I am a Chancellor's PostDoctoral Research Fellow in Tuberculosis and Host Immunity in the Faculty of Science, University of Technology Sydney, Australia. I have a keen interest in understanding the human immune system and devising applications for clinical use. My work is aimed at developing novel, miRNA based therapeutics for the treatment of inflammation during Tuberculosis infection. In addition to a PhD in immunology I am a medical doctor and a biotechnologist. This melding of clinical medicine and medical research with a grounding in the commercial aspects of biotechnology puts me in a unique position to be able to understand the research, clinical and commercial sides of the therapeutic divide.
- Australiasian society of immunology (ASI)
- ARCS Australia
Can supervise: YES
- Immunology and Infectious diseases
- Innate immune system
- Intracellular Pathogens
- Biomarker discovery
- Early diagnosis and treatment
- Bacterial Pathogenesis
- Infectious diseases
Luo, L, Bokil, NJ, Wall, AA, Kapetanovic, R, Lansdaal, NM, Marceline, F, Burgess, BJ, Tong, SJ, Guo, Z, Alexandrov, K, Ross, IL, Hibbs, ML, Stow, JL & Sweet, MJ 2017, 'SCIMP is a transmembrane non-TIR TLR adaptor that promotes proinflammatory cytokine production from macrophages.', Nature Communications, vol. 8, pp. 1-14.View/Download from: UTS OPUS or Publisher's site
Danger signals activate Toll-like receptors (TLRs), thereby initiating inflammatory responses. Canonical TLR signalling, via Toll/Interleukin-1 receptor domain (TIR)-containing adaptors and proinflammatory transcription factors such as NF-B, occurs in many cell types; however, additional mechanisms are required for specificity of inflammatory responses in innate immune cells. Here we show that SCIMP, an immune-restricted, transmembrane adaptor protein (TRAP), promotes selective proinflammatory cytokine responses by direct modulation of TLR4. SCIMP is a non-TIR-containing adaptor, binding directly to the TLR4-TIR domain in response to lipopolysaccharide. In macrophages, SCIMP is constitutively associated with the Lyn tyrosine kinase, is required for tyrosine phosphorylation of TLR4, and facilitates TLR-inducible production of the proinflammatory cytokines IL-6 and IL-12p40. Point mutations in SCIMP abrogating TLR4 binding also prevent SCIMP-mediated cytokine production. SCIMP is, therefore, an immune-specific TLR adaptor that shapes host defence and inflammation.
D'Souza, C, Henriques, ST, Wang, CK, Cheneval, O, Chan, LY, Bokil, NJ, Sweet, MJ & Craik, DJ 2016, 'Using the MCoTI-II Cyclotide Scaffold To Design a Stable Cyclic Peptide Antagonist of SET, a Protein Overexpressed in Human Cancer.', Biochemistry, vol. 55, no. 2, pp. 396-405.View/Download from: Publisher's site
The SET protein is a promising drug target in cancer therapy, because of its ability to inhibit the function of the tumor suppressor gene protein phosphatase 2A (PP2A). COG peptides, derived from apolipoprotein E (apoE), are potent antagonists of SET; they induce cytotoxicity in cancer cells upon binding to intracellular SET and modulate the nuclear factor kappa B (NF-B) signaling pathway. However, the therapeutic potential of COG peptides is limited, because of their poor proteolytic stability and low bioavailability. In this study, the COG peptide, COG1410, was stabilized by grafting it onto the ultrastable cyclic peptide scaffold, Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II). The grafted MCoTI-II peptides were cytotoxic to a cancer cell line and showed high stability in human serum. The most potent grafted MCoTI-II peptide inhibited lipopolysaccharide (LPS)-mediated activation of NF-B in murine macrophages. Overall, this study demonstrates the application of the MCoTI-II scaffold for the development of stable peptide drugs for cancer therapy.
