Shubin Zhang

TAK1 is required for the survival of hematopoietic cells and hepatocytes in mice.

Transforming growth factor β–activated kinase 1 (TAK1), a member of the MAPKKK family, is a key mediator of proinflammatory and stress signals. Activation of TAK1 by proinflammatory cytokines and T and B cell receptors induces the nuclear localization of nuclear factor κB (NF-κB) and the activation of c-Jun N-terminal kinase (JNK)/AP1 and P38, which play important roles in mediating inflammation, immune responses, T and B cell activation, and epithelial cell survival. Here, we report that TAK1 is critical for the survival of both hematopoietic cells and hepatocytes. Deletion of TAK1 results in bone marrow (BM) and liver failure in mice due to the massive apoptotic death of hematopoietic cells and hepatocytes. Hematopoietic stem cells and progenitors were among those hematopoietic cells affected by TAK1 deletion–induced cell death. This apoptotic cell death is autonomous, as demonstrated by reciprocal BM transplantation. Deletion of TAK1 resulted in the inactivation of both JNK and NF-κB signaling, as well as the down-regulation of expression of prosurvival genes.

Overexpression of Foxn1 attenuates age-associated thymic involution and prevents the expansion of peripheral CD4 memory T cells

The forkhead box n1 (Foxn1) transcription factor is essential for thymic organogenesis during embryonic development; however, a functional role of Foxn1 in the postnatal thymus is less well understood. We developed Foxn1 transgenic mice (Foxn1Tg), in which overexpression of Foxn1 is driven by the human keratin-14 promoter. Expression of the Foxn1 transgene increased the endogenous Foxn1 levels. In aged mice, overexpression of Foxn1 in the thymus attenuated the decline in thymocyte numbers, prevented the decline in frequency of early thymic progenitors, and generated a higher number of signal joint TCR excised circle. Histologic studies revealed that structural alterations associated with thymic involution were diminished in aged Foxn1 Tg. Total numbers of EpCAM+ MHC II+ and MHC II(hi) thymic epithelial cells were higher in young and old Foxn1Tg and more EpCAM+ MHC II(hi) TEC expressed Ki-67 in aged Foxn1Tg compared with WT. Furthermore, Foxn1Tg displayed a significant reduction in the expansion of splenic CD4+ memory compartments and attenuated the decline in CD4+ and CD8+ naive compartments. Our data indicate that manipulation of Foxn1 expression in the thymus ameliorates thymopoiesis in aged mice and offer a strategy to combat the age-associated decline in naive T-cell production and CD4 naive/memory ratios in the elderly.

c-Myc-mediated control of cell fate in megakaryocyte-erythrocyte progenitors.

It has been found that c-Myc protein plays a critical role in controlling self-renewal versus differentiation in hematopoietic stem cells. We report that c-Myc also controls the fate of megakaryocyte-erythrocyte progenitors through regulating the differentiation of erythroid and megakaryocytic progenitors. In addition to the significant reduction of granulocytes/macrophages and B and T lymphocytes because of the reduction of their corresponding progenitors, we found significantly increased numbers of megakaryocytic progenitors and mature megakaryocytes in bone marrow and spleens of c-Myc-knockout (c-Myc(-/-)) mice. Differentiation of erythrocytes was blocked at the erythroid progenitor stage. This increased megakaryocytopoiesis is a cell-intrinsic defect of c-Myc-mutant hematopoietic stem cells, as shown by transplantation studies. Furthermore, we found that c-Myc is required for polyploidy formation but not for cytoplasmic maturation of megakaryocytes. Megakaryocytes from c-Myc(-/-) mice are significantly smaller in size and lower in ploidy than those of control mice; however, because of the dramatic increase in megakaryocyte number, although fewer platelets are produced by each megakaryocyte, a greater than 3-fold increase in platelet number was consistently observed in c-Myc(-/-) mice. Thus, c-Myc(-/-) mice develop a syndrome of severe thrombocytosis-anemia-leukopenia because of significant increases in megakaryocytopoiesis and concomitant blockage of erythrocyte differentiation and reductions in myelolymphopoiesis.

