Eleonora Ottina

Bcl-2-regulated cell death signalling in the prevention of autoimmunity

Cell death mediated through the intrinsic, Bcl-2-regulated mitochondrial apoptosis signalling pathway is critical for lymphocyte development and the establishment of central and maintenance of peripheral tolerance. Defects in Bcl-2-regulated cell death signalling have been reported to cause or correlate with autoimmunity in mice and men. This review focuses on the role of Bcl-2 family proteins implicated in the development of autoimmune disorders and their potential as targets for therapeutic intervention.

Targeting antiapoptotic A1/Bfl-1 by in vivo RNAi reveals multiple roles in leukocyte development in mice.

Gene-targeting studies in mice have identified the essential roles of most prosurvival Bcl-2 family members in normal physiology and under conditions of stress. The function of one member, Bcl2a1/Bfl-1/A1, is only poorly understood because of quadruplication of its gene locus in mice, hindering conventional knockout studies. To overcome this problem, we generated mouse models allowing traceable constitutive or reversible ablation of A1 in the hematopoietic system by RNA interference. Knockdown of A1 impaired early stages of T-cell differentiation, B-cell homeostasis, and sensitized transitional as well as follicular B cells to apoptosis induced by ligation of the B-cell receptor. As a consequence, B-cell proliferation in response to mitogens was severely impaired, whereas that of T cells appeared unaffected. Furthermore, depending on the extent of A1 knockdown, granulocytes showed increased spontaneous death in culture or failed to accumulate in significant numbers in vivo. These models highlight the critical role of A1 in leukocyte development and homeostasis, constituting valuable tools for investigating presumed roles of this Bcl-2 family member in immunity, tumorigenesis, and drug resistance.

A1/Bfl-1 in leukocyte development and cell death

The function of the anti-apoptotic Bcl-2 family member Bcl2a1/Bfl-1/A1 is poorly understood due to the lack of appropriate loss-of-function mouse models and redundant effects with other Bcl-2 pro-survival proteins upon overexpression. Expression analysis of A1 suggests predominant roles in leukocyte development, their survival upon viral or bacterial infection, as well as during allergic reactions. In addition, A1 has been implicated in autoimmunity and the pathology and therapy resistance of hematological as well as solid tumors that may aberrantly express this protein. In this review, we aim to summarize current knowledge on A1 biology, focusing on its role in the immune system and compare it to that of other pro-survival Bcl-2 proteins.

Prosurvival Bcl-2 family members reveal a distinct apoptotic identity between conventional and plasmacytoid dendritic cells

Significance Dendritic cells (DCs) are pivotal for immune responses as they present antigens to T cells. Different DC subsets have different functions and are associated with different diseases. For example, plasmacytoid DCs (pDCs) produce type 1 interferons and are associated with the autoimmune disease, systemic lupus erythematosus. Understanding control of survival/apoptosis in different DC subsets may not only provide a molecular basis for their homeostasis but also guide therapeutic intervention of immunopathology. We revealed that two major DC subsets (pDCs and conventional DCs) express distinct BCL-2 family proteins at different levels and this correlated with their survival requirements. Accordingly, clinically applicable antagonist drugs killed the appropriate DC subsets, informing on the future use of these compounds for treating immune-mediated damage. Dendritic cells (DCs) are heterogeneous, comprising subsets with functional specializations that play distinct roles in immunity as well as immunopathology. We investigated the molecular control of cell survival of two main DC subsets: plasmacytoid DCs (pDCs) and conventional DCs (cDCs) and their dependence on individual antiapoptotic BCL-2 family members. Compared with cDCs, pDCs had higher expression of BCL-2, lower A1, and similar levels of MCL-1 and BCL-XL. Transgenic overexpression of BCL-2 increased the pDC pool size in vivo with only minor impact on cDCs. With a view to immune intervention, we tested BCL-2 inhibitors and found that ABT-199 (the BCL-2 specific inhibitor) selectively killed pDCs but not cDCs. Conversely, genetic knockdown of A1 profoundly reduced the proportion of cDCs but not pDCs. We also found that conditional ablation of MCL-1 significantly reduced the size of both DC populations in mice and impeded DC-mediated immune responses. Thus, we revealed that the two DC types have different cell survival requirements. The molecular basis of survival of different DC subsets thus advocates the antagonism of selective BCL-2 family members for treating diseases pertaining to distinct DC subsets.