Kapetanovic, R, Bokil, NJ, Achard, ME, Ong, CL, Peters, KM, Stocks, CJ, Phan, MD, Monteleone, M, Schroder, K, Irvine, KM, Saunders, BM, Walker, MJ, Stacey, KJ, McEwan, AG, Schembri, MA & Sweet, MJ 2016, 'Salmonella employs multiple mechanisms to subvert the TLR-inducible zinc-mediated antimicrobial response of human macrophages.', FASEB journal : official publication of the Federation of American Societies for Experimental Biology, vol. 30, no. 5, pp. 1901-1912.View/Download from: UTS OPUS or Publisher's site
We aimed to characterize antimicrobial zinc trafficking within macrophages and to determine whether the professional intramacrophage pathogen Salmonella enterica serovar Typhimurium (S Typhimurium) subverts this pathway. Using both Escherichia coli and S Typhimurium, we show that TLR signaling promotes the accumulation of vesicular zinc within primary human macrophages. Vesicular zinc is delivered to E. coli to promote microbial clearance, whereas S. Typhimurium evades this response via Salmonella pathogenicity island (SPI)-1. Even in the absence of SPI-1 and the zinc exporter ZntA, S Typhimurium resists the innate immune zinc stress response, implying the existence of additional host subversion mechanisms. We also demonstrate the combinatorial antimicrobial effects of zinc and copper, a pathway that S. Typhimurium again evades. Our use of complementary tools and approaches, including confocal microscopy, direct assessment of intramacrophage bacterial zinc stress responses, specific E. coli and S Typhimurium mutants, and inductively coupled plasma mass spectroscopy, has enabled carefully controlled characterization of this novel innate immune antimicrobial pathway. In summary, our study provides new insights at the cellular level into the well-documented effects of zinc in promoting host defense against infectious disease, as well as the complex host subversion strategies employed by S Typhimurium to combat this pathway.-Kapetanovic, R., Bokil, N. J., Achard, M. E. S., Ong, C.-L. Y., Peters, K. M., Stocks, C. J., Phan, M.-D., Monteleone, M., Schroder, K., Irvine, K. M., Saunders, B. M., Walker, M. J., Stacey, K. J., McEwan, A. G., Schembri, M. A., Sweet, M. J. Salmonella employs multiple mechanisms to subvert the TLR-inducible zinc-mediated antimicrobial response of human macrophages.
Arnež, K.H., Kindlova, M., Bokil, N.J., Murphy, J.M., Sweet, M.J. & Gunčar, G. 2015, 'Analysis of the N-terminal region of human MLKL, as well as two distinct MLKL isoforms, reveals new insights into necroptotic cell death.', Bioscience reports, vol. 36, no. 1, p. e00291.View/Download from: UTS OPUS or Publisher's site
The pseudokinase mixed lineage kinase domain-like (MLKL) is an essential effector of necroptotic cell death. Two distinct human MLKL isoforms have previously been reported, but their capacities to trigger cell death have not been compared directly. Herein, we examine these two MLKL isoforms, and further probe the features of the human MLKL N-terminal domain that are required for cell death. Expression in HEK293T cells of the N-terminal 201 amino acids (aa) of human MLKL is sufficient to cause cell death, whereas expression of the first 154 aa is not. Given that aa 1-125 are able to initiate necroptosis, our findings indicate that the helix that follows this region restrains necroptotic activity, which is again restored in longer constructs. Furthermore, MLKL isoform 2 (MLKL2), which lacks much of the regulatory pseudokinase domain, is a much more potent inducer of cell death than MLKL isoform 1 (MLKL1) in ectopic expression studies in HEK293T cells. Modelling predicts that a C-terminal helix constrains the activity of MLKL1, but not MLKL2. Although both isoforms are expressed by human monocyte-derived macrophages at the mRNA level, MLKL2 is expressed at much lower levels. We propose that it may have a regulatory role in controlling macrophage survival, either in the steady state or in response to specific stimuli.
