Benjamin T Kile

University of Melbourne, Melbourne, Victoria, Australia

Are you Benjamin T Kile?

Claim your profile

Publications (78)703.79 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: A current paradigm proposes that mitochondrial damage is a critical determinant of NLRP3 inflammasome activation. Here, we genetically assess whether mitochondrial signalling represents a unified mechanism to explain how NLRP3 is activated by divergent stimuli. Neither co-deletion of the essential executioners of mitochondrial apoptosis BAK and BAX, nor removal of the mitochondrial permeability transition pore component cyclophilin D, nor loss of the mitophagy regulator Parkin, nor deficiency in MAVS affects NLRP3 inflammasome function. In contrast, caspase-8, a caspase essential for death-receptor-mediated apoptosis, is required for efficient Toll-like-receptor-induced inflammasome priming and cytokine production. Collectively, these results demonstrate that mitochondrial apoptosis is not required for NLRP3 activation, and highlight an important non-apoptotic role for caspase-8 in regulating inflammasome activation and pro-inflammatory cytokine levels.
    EMBO Reports 07/2014; · 7.19 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: A current paradigm proposes that mitochondrial damage is a critical determinant of NLRP3 inflammasome activation. Here, we genetically assess whether mitochondrial signalling represents a unified mechanism to explain how NLRP3 is activated by divergent stimuli. Neither co-deletion of the essential executioners of mitochondrial apoptosis BAK and BAX, nor removal of the mitochondrial permeability transition pore component cyclophilin D, nor loss of the mitophagy regulator Parkin, nor deficiency in MAVS affects NLRP3 inflammasome function. In contrast, caspase-8, a caspase essential for death-receptor-mediated apoptosis, is required for efficient Toll-like-receptor-induced inflammasome priming and cytokine production. Collectively, these results demonstrate that mitochondrial apoptosis is not required for NLRP3 activation, and highlight an important non-apoptotic role for caspase-8 in regulating inflammasome activation and pro-inflammatory cytokine levels.
    EMBO Reports 06/2014; · 7.19 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Suppressor of cytokine signaling (SOCS) proteins are key regulators of innate and adaptive immunity. There is no described biological role for SOCS4, despite broad expression in the hematopoietic system. We demonstrate that mice lacking functional SOCS4 protein rapidly succumb to infection with a pathogenic H1N1 influenza virus (PR8) and are hypersusceptible to infection with the less virulent H3N2 (X31) strain. In SOCS4-deficient animals, this led to substantially greater weight loss, dysregulated pro-inflammatory cytokine and chemokine production in the lungs and delayed viral clearance. This was associated with impaired trafficking of influenza-specific CD8 T cells to the site of infection and linked to defects in T cell receptor activation. These results demonstrate that SOCS4 is a critical regulator of anti-viral immunity.
    PLoS Pathogens 05/2014; 10(5):e1004134. · 8.14 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Canavan disease is a leukodystrophy caused by mutations in the ASPA gene. This gene encodes the enzyme that converts N-acetylaspartate into acetate and aspartic acid. In Canavan disease spongiform encephalopathy of the brain causes progressive mental retardation, motor deficit and death. We have isolated a mouse with a novel ethylnitrosourea-induced mutation in Aspa. This mutant, named deaf14, carries a c.516T>A mutation that is predicted to cause a p.Y172X protein truncation. No full-length ASPA protein is produced in deaf14 brain and there is extensive spongy degeneration. Interestingly, we found that deaf14 mice have an attenuated startle in response to loud noise. The first auditory brainstem response peak has normal latency and amplitude but peaks II, III, IV and V have increased latency and decreased amplitude in deaf14 mice. Our work reveals a hitherto unappreciated pathology in a mouse model of Canavan disease, implying that auditory brainstem response testing could be used in diagnosis and to monitor the progression of this disease.
    Disease Models and Mechanisms 03/2014; · 4.96 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The fetal/neonatal hematopoietic system must generate enough blood cells to meet the demands of rapid growth. This unique challenge might underlie the high incidence of thrombocytopenia among preterm neonates. In this study, neonatal platelet production and turnover were investigated in newborn mice. Based on a combination of blood volume expansion and increasing platelet counts, the platelet mass increased 7-fold during the first two weeks of murine life, a time during which thrombopoiesis shifted from liver to bone marrow. Studies applying in-vivo biotinylation and mathematical modeling revealed that newborn and adult mice had similar platelet production rates, but neonatal platelets survived one day longer in circulation. This prolonged lifespan fully accounted for the rise in platelet counts observed during the second week of murine postnatal life. A study of pro-apoptotic and anti-apoptotic Bcl-2 family proteins found that neonatal platelets had higher levels of the anti-apoptotic protein Bcl-2 and were more resistant to apoptosis induced by the Bcl-2/Bcl-xL inhibitor ABT-737 than adult platelets. However, genetic ablation or pharmacologic inhibition of Bcl-2 alone did not shorten neonatal platelet survival or reduce platelet counts in newborn mice, indicating the existence of redundant or alternative mechanisms mediating the prolonged lifespan of neonatal platelets.