PARP-2 sustains erythropoiesis in mice by limiting replicative stress in erythroid progenitors

Erythropoiesis is a tightly regulated process in which multipotential hematopoietic stem cells produce mature red blood cells. Here we show that deletion of poly(ADP-ribose) polymerase-2 (PARP-2) in mice leads to chronic anemia at steady state, despite increased erythropoietin plasma levels, a phenomenon not observed in mice lacking PARP-1. Loss of PARP-2 causes shortened lifespan of erythrocytes and impaired differentiation of erythroid progenitors. In erythroblasts, PARP-2 deficiency triggers replicative stress, as indicated by the presence of micronuclei, the accumulation of γ-H2AX (phospho-histone H2AX) in S-phase cells and constitutive CHK1 and replication protein A phosphorylation. Transcriptome analyses revealed the activation of the p53-dependent DNA-damage response pathways in PARP-2-deficient cells, culminating in the upregulation of cell-cycle and cell death regulators, concomitant with G2/M arrest and apoptosis. Strikingly, while loss of the proapoptotic p53 target gene Puma restored hematocrit levels in the PARP-2-deficient mice, loss of the cell-cycle regulator and CDK inhibitor p21 leads to perinatal death by exacerbating impaired fetal liver erythropoiesis in PARP-2-deficient embryos. Although the anemia displayed by PARP-2-deficient mice is compatible with life, mice die rapidly when exposed to stress-induced enhanced hemolysis. Our results pinpoint an essential role for PARP-2 in erythropoiesis by limiting replicative stress that becomes essential in the absence of p21 and in the context of enhanced hemolysis, highlighting the potential effect that might arise from the design and use of PARP inhibitors that specifically inactivate PARP proteins.

Knockdown of the antiapoptotic Bcl-2 family member A1/Bfl-1 protects mice from anaphylaxis.

Many forms of hypersensitivity reactions and allergic responses depend on deregulated mast cell activity. Several reports suggested that the antiapoptotic Bcl-2 family protein Bcl2a1/Bfl-1/A1 plays a critical role in mast cell survival upon activation. However, its in vivo relevance is poorly understood because of quadruplication of the Bcl2a1 gene locus in mice, hindering conventional knockout studies. In this study, we used a mouse model allowing traceable constitutive knockdown of all A1 isoforms expressed in the hematopoietic system by RNA interference. Knockdown of A1 reduced mast cell numbers in the skin and impaired connective tissue–like mast cell survival upon FcεRI-mediated activation in vitro. In contrast, A1 was dispensable for mucosa-like mast cell differentiation and survival. Moreover, knockdown of A1 prevented IgE-mediated passive systemic and cutaneous anaphylaxis in vivo. Our findings demonstrate that A1 is essential for the homeostasis of connective tissue mast cells, identifying A1 as a possible therapeutic target for therapy of certain types of mast cell–driven allergy symptoms.

Conditional knockdown of BCL2A1 reveals rate-limiting roles in BCR-dependent B-cell survival