Kapetanovic, R, Bokil, NJ & Sweet, MJ 2015, 'Innate immune perturbations, accumulating DAMPs and inflammasome dysregulation: A ticking time bomb in ageing.', Ageing research reviews, vol. 24, no. Pt A, pp. 40-53.View/Download from: Publisher's site
Ageing has pronounced effects on the immune system, including on innate immune cells. Whilst most studies suggest that total numbers of different innate immune cell populations do not change dramatically during ageing, many of their functions such as phagocytosis, antigen presentation and inflammatory molecule secretion decline. In contrast, many endogenous damage-associated molecular patterns (DAMPs) accumulate during ageing. These include reactive oxygen species (ROS) released from damaged mitochondria, extracellular nucleotides like ATP, high mobility group box (HMGB) 1 protein, oxidized low density lipoprotein, amyloid-beta (A), islet amyloid polypeptide and particulates like monosodium urate (MSU) crystals and cholesterol crystals. Some of these DAMPs trigger the activation of inflammasomes, cytosolic danger sensing signalling platforms that drive both the maturation of specific pro-inflammatory mediators such as IL-1, as well as the initiation of pro-inflammatory pyroptotic cell death. Herein, we review the evidence that dysregulated inflammasome activation, via altered innate immune cell functions and elevated levels of DAMPs, contributes to the establishment of chronic, low-grade inflammation (characterized by elevated levels of IL-6 and C-reactive protein) and the development of age-related pathological processes.
Mavromatis, C.H., Bokil, N.J., Totsika, M., Kakkanat, A., Schaale, K., Cannistraci, C.V., Ryu, T., Beatson, S.A., Ulett, G.C., Schembri, M.A., Sweet, M.J. & Ravasi, T. 2015, 'The co-transcriptome of uropathogenic Escherichia coli-infected mouse macrophages reveals new insights into host-pathogen interactions.', Cellular microbiology, vol. 17, no. 5, pp. 730-746.View/Download from: UTS OPUS or Publisher's site
Urinary tract infections (UTI) are among the most common infections in humans. Uropathogenic Escherichia coli (UPEC) can invade and replicate within bladder epithelial cells, and some UPEC strains can also survive within macrophages. To understand the UPEC transcriptional programme associated with intramacrophage survival, we performed host-pathogen co-transcriptome analyses using RNA sequencing. Mouse bone marrow-derived macrophages (BMMs) were challenged over a 24 h time course with two UPEC reference strains that possess contrasting intramacrophage phenotypes: UTI89, which survives in BMMs, and 83972, which is killed by BMMs. Neither of these strains caused significant BMM cell death at the low multiplicity of infection that was used in this study. We developed an effective computational framework that simultaneously separated, annotated and quantified the mammalian and bacterial transcriptomes. Bone marrow-derived macrophages responded to the two UPEC strains with a broadly similar gene expression programme. In contrast, the transcriptional responses of the UPEC strains diverged markedly from each other. We identified UTI89 genes up-regulated at 24 h post-infection, and hypothesized that some may contribute to intramacrophage survival. Indeed, we showed that deletion of one such gene (pspA) significantly reduced UTI89 survival within BMMs. Our study provides a technological framework for simultaneously capturing global changes at the transcriptional level in co-cultures, and has generated new insights into the mechanisms that UPEC use to persist within the intramacrophage environment.
Irvine, KM, Skoien, R, Bokil, NJ, Melino, M, Thomas, GP, Loo, D, Gabrielli, B, Hill, MM, Sweet, MJ, Clouston, AD & Powell, EE 2014, 'Senescent human hepatocytes express a unique secretory phenotype and promote macrophage migration.', World Journal of Gastroenterology, vol. 20, no. 47, pp. 17851-17862.View/Download from: UTS OPUS or Publisher's site
To develop a model of stress-induced senescence to study the hepatocyte senescence associated secretory phenotype (SASP).Hydrogen peroxide treatment was used to induce senescence in the human HepG2 hepatocyte cell line. Senescence was confirmed by cytochemical staining for a panel of markers including Ki67, p21, heterochromatin protein 1, and senescence-associated--galactosidase activity. Senescent hepatocytes were characterised by gene expression arrays and quantitative polymerase chain reaction (qPCR), and conditioned media was used in proteomic analyses, a human chemokine protein array, and cell migration assays to characterise the composition and function of the hepatocyte SASP.Senescent hepatocytes induced classical markers of senescence (p21, heterochromatin protein 1, and senescence-associated--galactosidase activity); and downregulated the proliferation marker, Ki67. Hepatocyte senescence induced a 4.6-fold increase in total secreted protein (P = 0.06) without major alterations in the protein profile. Senescence-induced genes were identified by microarray (Benjamini Hochberg-corrected P < 0.05); and, consistent with the increase in secreted protein, gene ontology analysis revealed a significant enrichment of secreted proteins among inducible genes. The hepatocyte SASP included characteristic factors such as interleukin (IL)-8 and IL-6, as well as novel components such as SAA4, IL-32 and Fibrinogen, which were validated by qPCR and/or chemokine protein array. Senescent hepatocyte-conditioned medium elicited migration of inflammatory (granulocyte-macrophage colony stimulating factor, GM-CSF-derived), but not non-inflammatory (CSF-1-derived) human macrophages (P = 0.022), which could contribute to a pro-inflammatory microenvironment in vivo, or facilitate the clearance of senescent cells.Our novel model of hepatocyte senescence provides insights into mechanisms by which senescent hepatocytes may promote chronic liver disease pathogenesis.
