Erythroid cells undergo enucleation and the removal of organelles during terminal differentiation 1-3 . Although autophagy has been suggested to mediate the elimination of organelles for erythroid maturation 2-6 , the molecular mechanisms underlying this process remain undefined. Here we report a role for a Bcl-2 family member, Nix (also called Bnip3L) 7-9 , in the regulation of erythroid maturation through mitochondrial autophagy. Nix −/− mice developed anaemia with reduced mature erythrocytes and compensatory expansion of erythroid precursors. Erythrocytes in the peripheral blood of Nix −/− mice exhibited mitochondrial retention and reduced lifespan in vivo. Although the clearance of ribosomes proceeded normally in the absence of Nix, the entry of mitochondria into autophagosomes for clearance was defective. Deficiency in Nix inhibited the loss of mitochondrial membrane potential (ΔΨ m ), and treatment with uncoupling chemicals or a BH3 mimetic induced the loss of ΔΨ m and restored the sequestration of mitochondria into autophagosomes in Nix −/− erythroid cells. These results suggest that Nix-dependent loss of ΔΨ m is important for targeting the mitochondria into autophagosomes for clearance during erythroid maturation, and interference with this function impairs erythroid maturation and results in anaemia. Our study may also provide insights into molecular mechanisms underlying mitochondrial quality control involving mitochondrial autophagy.Nix, a BH3-only member of the Bcl-2 family, is upregulated in erythroid cells undergoing terminal differentiation 10 . To determine the potential function for Nix in erythroid maturation, we generated Nix −/− mice using embryonic stem (ES) cells with a gene trap insertion between exons 3 and 4 of Nix ( Supplementary Fig. 2). We first examined red blood cells in the peripheral blood (RBCs), including reticulocytes and erythrocytes, in Nix −/− mice. Although RBC counts were decreased (Supplementary Table 1), polychromasia and increased reticulocytes were observed in Nix −/− mice ( Fig. 1a and Supplementary Fig. 3a). We also examined RBCs for the expression of an erythroid cell marker, glycophorin-A-associated Ter119, and for transferrin receptor CD71, which is downregulated during terminal erythroid differentiation 11,12 . Although Ter119 low CD71 high and Ter119 + CD71 high early erythroblasts 13 were absent in the peripheral blood, a significant increase in Ter119 + CD71 + reticulocytes was observed in Nix −/− mice (Fig. 1b). Electron microscopy also showed more irregularly shaped cellsCorrespondence and requests for materials should be addressed to M.C. (minc@bcm.tmc.edu) or J.W. (jinwang@bcm.tmc.edu). Author Contributions H.S. conducted the majority of the experiments, supervised by J.W. and M.C.; P.T. stained spleen sections and blood smears; S.K.D. measured osmotic fragility and assisted with biotin and CMFDA labelling; A.S. performed RT-PCR for Epo; J.T.P. and P.T. provided experimental advice; M.C. and J.W. generated the Nix −/− mice, designed experiments and...

Apoptosis in the immune system is critical for maintaining self-tolerance and preventing autoimmunity. Nevertheless, inhibiting apoptosis in lymphocytes is not alone sufficient to break self-tolerance, suggesting the involvement of other cell types. We investigated whether apoptosis in dendritic cells (DCs) helps regulate self-tolerance by generating transgenic mice expressing the baculoviral caspase inhibitor, p35, in DCs (DC-p35). DC-p35 mice displayed defective DC apoptosis, resulting in their accumulation and, in turn, chronic lymphocyte activation and systemic autoimmune manifestations. The observation that a defect in DC apoptosis can independently lead to autoimmunity is consistent with a central role for these cells in maintaining immune self-tolerance.

Vaccination has been the most widely used strategy to protect against viral infections for centuries. However, the molecular mechanisms governing the long-term persistence of immunological memory in response to vaccines remain unclear. Here we show that autophagy plays a critical role in the maintenance of memory B cells against influenza virus infection. Memory B cells displayed elevated levels of basal autophagy with increased expression of genes that regulate autophagy initiation or autophagosome maturation. Mice with B cell-specific deletion of Atg7 (B/Atg7−/−) showed normal primary antibody responses after immunization against influenza, but failed to generate protective secondary antibody responses when challenged with influenza viruses, resulting in high viral loads, widespread lung destruction and increased fatality. Our results suggest that autophagy is essential for the survival of virus-specific memory B cells and the maintenance of protective antibody responses required to combat infections.