    Blood 03/2014; · 9.06 Impact Factor
  • Cell metabolism 03/2014; 19(3):345-346. · 17.35 Impact Factor
  • Benjamin T Kile
    [Show abstract] [Hide abstract]
    ABSTRACT: The role of apoptotic pathways in the development and function of the megakaryocyte lineage has generated renewed interest in recent years. This has been driven by the advent of BH3 mimetic drugs that target BCL2 family proteins to induce apoptosis in tumour cells: agents such as ABT-263 (navitoclax, which targets BCL2, BCL-XL [BCL2L1] and BCL2L2) and ABT-199 (a BCL2-specific agent) are showing great promise in early stage clinical trials. However, the major dose-limiting toxicity of navitoclax has proven to be thrombocytopenia, an on-target effect of inhibiting BCL-XL . It transpires that the anucleate platelet contains a classical intrinsic apoptosis pathway, which at steady state regulates its life span in the circulation. BCL-XL is the critical pro-survival protein that restrains apoptosis and maintains platelet viability. These findings have paved the way to a deeper understanding of apoptotic pathways and processes in platelets, and their precursor cell, the megakaryocyte.
    British Journal of Haematology 01/2014; · 4.94 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Bax and Bak are critical effectors of apoptosis. Although both are widely expressed and usually functionally redundant, recent studies suggest that Bak has particular importance in certain cell types. Genetic and biochemical studies indicate that Bak activation is prevented primarily by Mcl-1 and Bcl-xL, whereas Bax is held in check by all pro-survival Bcl-2 homologues, including Bcl-2 itself. In this study, we have investigated whether loss of Bak or elevated Mcl-1 modulates haemopoietic abnormalities provoked by overexpression of Bcl-2. The Mcl-1 transgene had little impact, probably because the expression level was insufficient to effectively reduce Bak activation. However, loss of Bak enhanced lymphocytosis in vavP-BCL-2 transgenic mice and increased resistance of their thymocytes to some cytotoxic agents, implying that Bak-specific signals can be triggered in certain lymphoid populations. Nevertheless, lack of Bak had no significant impact on thymic abnormalities in vavP-BCL-2tg mice, which kinetic analysis suggested was due to accumulation of self-reactive thymocytes that resist deletion. Intriguingly, although Bak(-/-) mice have elevated platelet counts, Bak(-/-)vavP-BCL-2 mice, like vavP-BCL-2 littermates, were thrombocytopaenic. To clarify why, the vavP-BCL-2 platelet phenotype was scrutinised more closely. Platelet life span was found to be elevated in vavP-BCL-2 mice, which should have provoked thrombocytosis, as in Bak(-/-) mice. Analysis of bone marrow chimaeric mice suggested the low platelet phenotype was due principally to extrinsic factors. Following splenectomy, blood platelets remained lower in vavP-BCL-2 than wild-type mice. However, in Rag1(-/-) BCL-2tg mice, platelet levels were normal, implying that elevated lymphocytes are primarily responsible for BCL-2tg-induced thrombocytopaenia.Cell Death and Differentiation advance online publication, 24 January 2014; doi:10.1038/cdd.2013.201.
    Cell death and differentiation 01/2014; · 8.24 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: BH3 mimetic drugs that target BCL-2 family pro-survival proteins to induce tumour cell apoptosis represent a new era in cancer therapy. Clinical trials of navitoclax (ABT-263, which targets BCL-2, BCL-XL and BCL-W) have shown great promise, but encountered dose-limiting thrombocytopenia. Recent work has demonstrated that this is due to the inhibition of BCL-XL, which is essential for platelet survival. These findings raise new questions about the established model of platelet shedding by megakaryocytes, which is thought to be an apoptotic process. Here we generate mice with megakaryocyte-specific deletions of the essential mediators of extrinsic (Caspase-8) and intrinsic (BAK/BAX) apoptosis. We show that megakaryocytes possess a Fas ligand-inducible extrinsic apoptosis pathway. However, Fas activation does not stimulate platelet production, rather, it triggers Caspase-8-mediated killing. Combined loss of Caspase-8/BAK/BAX does not impair thrombopoiesis, but can protect megakaryocytes from death in mice infected with lymphocytic choriomeningitis virus. Thus, apoptosis is dispensable for platelet biogenesis.