Stafford, S.L., Bokil, N.J., Achard, M.E.S., Kapetanovic, R., Schembri, M.A., Mcewan, A.G. & Sweet, M.J. 2013, 'Metal ions in macrophage antimicrobial pathways: Emerging roles for zinc and copper', Bioscience Reports, vol. 33, no. 4, pp. 541-554.View/Download from: Publisher's site
The immunomodulatory and antimicrobial properties of zinc and copper have long been appreciated. In addition, these metal ions are also essential for microbial growth and survival. This presents opportunities for the host to either harness their antimicrobial properties or limit their availability as defence strategies. Recent studies have shed some light on mechanisms by which copper and zinc regulation contribute to host defence, but there remain many unanswered questions at the cellular and molecular levels. Here we review the roles of these two metal ions in providing protection against infectious diseases in vivo, and in regulating innate immune responses. In particular, we focus on studies implicating zinc and copper in macrophage antimicrobial pathways, as well as the specific host genes encoding zinc transporters (SLC30A, SLC39A family members) and CTRs (copper transporters, ATP7 family members) that may contribute to pathogen control by these cells. © 2013 The Author(s).
Stafford, SL, Bokil, NJ, Achard, MES, Kapetanovic, R, Schembri, MA, McEwan, AG & Sweet, MJ 2013, 'Metal ions in macrophage antimicrobial pathways: emerging roles for zinc and copper.', Bioscience reports, vol. 33, no. 4.View/Download from: UTS OPUS or Publisher's site
The immunomodulatory and antimicrobial properties of zinc and copper have long been appreciated. In addition, these metal ions are also essential for microbial growth and survival. This presents opportunities for the host to either harness their antimicrobial properties or limit their availability as defence strategies. Recent studies have shed some light on mechanisms by which copper and zinc regulation contribute to host defence, but there remain many unanswered questions at the cellular and molecular levels. Here we review the roles of these two metal ions in providing protection against infectious diseases in vivo, and in regulating innate immune responses. In particular, we focus on studies implicating zinc and copper in macrophage antimicrobial pathways, as well as the specific host genes encoding zinc transporters (SLC30A, SLC39A family members) and CTRs (copper transporters, ATP7 family members) that may contribute to pathogen control by these cells.