Bcl2 family proteins control mitochondrial apoptosis and its members exert critical cell type and differentiation stage-specific functions, acting as barriers against autoimmunity or transformation. Anti-apoptotic Bcl2a1/Bfl1/A1 is frequently deregulated in different types of blood cancers in humans but its physiological role is poorly understood as quadruplication of the Bcl2a1 gene locus in mice hampers conventional gene targeting strategies. Transgenic overexpression of A1, deletion of the A1-a paralogue or constitutive knockdown in the hematopoietic compartment of mice by RNAi suggested rate-limiting roles in lymphocyte development, granulopoiesis and mast cell activation. Here we report on the consequences of conditional knockdown of A1 protein expression using a reverse transactivator (rtTA)-driven approach that highlights a critical role for this Bcl2 family member in the maintenance of mature B-cell homeostasis. Furthermore, we define the A1/Bim (Bcl-2 interacting mediator of cell death) axis as a target of key kinases mediating B-cell receptor (BCR)-dependent survival signals, such as, spleen tyrosine kinase (Syk) and Brutons tyrosine kinase (Btk). As such, A1 represents a putative target for the treatment of B-cell-related pathologies depending on hyperactivation of BCR-emanating survival signals and loss of A1 expression accounts, in part, for the pro-apoptotic effects of Syk- or Btk inhibitors that rely on the ‘BH3-only’ protein Bim for cell killing.

Characterisation of a novel A1-specific monoclonal antibody.

Dear Editor, A1/BFL-1 is the least studied pro-survival BCL-2 family member. This can be largely attributed to the lack of proper tools to study A1/BFL-1 function. Owing to the genomic organisation of the A1 locus in mice (three expressed A1 genes and one pseudo-gene, interspersed by unrelated genes)1 a knockout is challenging. We generated shRNA transgenic mice in which all functional A1 isoforms were knocked down. In accordance with A1 mRNA expression studies, we found that A1 is critical for the development and survival of lymphocytes and granulocytes.2 As the A1/BFL-1 protein is regulated by ubiquitin-dependent proteasomal degradation, the A1 mRNA expression data may not truly reflect the A1/BFL-1 protein levels. Previous attempts to generate A1-specific antibodies have failed and commercially available antibodies do not reliably detect the endogenous protein. To generate A1-specific monoclonal antibodies, we immunised rats with a truncated/mutated A1 protein (delta-C20, P104K)3 together with two KLH-conjugated peptides corresponding to central and C-terminal residues of the A1 protein (aa71–84; aa129–154). Screening by ELISA and western blotting identified one monoclonal antibody that detected overexpressed A1-a, A1-b and A1-d, and to a lesser extent overexpressed human homologue BFL-1 (data not shown and Figure 1a). To test whether this antibody could reliably detect endogenous A1, we used the mouse WEHI-231 B lymphoma cells, known to express high levels of this protein.4 Western blotting revealed a single band of the molecular weight expected for A1 in untreated WEHI-231 cells (Figure 1b, first lane). Overexpressed A1 protein is highly unstable due to ubiquitin-dependent proteasomal degradation.5 To further verify the specificity of the A1 antibody, we tested the impact of protein synthesis inhibition or proteasome inhibition on the protein detected in WEHI-231 cells. As expected, the protein synthesis inhibitor cyclohexamide (CHX) decreased the intensity of the protein band, whereas the proteasome inhibitor (MG132) increased it substantially (Figure 1b). Furthermore, we were able to show that this antibody can be used to immunoprecipitate endogenous A1 protein from lysates of WEHI-231 cells (Figure 1c). Next we examined whether this antibody could also detect endogenous A1 in primary mouse cells. In accordance with previous reports on A1 mRNA expression,1 we could reliably detect A1 protein in haematopoietic tissues, such as the lymph nodes and spleen but not in the heart, kidney, liver or lungs (Figure 1d). Immunohistochemical staining using this antibody showed strong A1 protein staining within cell foci in the germinal centres of lymph nodes of non-immunised mice (Figure 1e). No staining with this antibody against A1 was observed in non-haematopoietic tissues, such as the pancreas or the heart (data not shown). To further validate the specificity of this A1 antibody in primary cells, mouse spleen cells were treated with crosslinking IgM antibodies, a stimulus known to upregulate A1 mRNA levels in B lymphocytes.6 Such BCR (B-cell receptor) stimulation increased the protein band detected by our A1 antibody and its density was further augmented when cells were additionally treated with the proteasome inhibitor MG132 during the last hour of the stimulation (Figure 1f). A1 mRNA levels are upregulated when bone marrow cells are treated with GM-CSF or when mast cells are stimulated with the calcium ionophore ionomycin.7, 8 These stimuli caused strong upregulation of the protein band detected by the A1 antibody and the density of this protein band was further increased by the addition of MG132 during the last hour of stimulation (Figures 1g and h). Finally, we validated the specificity of the antibody by using our A1 knockdown mice. In cells from these animals high GFP levels indicate high levels of A1 shRNA expression and thus low levels of endogenous A1 protein.2 We therefore FACS-sorted GFP-positive and GFP-negative spleen cells and treated them with concanavalin A (ConA), a stimulus known to upregulate A1 mRNA levels in T cells.9 As expected, our antibody detected a protein band of the molecular weight predicted for A1 in ConA-stimulated GFP-negative cells but not in the GFP-positive (i.e. A1 shRNA expressing) splenocytes (Figure 1i). This confirms the specificity of our A1 antibody. Figure 1 The newly developed A1 antibody reliably detects the endogenous levels of the pro-survival BCL-2 family member A1. (a) EYZ (control), A1-a, -b, -d and BFL-1 expression vectors were transiently transfected into 293T cells and protein lysates (total protein ... In conclusion, we present here for the first time a mouse A1-specific monoclonal antibody capable of detecting endogenous A1 protein in cell lines as well as in primary mouse cells. Unfortunately, this antibody does not recognise endogenous levels of human BFL-1 (data not shown). This antibody will be made available commercially.

