Latest Articles

Control of hematopoietic stem cell quiescence by the E3 ubiquitin ligase Fbw7

2008-05-12

Ubiquitination is a posttranslational mechanism that controls diverse cellular processes. We focus here on the ubiquitin ligase Fbw7, a recently identified hematopoietic tumor suppressor that can target for degradation several important oncogenes, including Notch1, c-Myc, and cyclin E. We have generated conditional Fbw7 knockout animals and inactivated the gene in hematopoietic stem cells (HSCs), progenitors, and their differentiated progeny. Deletion of Fbw7 specifically and rapidly affects hematopoiesis in a cell-autonomous manner. Fbw7^–/– HSCs show defective maintenance of quiescence, leading to impaired self-renewal and a severe loss of competitive repopulating capacity. Furthermore, Fbw7^–/– progenitors are unable to colonize the thymus, leading to a profound depletion of T cell progenitors. Deletion of Fbw7 in bone marrow (BM) stem cells and progenitors leads to the stabilization of c-Myc, a transcription factor previously implicated in HSC self-renewal. On the other hand, neither Notch1 nor cyclin E is visibly stabilized in the BM of Fbw7-deficient mice. Gene expression studies of Fbw7^–/– HSCs and hematopoietic progenitors indicate that Fbw7 regulates, through the regulation of HSC cycle entry, the transcriptional "signature" that is associated with the quiescent, self-renewing HSC phenotype.

Toll-like receptor 9 inhibition reduces mortality in polymicrobial sepsis

Plitas, G., Burt, B. M., Nguyen, H. M., Bamboat, Z. M., DeMatteo, R. P. — 2008-05-12

The high rate of mortality in patients with sepsis results from an inappropriately amplified systemic inflammatory response to infection. Toll-like receptors (TLRs) are important for the activation of innate immunity against microbial pathogens. We demonstrate a critical role of TLR9 in the dysregulated immune response and death associated with sepsis. Compared with wild-type (WT) mice, TLR9^–/– mice exhibited lower serum inflammatory cytokine levels, higher bacterial clearance, and greater survival after experimental peritonitis induced by cecal ligation and puncture (CLP). Protection of TLR9^–/– mice after CLP was associated with a greater number of peritoneal dendritic cells (DCs) and granulocytes than in WT controls. Adoptive transfer of TLR9^–/– DCs was sufficient to protect WT mice from CLP and increased the influx of peritoneal granulocytes. Subsequent experiments with a depleting antibody revealed that granulocytes were required for survival in TLR9^–/– mice. Remarkably, a single injection of an inhibitory CpG sequence that blocks TLR9 protected WT mice, even when administered as late as 12 h after CLP. Our findings demonstrate that the detrimental immune response to bacterial sepsis occurs via TLR9 stimulation. TLR9 blockade is a potential strategy for the treatment of human sepsis.

Development and characterization of IL-21-producing CD4+ T cells

2008-05-12

It has recently been shown that interleukin (IL)-21 is produced by Th17 cells, functions as an autocrine growth factor for Th17 cells, and plays critical roles in autoimmune diseases. In this study, we investigated the differentiation and characteristics of IL-21–producing CD4⁺ T cells by intracellular staining. Unexpectedly, we found that under Th17-polarizing conditions, the majority of IL-21–producing CD4⁺ T cells did not produce IL-17A and -17F. We also found that IL-6 and -21 potently induced the development of IL-21–producing CD4⁺ T cells without the induction of IL-4, IFN-, IL-17A, or IL-17F production. On the other hand, TGF-β inhibited IL-6– and IL-21–induced development of IL-21–producing CD4⁺ T cells. IL-2 enhanced the development of IL-21–producing CD4⁺ T cells under Th17-polarizing conditions. Finally, IL-21–producing CD4⁺ T cells exhibited a stable phenotype of IL-21 production in the presence of IL-6, but retained the potential to produce IL-4 under Th2-polarizing conditions and IL-17A under Th17-polarizing conditions. These results suggest that IL-21–producing CD4⁺ T cells exhibit distinct characteristics from Th17 cells and develop preferentially in an IL-6–rich environment devoid of TGF-β, and that IL-21 functions as an autocrine growth factor for IL-21–producing CD4⁺ T cells.