Severe acute respiratory syndrome (SARS) is a newly emerging infectious disease. The potential recurrence of the disease from animal reservoirs highlights the significance of development of safe and efficient vaccines to prevent a future SARS epidemic. In this study, we expressed the recombinant receptor-binding domain (rRBD) in mammalian (293T) cells, insect (Sf9) cells, and E. coli, respectively, and compared their immunogenicity and protection against SARS-CoV infection in an established mouse model. Our results show that all rRBD proteins expressed in the above systems maintained intact conformation, being able to induce highly potent neutralizing antibody responses and complete protective immunity against SARS-CoV challenge in mice, albeit the rRBD expressed in 293T cells elicited stronger humoral immune responses with significantly higher neutralizing activity (P < 0.05) than those expressed in Sf9 and E. coli cells. These results suggest that all three rRBDs are effective in eliciting immune responses and protection against SARS-CoV and any of the above expression systems can be used for production of rRBD-based SARS subunit vaccines. Preference will be given to rRBD expressed in mammalian cells for future evaluation of the vaccine efficacy in a non-human primate model of SARS because of its ability to refold into a native conformation more readily and to induce higher level of neutralizing antibody responses than those expressed in E. coli and insect cells.

IntroductionDendritic cells (DCs) represent the most efficient antigenpresenting cells in capturing, processing, and presenting antigens for lymphocyte activation. [1][2][3][4][5] Several studies have shown that DCs undergo rapid turnover in vivo. [6][7][8][9] DCs may also undergo accelerated clearance from the lymphoid organs after interacting with antigen-specific T cells. 6 It is possible that the life span of DCs can influence their duration for stimulating lymphocytes, thereby affecting the outcome of lymphocyte activation and immune responses. In support of this possibility, ablation of DCs with diphtheria toxin in transgenic mice has been shown to impair the priming of antigen-specific cytotoxic T cells, 10 while inhibition of apoptosis in DCs enhances the antigen-specific immune responses. 8 Apoptosis plays essential roles in multiple cellular processes, including development, tissue homeostasis, immune tolerance, and immune surveillance. [11][12][13] The critical role for apoptosis in maintaining peripheral tolerance is demonstrated by systemic autoimmune diseases that result from mutations in the proapoptotic Fas receptor or Fas ligand genes, in both humans and mice. [12][13][14] DCs may contribute to the maintenance of immune tolerance. 5,15,16 We have observed that targeted inhibition of apoptosis in DCs with p35, a caspase inhibitor that preferentially targets caspase-8 in the Fassignaling pathway, 17 can induce spontaneous T-cell activation and the development of systemic autoimmunity in transgenic mice. 18 However, whether other apoptosis pathways in DCs help to regulate self-tolerance remains to be tested.The Bcl-2 family proteins are critical regulators of mitochondrial apoptosis pathway. 19,20 They share one or more Bcl-2 homology (BH) domains and can be divided into 3 subfamilies, 19,20 including the antiapoptotic subfamily proteins, such as Bcl-2, Bcl-xL, and Mcl-1; the proapoptotic Bax-and Bak-like proteins; and the proapoptotic BH3-only subfamily, such as Bim and Bid. In particular, BH3-only proteins emerge as the upstream sensors for different apoptosis signaling in specific cell types. 21 BH3-only proteins either inhibit the antiapoptotic molecules as "derepressors" or directly activate proapoptotic Bax or Bak to induce apoptosis. 21,22 Bcl-2 family proteins may also play important roles in the regulation of apoptosis in DCs. 8,9 It has been shown that overexpression of Bcl-2 in DCs can prolong DC survival and enhance the immunogenicity of DCs in transgenic mice. 8 This suggests that the mitochondrion-dependent apoptosis regulated by Bcl-2 family proteins may play an important role in regulating DC survival and functions.Bim is a proapoptotic BH3-only protein in the Bcl-2 family that has been shown to play a critical role in regulating homeostasis of lymphocytes. 21,23 Although Bim can be rapidly induced in DCs by different stimuli, 9 whether apoptosis and homeostasis of DCs can be regulated by Bim has not been determined. Deficiency in Bim causes significant expansion of lymphocytes and ...