Achard, M.E.S., Stafford, S.L., Bokil, N.J., Chartres, J., Bernhardt, P.V., Schembri, M.A., Sweet, M.J. & McEwan, A.G. 2012, 'Copper redistribution in murine macrophages in response to Salmonella infection.', The Biochemical journal, vol. 444, no. 1, pp. 51-57.View/Download from: UTS OPUS or Publisher's site
The movement of key transition metal ions is recognized to be of critical importance in the interaction between macrophages and intracellular pathogens. The present study investigated the role of copper in mouse macrophage responses to Salmonella enterica sv. Typhimurium. The copper chelator BCS (bathocuproinedisulfonic acid, disodium salt) increased intracellular survival of S. Typhimurium within primary mouse BMM (bone-marrow-derived macrophages) at 24 h post-infection, implying that copper contributed to effective host defence against this pathogen. Infection of BMM with S. Typhimurium or treatment with the TLR (Toll-like receptor) 4 ligand LPS (lipopolysaccharide) induced the expression of several genes encoding proteins involved in copper transport [Ctr (copper transporter) 1, Ctr2 and Atp7a (copper-transporting ATPase 1)], as well as the multi-copper oxidase Cp (caeruloplasmin). Both LPS and infection with S. Typhimurium triggered copper accumulation within punctate intracellular vesicles (copper 'hot spots') in BMM as indicated by the fluorescent reporter CS1 (copper sensor 1). These copper hot spots peaked in their accumulation at approximately 18 h post-stimulation and were dependent on copper uptake into cells. Localization studies indicated that the copper hot spots were in discrete vesicles distinct from Salmonella containing vacuoles and lysosomes. We propose that copper hot spot formation contributes to antimicrobial responses against professional intracellular bacterial pathogens.
Schroder, K, Irvine, KM, Taylor, MS, Bokil, NJ, Le Cao, K-A, Masterman, K-A, Labzin, LI, Semple, CA, Kapetanovic, R, Fairbairn, L, Akalin, A, Faulkner, GJ, Baillie, JK, Gongora, M, Daub, CO, Kawaji, H, McLachlan, GJ, Goldman, N, Grimmond, SM, Carninci, P, Suzuki, H, Hayashizaki, Y, Lenhard, B, Hume, DA & Sweet, MJ 2012, 'Conservation and divergence in Toll-like receptor 4-regulated gene expression in primary human versus mouse macrophages.', Proceedings of the National Academy of Sciences of the United States of America, vol. 109, no. 16, pp. E944-E953.View/Download from: UTS OPUS or Publisher's site
Evolutionary change in gene expression is generally considered to be a major driver of phenotypic differences between species. We investigated innate immune diversification by analyzing interspecies differences in the transcriptional responses of primary human and mouse macrophages to the Toll-like receptor (TLR)-4 agonist lipopolysaccharide (LPS). By using a custom platform permitting cross-species interrogation coupled with deep sequencing of mRNA 5' ends, we identified extensive divergence in LPS-regulated orthologous gene expression between humans and mice (24% of orthologues were identified as "divergently regulated"). We further demonstrate concordant regulation of human-specific LPS target genes in primary pig macrophages. Divergently regulated orthologues were enriched for genes encoding cellular "inputs" such as cell surface receptors (e.g., TLR6, IL-7R) and functional "outputs" such as inflammatory cytokines/chemokines (e.g., CCL20, CXCL13). Conversely, intracellular signaling components linking inputs to outputs were typically concordantly regulated. Functional consequences of divergent gene regulation were confirmed by showing LPS pretreatment boosts subsequent TLR6 responses in mouse but not human macrophages, in keeping with mouse-specific TLR6 induction. Divergently regulated genes were associated with a large dynamic range of gene expression, and specific promoter architectural features (TATA box enrichment, CpG island depletion). Surprisingly, regulatory divergence was also associated with enhanced interspecies promoter conservation. Thus, the genes controlled by complex, highly conserved promoters that facilitate dynamic regulation are also the most susceptible to evolutionary change.