    Nature Communications 01/2014; 5:3455. · 10.02 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: CHARGE syndrome is a rare human disorder caused by mutations in the gene encoding chromodomain helicase DNA binding protein 7 (CHD7). Characteristics of CHARGE are varied and include developmental ear and hearing anomalies. Here we report a novel mouse model of CHD7 dysfunction, termed Looper. The Looper strain harbours a nonsense mutation (c.5690C>A, p.S1897X) within the Chd7 gene. Looper mice exhibit many of the clinical features of the human syndrome, consistent with previously reported CHARGE models, including growth retardation, facial asymmetry, vestibular defects, eye anomalies, hyperactivity, ossicle malformation, hearing loss and vestibular dysfunction. Looper mice display an otosclerosis-like fusion of the stapes footplate to the cochlear oval window and blepharoconjunctivitis but not coloboma. Looper mice are hyperactive and have vestibular dysfunction but do not display motor impairment.
    PLoS ONE 01/2014; 9(5):e97559. · 3.73 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Among the cytokines linked to inflammation-associated cancer, interleukin (IL)-6 drives many of the cancer "hallmarks" through downstream activation of the gp130/STAT3 signaling pathway. However, we show that the related cytokine IL-11 has a stronger correlation with elevated STAT3 activation in human gastrointestinal cancers. Using genetic mouse models, we reveal that IL-11 has a more prominent role compared to IL-6 during the progression of sporadic and inflammation-associated colon and gastric cancers. Accordingly, in these models and in human tumor cell line xenograft models, pharmacologic inhibition of IL-11 signaling alleviated STAT3 activation, suppressed tumor cell proliferation, and reduced the invasive capacity and growth of tumors. Our results identify IL-11 signaling as a potential therapeutic target for the treatment of gastrointestinal cancers.
    Cancer cell 08/2013; 24(2):257-71. · 25.29 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: ABCA12 is involved in the transport of ceramides in skin, but it may play a wider role in lipid metabolism. We show that, in Abca12-deficient macrophages, cholesterol efflux failed to respond to activation with LXR agonists. Abca12 deficiency caused a reduction in the abundance of Abca1, Abcg1, and Lxrβ. Overexpression of Lxrβ reversed the effects. Mechanistically, Abca12 deficiency did not affect expression of genes involved in cholesterol metabolism. Instead, a physical association between Abca1, Abca12, and Lxrβ proteins was established. Abca12 deficiency enhanced interaction between Abca1 and Lxrβ and the degradation of Abca1. Overexpression of ABCA12 in HeLa-ABCA1 cells increased the abundance and stability of ABCA1. Abca12 deficiency caused an accumulation of cholesterol in macrophages and the formation of foam cells, impaired reverse cholesterol transport in vivo, and increased the development of atherosclerosis in irradiated Apoe(-/-) mice reconstituted with Apoe(-/-)Abca12(-/-) bone marrow. Thus, ABCA12 regulates the cellular cholesterol metabolism via an LXRβ-dependent posttranscriptional mechanism.
    Cell metabolism 08/2013; 18(2):225-38. · 17.35 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: To define genetic lesions driving leukemia, we targeted cre-dependent Sleeping Beauty (SB) transposon mutagenesis to the blood-forming system using a hematopoietic-selective vav 1 oncogene (vav1) promoter. Leukemias of diverse lineages ensued, most commonly lymphoid leukemia and erythroleukemia. The inclusion of a transgenic allele of Janus kinase 2 (JAK2)V617F resulted in acceleration of transposon-driven disease and strong selection for erythroleukemic pathology with transformation of bipotential erythro-megakaryocytic cells. The genes encoding the E-twenty-six (ETS) transcription factors Ets related gene (Erg) and Ets1 were the most common sites for transposon insertion in SB-induced JAK2V617F-positive erythroleukemias, present in 87.5% and 65%, respectively, of independent leukemias examined. The role of activated Erg was validated by reproducing erythroleukemic pathology in mice transplanted with fetal liver cells expressing translocated in liposarcoma (TLS)-ERG, an activated form of ERG found in human leukemia. Via application of SB mutagenesis to TLS-ERG-induced erythroid transformation, we identified multiple loci as likely collaborators with activation of Erg. Jak2 was identified as a common transposon insertion site in TLS-ERG-induced disease, strongly validating the cooperation between JAK2V617F and transposon insertion at the Erg locus in the JAK2V617F-positive leukemias. Moreover, loci expressing other regulators of signal transduction pathways were conspicuous among the common transposon insertion sites in TLS-ERG-driven leukemia, suggesting that a key mechanism in erythroleukemia may be the collaboration of lesions disturbing erythroid maturation, most notably in genes of the ETS family, with mutations that reduce dependence on exogenous signals.