Breaking tolerance to the natural human liver autoantigen cytochrome P450 2D6 by virus infection

2008-05-12

Autoimmune liver diseases, such as autoimmune hepatitis (AIH) and primary biliary cirrhosis, often have severe consequences for the patient. Because of a lack of appropriate animal models, not much is known about their potential viral etiology. Infection by liver-tropic viruses is one possibility for the breakdown of self-tolerance. Therefore, we infected mice with adenovirus Ad5 expressing human cytochrome P450 2D6 (Ad-2D6). Ad-2D6–infected mice developed persistent autoimmune liver disease, apparent by cellular infiltration, hepatic fibrosis, "fused" liver lobules, and necrosis. Similar to type 2 AIH patients, Ad-2D6–infected mice generated type 1 liver kidney microsomal–like antibodies recognizing the immunodominant epitope WDPAQPPRD of cytochrome P450 2D6 (CYP2D6). Interestingly, Ad-2D6–infected wild-type FVB/N mice displayed exacerbated liver damage when compared with transgenic mice expressing the identical human CYP2D6 protein in the liver, indicating the presence of a stronger immunological tolerance in CYP2D6 mice. We demonstrate for the first time that infection with a virus expressing a natural human autoantigen breaks tolerance, resulting in a chronic form of severe, autoimmune liver damage. Our novel model system should be instrumental for studying mechanisms involved in the initiation, propagation, and precipitation of virus-induced autoimmune liver diseases.

Divergent roles of endothelial NF-{kappa}B in multiple organ injury and bacterial clearance in mouse models of sepsis

Ye, X., Ding, J., Zhou, X., Chen, G., Liu, S. F. — 2008-05-12

To define the roles of endothelial-intrinsic nuclear factor B (NF-B) activity in host defense and multiple organ injury in response to sepsis, we generated double transgenic (TG) mice (EC-rtTA/I-Bmt) that conditionally overexpress a degradation-resistant form of the NF-B inhibitor I-B (I-Bmt) selectively on vascular endothelium. The EC-rtTA/I-Bmt mice had no basal, but a relatively high level of doxycycline-inducible, I-Bmt expression. I-Bmt expression was detected in endothelial cells, but not in fibroblasts, macrophages, and whole blood cells, confirming that transgene expression was restricted to the endothelium. When subjected to endotoxemia, EC-rtTA/I-Bmt mice showed endothelial-selective blockade of NF-B activation, repressed expression of multiple endothelial adhesion molecules, reduced neutrophil infiltration into multiple organs, decreased endothelial permeability, ameliorated multiple organ injury, reduced systemic hypotension, and abrogated intravascular coagulation. When subjected to cecal ligation and puncture–induced sepsis, the TG mice had less severe multiple organ injury and improved survival compared with wild-type (WT) mice. WT and EC-rtTA/I-Bmt mice had comparable capacity to clear three different pathogenic bacteria. Our data demonstrate that endothelial NF-B activity is an essential mediator of septic multiple organ inflammation and injury but plays little role in the host defense response to eradicate invading pathogenic bacteria.

Proteasomal degradation restricts the nuclear lifespan of AID

2008-05-12

Activation-induced cytidine deaminase (AID) initiates all postrearrangement processes that diversify the immunoglobulin repertoire by specific deamination of cytidines at the immunoglobulin (Ig) locus. As uncontrolled expression of AID is potentially mutagenic, different types of regulation, particularly nucleocytoplasmic shuttling, restrict the likelihood of AID–deoxyribonucleic acid encounters. We studied additional mechanisms of regulation affecting the stability of the AID protein. No modulation of protein accumulation according to the cell cycle was observed in a Burkitt's lymphoma cell line. In contrast, the half-life of AID was markedly reduced in the nucleus, and this destabilization was accompanied by a polyubiquitination that was revealed in the presence of proteasome inhibitors. The same compartment-specific degradation was observed in activated mouse B cells, and also in a non–B cell line. No specific lysine residues could be linked to this degradation, so it remains unclear whether polyubiquitination proceeds through several alternatives sites or through the protein N terminus. The nuclear-restricted form of AID displayed enhanced mutagenicity at both Ig and non-Ig loci, most notably at TP53, suggesting that modulation of nuclear AID content through proteasomal degradation may represent another level of control of AID activity.