Bokil, NJ, Totsika, M, Carey, AJ, Stacey, KJ, Hancock, V, Saunders, BM, Ravasi, T, Ulett, GC, Schembri, MA & Sweet, MJ 2011, 'Intramacrophage survival of uropathogenic Escherichia coli: differences between diverse clinical isolates and between mouse and human macrophages.', Immunobiology, vol. 216, no. 11, pp. 1164-1171.View/Download from: UTS OPUS or Publisher's site
Uropathogenic E. coli (UPEC) are the primary cause of urinary tract infections. Recent studies have demonstrated that UPEC can invade and replicate within epithelial cells, suggesting that this bacterial pathogen may occupy an intracellular niche within the host. Given that many intracellular pathogens target macrophages, we assessed the interactions between UPEC and macrophages. Colonization of the mouse bladder by UPEC strain CFT073 resulted in increased expression of myeloid-restricted genes, consistent with the recruitment of inflammatory macrophages to the site of infection. In in vitro assays, CFT073 was able to survive within primary mouse bone marrow-derived macrophages (BMM) up to 24h post-infection. Three additional well-characterized clinical UPEC isolates associated with distinct UTI symptomatologies displayed variable long-term survival within BMM. UPEC strains UTI89 and VR50, originally isolated from patients with cystitis and asymptomatic bacteriuria respectively, showed elevated bacterial loads in BMM at 24h post-infection as compared to CFT073 and the asymptomatic bacteriuria strain 83972. These differences did not correlate with differential effects on macrophage survival or initial uptake of bacteria. E. coli UTI89 localized to a Lamp1(+) vesicular compartment within BMM. In contrast to survival within mouse BMM, intracellular bacterial loads of VR50 were low in both human monocyte-derived macrophages (HMDM) and in human T24 bladder epithelial cells. Collectively, these data suggest that some UPEC isolates may subvert macrophage anti-microbial pathways, and that host species differences may impact on intracellular UPEC survival.
Long QT syndrome (LQTS) is a cardiac disorder associated with sudden death especially in young, seemingly healthy individuals. It is characterised by abnormalities of the heart beat detected as lengthening of the QT interval during cardiac repolarisation. The incidence of LQTS is given as 1 in 2000 but this may be an underestimation as many cases go undiagnosed, due to the rarity of the condition and the wide spectrum of symptoms. Presently 12 genes associated with LQTS have been identified with differing signs and symptoms, depending on the locus involved. The majority of cases have mutations in the KCNQ1 (LQT1), KCNH2 (LQT2) and SCN5A (LQT3) genes. Genetic testing is increasingly used when a clearly affected proband has been identified, to determine the nature of the mutation in that family. Unfortunately tests on probands may be uninformative, especially if the defect does not lie in the set of genes which are routinely tested. Novel mutations in these known LQTS genes and additional candidate genes are still being discovered. The functional implications of these novel mutations need to be assessed before they can be accepted as being responsible for LQTS. Known epigenetic modification affecting KCNQ1 gene expression may also be involved in phenotypic variability of LQTS. Genetic diagnosis of LQTS is thus challenging. However, where a disease associated mutation is identified, molecular diagnosis can be important in guiding therapy, in family testing and in determining the cause of sudden cardiac death. New developments in technology and understanding offer increasing hope to families with this condition.
Summers, K.M., Bokil, N.J., Lu, F.T., Low, J.T., Baisden, J.M., Duffy, D. & Radford, D.J. 2010, 'Mutations at KCNQ1 and an unknown locus cause long QT syndrome in a large Australian family: implications for genetic testing.', American journal of medical genetics. Part A, vol. 152A, no. 3, pp. 613-621.View/Download from: Publisher's site
A large Australian family affected with long QT syndrome (LQTS) was studied. The medical characteristics of the 16 clinically affected members were consistent with LQT1. A previously identified mutation in KCNQ1 was found in 12 affected individuals and 1 unaffected infant but absent in 4 affected family members. A haplotype consisting of specific alleles for microsatellites flanking in KCNQ1 was associated with the mutation. This was absent from the four affected individuals without the mutation, who had three different haplotypes in this region, indicating that LQTS is unlikely to be segregating with KCNQ1 in these anomalous family members. A genome scan revealed 12 regions where all four of these individuals shared alleles. One region on chromosome 21 contained the KCNE1, KCNE2, KCNJ6, and KCNJ15 genes. A common variant of KCNE1 was segregating in the family but did not explain the anomalous cases. A candidate region on chromosome 7 contained the AKAP9 and KCND2 genes. A previously reported mutation in the N-terminal Yotiao region of AKAP9 was absent from the family. No evidence was found implicating any other known or suspected LQTS gene. This family shows that there remain unidentified genetic causes of LQTS which are clinically significant and highlights the difficulties associated with genetic testing in LQTS, since we cannot rule out risk in individuals who are negative for the known mutation in KCNQ1 without knowing the second disease locus.
