in the liver, chronic liver inflammation and liver fibrosis in infected animals. An HBV neutralizing antibody efficiently inhibited HBV infection and associated liver diseases in humanized mice. In addition, we found that the HBV mediated liver disease was associated with high level of infiltrated human macrophages with M2-like activation phenotype. Importantly, similar M2-like macrophage accumulation was confirmed in chronic hepatitis B patients with liver diseases. Furthermore, gene expression analysis showed that induction of M2-like macrophage in the liver is associated with accelerated liver fibrosis and necrosis in patients with acute HBV-induced liver failure. Lastly, we demonstrate that HBV promotes M2-like activation in both M1 and M2 macrophages in cell culture studies. Our study demonstrates that the A2/NSG-hu HSC/Hep mouse model is valuable in studying HBV infection, human immune responses and associated liver diseases. Furthermore, results from this study suggest a critical role for macrophage polarization in hepatitis B virus-induced immune impairment and liver pathology.