    Proceedings of the National Academy of Sciences 03/2013; · 9.74 Impact Factor
  • Source
    Blood 02/2013; 121(8):1479-81. · 9.06 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Successful replication and transmission of large DNA viruses such as the cytomegaloviruses (CMV) family of viruses depends on the ability to interfere with multiple aspects of the host immune response. Apoptosis functions as a host innate defence mechanism against viral infection, and the capacity to interfere with this process is essential for the replication of many viruses. The Bcl-2 family of proteins are the principle regulators of apoptosis, with two pro-apoptotic members, Bax and Bak, essential for apoptosis to proceed. The m38.5 protein encoded by murine CMV (MCMV) has been identified as Bax-specific inhibitor of apoptosis. Recently, m41.1, a protein product encoded by the m41 open reading frame (ORF) of MCMV, has been shown to inhibit Bak activity . Here we show that m41.1 is critical for optimal MCMV replication . Growth of a m41.1 mutant was attenuated in multiple organs, a defect that was not apparent in mice. Thus, m41.1 promotes MCMV replication by inhibiting Bak-dependent apoptosis during infection. The results show that Bax and Bak mediate non-redundant functions during MCMV infection and that the virus produces distinct inhibitors for each protein to counter the activity of these proteins.
    PLoS Pathogens 02/2013; 9(2):e1003192. · 8.14 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Dysregulation of the "intrinsic" apoptotic pathway is associated with the development of cancer and autoimmune disease. Bak and Bax are two proapoptotic members of the Bcl-2 protein family with overlapping, essential roles in the intrinsic apoptotic pathway. Their activity is critical for the control of cell survival during lymphocyte development and homeostasis, best demonstrated by defects in thymic T-cell differentiation and peripheral lymphoid homeostasis caused by their combined loss. Because most bak(-/-)bax(-/-) mice die perinatally, the roles of Bax and Bak in immunological tolerance and prevention of autoimmune disease remain unclear. We show that mice reconstituted with a Bak/Bax doubly deficient hematopoietic compartment develop a fatal systemic lupus erythematosus-like autoimmune disease characterized by hypergammaglobulinemia, autoantibodies, lymphadenopathy, glomerulonephritis, and vasculitis. Importantly, these mice also develop a multiorgan autoimmune disease with autoantibodies against most solid glandular structures and evidence of glandular atrophy and necrotizing vasculitis. Interestingly, similar albeit less severe pathology was observed in mice containing a hematopoietic compartment deficient for only Bak, a phenotype reminiscent of the disease seen in patients with point mutations in BAK. These studies demonstrate a critical role for Bak and an ancillary role for Bax in safeguarding immunological tolerance and prevention of autoimmune disease. This suggests that direct activators of the intrinsic apoptotic pathway, such as BH3 mimetics, may be useful for treatment of diverse autoimmune diseases.
    Proceedings of the National Academy of Sciences 01/2013; · 9.74 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Over 120 loci are known to cause inherited hearing loss in humans. The deafness gene has been identified for only half of these loci. With the aim of identifying some of the remaining deafness genes, we performed an ethylnitrosourea mutagenesis screen for deaf mice. We isolated two mutants with semi-dominant hearing loss, Deaf11 and Deaf13. Both contained causative mutations in Atp2b2, which encodes the plasma membrane calcium ATPase 2. The Atp2b2 (Deaf11) mutation leads to a p. I1023S substitution in the tenth transmembrane domain. The Atp2b2 (Deaf13) mutation leads to a p. R561S substitution in the catalytic core. Mice homozygous for these mutations display profound hearing loss. Heterozygotes display mild to moderate, progressive hearing loss.