TNF-α/Fas-RIP-1–induced cell death signaling separates murine hematopoietic stem cells/progenitors into 2 distinct populations

We studied the effects of TNF-α and Fas-induced death signaling in hematopoietic stem and progenitor cells (HSPCs) by examining their contributions to the development of bone marrow failure syndromes in Tak1-knockout mice (Tak1(-/-)). We found that complete inactivation of TNF-α signaling by deleting both of its receptors, 1 and 2 (Tnfr1(-/-)r2(-/-)), can prevent the death of 30% to 40% of Tak1(-/-) HSPCs and partially repress the bone marrow failure phenotype of Tak1(-/-) mice. Fas deletion can prevent the death of 5% to 10% of Tak1(-/-) HSPCs but fails to further improve the survival of Tak1(-/-)Tnfr1(-/-)r2(-/-) HSPCs, suggesting that Fas might induce death within a subset of TNF-α-sensitive HSPCs. This TNF-α/Fas-induced cell death is a type of receptor-interacting protein-1 (RIP-1)-dependent programmed necrosis called necroptosis, which can be prevented by necrostatin-1, a specific RIP-1 inhibitor. In addition, we found that the remaining Tak1(-/-) HSPCs died of apoptosis mediated by the caspase-8-dependent extrinsic apoptotic pathway. This apoptosis can be converted into necroptosis by the inhibition of caspase-8 and prevented by inhibiting both caspase-8 and RIP-1 activities. We concluded that HSPCs are heterogeneous populations in response to death signaling stimulation. Tak1 mediates a critical survival signal, which protects against both TNF-α/Fas-RIP-1-dependent necroptosis and TNF-α/Fas-independent apoptosis in HSPCs.

IL22 Regulates Human Urothelial Cell Sensory and Innate Functions through Modulation of the Acetylcholine Response, Immunoregulatory Cytokines and Antimicrobial Peptides: Assessment of an In Vitro Model

Human urinary disorders are generally studied in rodent models due to limitations of functional in vitro culture models of primary human urothelial cells (HUCs). Current HUC culture models are often derived from immortalized cancer cell lines, which likely have functional characteristics differ from healthy human urothelium. Here, we described a simple explant culture technique to generate HUCs and assessed their in vitro functions. Using transmission electron microscopy, we assessed morphology and heterogeneity of the generated HUCs and characterized their intercellular membrane structural proteins relative to ex vivo urothelium tissue. We demonstrated that our cultured HUCs are free of fibroblasts. They are also heterogeneous, containing cells characteristic of both immature basal cells and mature superficial urothelial cells. The cultured HUCs expressed muscarinic receptors (MR1 and MR2), carnitine acetyltransferase (CarAT), immunoregulatory cytokines IL7, IL15, and IL23, as well as the chemokine CCL20. HUCs also expressed epithelial cell-specific molecules essential for forming intercellular structures that maintain the functional capacity to form the physiological barrier of the human bladder urothelium. A subset of HUCs, identified by the high expression of CD44, expressed the Toll-like receptor 4 (TLR4) along with its co-receptor CD14. We demonstrated that HUCs express, at the mRNA level, both forms of the IL22 receptor, the membrane-associated (IL22RA1) and the secreted soluble (IL22RA2) forms; in turn, IL22 inhibited expression of MR1 and induced expression of CarAT and two antimicrobial peptides (S100A9 and lipocalin-2). While the cellular sources of IL22 have yet to be identified, the HUC cytokine and chemokine profiles support the concept that IL22-producing cells are present in the human bladder mucosa tissue and that IL22 plays a regulatory role in HUC functions. Thus, the described explant technique is clearly capable of generating functional HUCs suitable for the study of human urinary tract disorders, including interactions between urothelium and IL22-producing cells.

A potential role for B cells in suppressed immune responses in cord blood transplant recipients

We evaluated immune reconstitution in 58 adults who received hematopoietic SCTs from allogeneic siblings (allosib), matched unrelated donors (MUD) or cord blood (CB) at 90-day intervals for 1 year post transplant. CB recipients had a higher incidence of infections in the first 100 days compared with allosib and MUD recipients. The number of circulating T cells was lower in CB recipients compared with MUD recipients at 90 days and compared with allosib recipients at 180 days. Spectratype analysis of the TCR Vβ complementarity determining region 3 (CDR3) of patient lymphocytes revealed that the TCR repertoire remained poorly diversified even at 360 days in nearly all patients. In contrast, the number of circulating B cells was significantly elevated in CB recipients compared with allosib recipients throughout the first year post transplant and compared with MUD recipients at 9–12 months. Spectratype analysis of the B-cell receptor VH CDR3 showed that the B-cell repertoire was diversified in most patients by 90 days. CD5pos B cells from assayed CB recipients expressed intracellular IL-10 early post transplant. Our data suggest that B cells, in addition to T cells, may have a role in impaired immune responses in CB transplant patients.