MicroRNAs (miRNAs) are a class of recently discovered noncoding RNA genes that post-transcriptionally regulate gene expression. It is becoming clear that miRNAs play an important role in the regulation of gene translation during development. However, in mammals, expression data are principally based on whole tissue analysis and are still very incomplete. We isolated CD34 þ CD38À hematopoietic stem cells (HSCs) from human umbilical cord blood, on the basis of cell-surface markers using fluorescence-activated cell sorting (FACS). Also, CD34þ subpopulation was FACS isolated as the control. Next, using microarray containing oligonucleotides corresponding to 517 miRNAs from human, mouse, and rat genomes, we obtained miRNA gene expression profiles of both subpopulations. We focused on the HSCs correlative miRNAs with comparison to the control. The miRNAs of particular interest were confirmed by real-time RT-PCR. HSCs-overexpressed hsa-miR-520h and underexpressed hsa-miR-129 were selected for target prediction and analysis. The result showed that EIF2C3 and CAMTA1, genes related to miRNAs processing or transcription regulation, were proved to be real targets for hsa-miR-129. And ABCG2, involved in stemness maintaining, a real target for hsa-miR520h. Finally, we chose hsa-miR-520h, enriched in HSCs but low in CD34 þ cells, for functional characterization, because of its possible role in differentiation of HSCs by regulating ABCG2. As a result, hsa-miR-520h transduction into CD34 þ cells greatly increased number of different progenitor colonies in Colony-Forming-Cell assays, suggesting that hsa-miR520h may promote differentiation of HSCs into progenitor cells by inhibiting ABCG2 expression. This study paves the way for identifying HSC-specific miRNAs and their roles in HSC development.

We have recently demonstrated that Epstein-Barr virus (EBV)-encoded deoxyuridine triphosphate nucleotidohydrolase (dUTPase) modulates innate immunity in human primary monocyte-derived macrophages through toll-like receptor (TLR) 2 leading to NF-κB activation and the production of pro-inflammatory cytokines. Our previous depletion studies indicated that dendritic cells (DCs) may also be a target of the EBV-encoded dUTPase. However, the role of EBV-encoded dUTPase in DC activation/function and its potential contribution to the inflammatory cellular milieu characteristic of EBV-associated diseases remains poorly understood. In the present study, we demonstrate that EBV-encoded dUTPase significantly altered the expression of genes involved in oncogenesis, inflammation and viral defense mechanisms in human primary DCs by microarray analysis. Proteome array studies revealed that EBV-encoded dUTPase modulates DC immune responses by inducing the secretion of pro-inflammatory TH1/TH17 cytokines. More importantly, we demonstrate that EBV-encoded dUTPase is secreted in exosomes from chemically induced Raji cells at sufficient levels to induce NF-κB activation and cytokine secretion in primary DCs and peripheral blood mononuclear cells (PBMCs). Interestingly, the production of pro-inflammatory cytokines in DCs and PBMCs was TLR2-dependent. Together these findings suggest that the EBV-encoded dUTPase may act as an intercellular signaling molecule capable of modulating the cellular microenvironment and thus, it may be important in the pathophysiology of EBV related diseases.

Janus kinases comprise carboxyterminal kinase, pseudokinase, SH2-like, and N-terminal FERM domains. We identified three patient-derived mutations in the FERM domain of Jak3 and investigated the functional consequences of these mutations. These mutations inhibited receptor binding and also abrogated kinase activity, suggesting interactions between the FERM and kinase domains. In fact, the domains were found to physically associate, and coexpression of the FERM domain enhanced activity of the isolated kinase domain. Conversely, staurosporine, which alters kinase domain structure, disrupted receptor binding, even though the catalytic activity of Jak3 is dispensable for receptor binding. Thus, the Jak FERM domain appears to have two critical functions: receptor interaction and maintenance of kinase integrity.