Generation and Evaluation of an IPTG-Regulated Version of Vav-Gene Promoter for Mouse Transgenesis

Different bacteria-derived systems for regulatable gene expression have been developed for the use in mammalian cells and some were also successfully adopted for in vivo use in vertebrate model organisms. However, certain limitations apply to most of these systems, including leakiness of transgene expression, inefficient transgene silencing or activation, as well as limited tissue accessibility of transgene-inducers or their unfavourable pharmacokinetics. In this study, we evaluated the suitability of the lac-operon/lac-repressor (lacO/lacI) system for the regulation of the well-established Vav-gene promoter that allows inducible transgene expression in different haematopoietic lineages in mice. Using the fluorescence marker protein Venus as a reporter, we observed that the lacO/lacI system could be amended to modulate transgene-expression in haematopoietic cells. However, reporter expression was not uniform and the lacO elements introduced into the Vav-gene promoter only conferred limited repression and reversion of lacI-mediated gene silencing after administration of IPTG. Although further optimization of the system is required, the lacO-modified version of the Vav-gene promoter may be adopted as a tool where low basal gene-expression and limited transient induction of protein expression are desired, e.g. for the activation of oncogenes or transgenes that act in a dominant-negative manner.

Replenishment of the B cell compartment after doxorubicin-induced hematopoietic toxicity is facilitated by STAT1

STAT1 serves as an important regulator in the response to pathogens, oncogenic transformation, and genotoxic insults. It exerts these effects by shaping the innate and adaptive immune response and by participating in genotoxic stress pathways, leading to apoptosis and inhibition of cell proliferation. We have investigated the role of STAT1 in hematopoietic toxicity induced by doxorubicin in STAT1‐proficient and ‐deficient mice. Whereas the early genotoxic effect of doxorubicin did not depend on STAT1, expression of STAT1 was required for efficient B lymphocyte repopulation in the recovery phase. We found a lower abundance of lymphocyte precursors in the BM of STAT1‐deficient animals, which was particularly evident after doxorubicin‐induced hematopoietic toxicity. In accordance, colony‐forming assays with STAT1‐deficient BM cells revealed a decreased number of pre‐B colonies. Differentiation from the pro‐B to the pre‐B stage was not affected, as demonstrated by unaltered differentiation of purified B cell precursors from BM in the presence of IL‐7. With the exception of Sca‐1, expression of genes implicated in early lymphocyte development in pro‐B cells did not depend on STAT1. Our findings indicate a specific requirement for STAT1 in lymphoid development before differentiation to pre‐B cells, which becomes particularly apparent in the recovery phase from doxorubicin‐induced hematopoietic toxicity.