Association between early promoter-specific DNA methylation changes and outcome in older acute myeloid leukemia patients.

Treatment options for older patients with acute myeloid leukemia (AML) range from supportive care alone to full-dose chemotherapy. Identifying factors that predict response to therapy may help increase efficacy and avoid toxicity. The phase II SWOG S0703 study investigated the use of hydroxyurea and azacitidine with gemtuzumab ozogamicin in the elderly AML population and found survival rates similar to those expected with standard AML regimens, with less toxicity. As part of this study, global DNA methylation along with promoter DNA methylation and expression analysis of six candidate genes (CDKN2A, CDKN2B, HIC1, RARB, CDH1 and APAF1) were determined before and during therapy to investigate whether very early changes are prognostic for clinical response. Global DNA methylation was not associated with a clinical response. Samples after 3 or 4 days of treatment with azacitidine showed significantly decreased CDKN2A promoter DNA methylation in patients achieving complete remission (CR) compared to those who did not. Samples from day 7 of treatment showed significantly decreased RARB, CDKN2B and CDH1 promoter DNA methylation in responders compared to nonresponders. Gene-specific DNA methylation analysis of peripheral blood samples may help early identification of those older AML patients most likely to benefit from demethylating agent therapy.

Enhancing T Lineage Production in Aged Mice: A Novel Function of Foxn1 in the Bone Marrow Niche

Foxn1 is essential for thymic organogenesis and T lymphopoiesis. Whereas reduced Foxn1 expression results in a decline in T lymphopoiesis, overexpression of Foxn1 in the thymus of a transgenic mouse model (Foxn1Tg) attenuates the age-associated decline in T lymphopoiesis. T lymphopoiesis begins with early T cell progenitors (ETP), derived from multipotent progenitors (MPP) in the bone marrow (BM). A decline in MPP and ETP numbers with age is thought to contribute to reduced T lymphopoiesis. Previously, we showed that reduced ETP number with age is attenuated in Foxn1 transgenic (Tg); whether the effect is initiated in the BM with MPP is not known. In this study, we report that Foxn1 is expressed in wild-type BM and overexpressed in Foxn1Tg. With age, the number of MPP in Foxn1Tg was not reduced, and Foxn1Tg also have a larger pool of hematopoietic stem cells. Furthermore, the Foxn1Tg BM is more efficient in generating MPP. In contrast to MPP, common lymphoid progenitors and B lineage cell numbers were significantly lower in both young and aged Foxn1Tg compared with wild type. We identified a novel population of lineageneg/low, CD45pos EpCAMpos, SCA1pos, CD117neg, CD138neg, MHCIIneg cells as Foxn1-expressing BM cells that also express Delta-like 4. Thus, Foxn1 affects both T lymphopoiesis and hematopoiesis, and the Foxn1 BM niche may function in skewing MPP development toward T lineage progenitors.

Importance of a specific amino acid pairing for murine MLL leukemias driven by MLLT1/3 or AFF1/4

Acute leukemias caused by translocations of the MLL gene at chromosome 11 band q23 (11q23) are characterized by a unique gene expression profile. More recently, data from several laboratories indicate that the most commonly encountered MLL fusion proteins, MLLT1, MLLT3, and AFF1 are found within a molecular complex that facilitates the elongation phase of mRNA transcription. Mutational analyses suggest that interaction between the MLLT1/3 proteins and AFF family proteins are required for experimental transformation of hematopoietic progenitor cells (HPCs). Here, we define a specific pairing of two amino acids that creates a salt bridge between MLLT1/3 and AFF proteins that is critically important for MLL-mediated transformation of HPCs. Our findings, coupled with the newly defined structure of MLLT3 in complex with AFF1, should facilitate the development of small molecules that block this amino acid interaction and interfere with the activity of the most common MLL oncoproteins.