    PLoS ONE 01/2013; 8(6):e67479. · 3.73 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Neuroinflammation is critical in the neural cell death seen in stroke. It has been shown that CNS and peripheral responses drive this neuroinflammatory response in the brain. The Toll-like receptors (TLRs) are important regulators of inflammation in response to both exogenous and endogenous stressors. Taking advantage of a downstream adapter molecule that controls the majority of TLR signalling, this study investigated the role of the TLR adaptor protein myeloid differentiation factor 88 (MyD88) in the control of CNS and peripheral inflammation. Reversible middle-cerebral artery occlusion was used as the model of stroke in vivo; in vitro primary cultured neurons and glia were subject to four hours of oxygen and glucose deprivation (OGD). Both in vitro and in vivo Myd88-/- animals or cells were compared with wild type (WT). We found that after stroke Myd88-/- animals have a larger infarct volume compared to WT animals. Interestingly, in vitro there was no difference between the survival of Myd88-/- and WT cells following OGD, suggesting that peripheral responses were influencing stroke outcome. We therefore generated bone marrow chimeras and found that Myd88-/- animals have a smaller stroke infarct than their radiation naive counterparts if their hematopoietic cells are WT. Furthermore, WT animals have a larger stroke than their radiation naive counterparts if the hematopoietic cells are Myd88-/- . We have demonstrated that MyD88-dependent signalling in the hematopoietic cell lineage reduces infarct size following stroke and that infiltrating cells to the site of neuroinflammation are neuroprotective following stroke.
    PLoS ONE 01/2013; 8(3):e57948. · 3.73 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The tetracycline (tet)-regulated expression system allows for the inducible overexpression of protein-coding genes, or inducible gene knockdown based on expression of short hairpin RNAs (shRNAs). The system is widely used in mice, however it requires robust expression of a tet transactivator protein (tTA or rtTA) in the cell type of interest. Here we used an in vivo tet-regulated fluorescent reporter approach to characterise inducible gene/shRNA expression across a range of hematopoietic cell types of several commonly used transgenic tet transactivator mouse strains. We find that even in strains where the tet transactivator is expressed from a nominally ubiquitous promoter, the efficiency of tet-regulated expression can be highly variable between hematopoietic lineages and between differentiation stages within a lineage. In some cases tet-regulated reporter expression differs markedly between cells within a discrete, immunophenotypically defined population, suggesting mosaic transactivator expression. A recently developed CAG-rtTA3 transgenic mouse displays intense and efficient reporter expression in most blood cell types, establishing this strain as a highly effective tool for probing hematopoietic development and disease. These findings have important implications for interpreting tet-regulated hematopoietic phenotypes in mice, and identify mouse strains that provide optimal tet-regulated expression in particular hematopoietic progenitor cell types and mature blood lineages.
    PLoS ONE 01/2013; 8(1):e54009. · 3.73 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A fundamental issue in understanding homeostasis of the hematopoietic system is to what extent intrinsic and extrinsic factors regulate cell fate. We recently revisited this issue for the case of blood platelets and concluded that platelet life span is largely regulated by internal factors, in contrast to the long-held view that accumulated damage from the environment triggers clearance. However, it is known that in humans there is an ongoing fixed requirement for platelets to maintain hemostasis and prevent bleeding; hence a proportion of platelets may be consumed in such processes before the end of their natural life span. Whether it is possible to detect this random loss of platelets in normal individuals at steady-state is unknown. To address this question, we have developed a mathematical model that independently incorporates age-independent random loss and age-dependent natural senescent clearance. By fitting to population survival curves, we illustrate the application of the model in quantifying the fixed requirement for platelets to maintain hemostasis in mice, and discuss the relationship with previous work in humans. Our results suggest a higher requirement for platelets in mice than in humans, however experimental uncertainty in the data limits our ability to constrain this quantity. We then explored the relationship between experimental uncertainty and parameter constraint using simulated data. We conclude that in order to provide useful constraint on the random loss fraction the standard error in the mean of the data must be reduced substantially, either through improving experimental uncertainty or increasing the number of experimental replicates to impractical levels. Finally we find that parameter constraint is improved at higher values of the random loss fraction; thus the model find utility in situations where the random loss fraction is expected to be high, for example during active bleeding or some types of thrombocytopenia.
    PLoS ONE 01/2013; 8(3):e57783. · 3.73 Impact Factor

Publication Stats

2k Citations
703.79 Total Impact Points

Institutions

  • 2007–2014
    • University of Melbourne
      Melbourne, Victoria, Australia
    • Sanford-Burnham Medical Research Institute
      La Jolla, California, United States
  • 2002–2014
    • The Walter and Eliza Hall Institute of Medical Research
      • • Division of Cancer and Haematology
      • • Division of Molecular Medicine
      Melbourne, Victoria, Australia
  • 2011
    • University of Washington Seattle
      • Department of Pediatrics
      Seattle, Washington, United States
  • 2008
    • Monash University (Australia)
      • Department of Biochemistry and Molecular Biology
      Melbourne, Victoria, Australia
  • 2003–2007
    • Baylor College of Medicine
      • Department of Molecular & Human Genetics
      Houston, TX, United States
  • 2000–2001
    • Royal Melbourne Hospital
      Melbourne, Victoria, Australia