Summers, KM, Bokil, NJ, Baisden, JM, West, MJ, Sweet, MJ, Raggatt, LJ & Hume, DA 2009, 'Experimental and bioinformatic characterisation of the promoter region of the Marfan syndrome gene, FBN1.', Genomics, vol. 94, no. 4, pp. 233-240.View/Download from: Publisher's site
Mutations in the FBN1 gene, encoding the extracellular matrix protein fibrillin-1, result in the dominant connective tissue disease Marfan syndrome. Marfan syndrome has a variable phenotype, even within families carrying the same FBN1 mutation. Differences in gene expression resulting from sequence differences in the promoter region of the FBN1 gene are likely to be involved in causing this phenotypic variability. In this report, we present an analysis of FBN1 transcription start site (TSS) use in mouse and human tissues. We found that transcription of FBN1 initiated primarily from a single CpG-rich promoter which was highly conserved in mammals. It contained potential binding sites for a number of factors implicated in mesenchyme differentiation and gene expression. The human osteosarcoma line MG63 had high levels of FBN1 mRNA and secreted fibrillin-1 protein to form extracellular matrix fibres. The human embryonic kidney line HEK293 and two breast cancer lines MCF7 and MDA-MB-231 had levels of FBN1 mRNA 1000 fold lower and produced negligible amounts of fibrillin-1 protein. Therefore MG63 appears to be the optimal cell line for examining tissue-specific, biologically relevant promoter activity for FBN1. In reporter assays, the conserved promoter region was more active in MG63 cells than in non-FBN1-expressing lines but additional elements outside the proximal promoter are probably required for optimal tissue-specific expression. Understanding the regulation of the FBN1 gene may lead to alternative therapeutic strategies for Marfan syndrome.
Trieu, A, Bokil, N, Dunn, JA, Roberts, TL, Xu, D, Liew, FY, Hume, DA, Stacey, KJ & Sweet, MJ 2009, 'TLR9-independent effects of inhibitory oligonucleotides on macrophage responses to S. typhimurium.', Immunology and cell biology, vol. 87, no. 3, pp. 218-225.View/Download from: Publisher's site
Detection of bacterial CpG-containing DNA (CpG DNA) by innate immune cells is dependent on toll-like receptor 9 (TLR9). Here we show that the expression of tlr9 mRNA was induced in mouse bone marrow-derived macrophages (BMMs) upon infection with the facultative Gram-negative intracellular bacterium Salmonella enterica serovar Typhimurium (S. typhimurium). Treatment of BMM with the inhibitory oligonucleotide (ODN) 2114, an antagonist of TLR9 signalling, enhanced intracellular S. typhimurium numbers approximately fivefold, whereas a control ODN (2310) had no significant effect. Surprisingly, 2114 also amplified S. typhimurium bacterial loads in TLR9-deficient BMM. Indeed, 2114 suppressed responses (nuclear factor-kappaB-dependent reporter gene expression and interleukin-12p40 secretion) to not only CpG DNA, but also the TLR2 ligand Pam(3)Cys, in BMM and RAW264 cells in a sequence-specific manner. Inhibitory ODNs, which have been proposed as therapeutic agents for the treatment of systemic lupus erythematosus because of their inhibitory effects on TLR9 signalling, may thus compromise the host response to bacterial pathogens through TLR9-independent mechanisms.
Ravasi, T., Mavromatis, C.H., Bokil, N.J., Schembri, M.A. & Sweet, M.J. 2016, 'Co-transcriptomic analysis by rna sequencing to simultaneously measure regulated gene expression in host and bacterial pathogen' in McCoy, C.E. (ed), Toll-Like Receptors: Practice and Methods, Springer, New York, pp. 145-156.View/Download from: UTS OPUS or Publisher's site
© Springer Science+Business Media New York 2016.Intramacrophage pathogens subvert antimicrobial defence pathways using various mechanisms, including the targeting of host TLR-mediated transcriptional responses. Conversely, TLR-inducible host defence mechanisms subject intramacrophage pathogens to stress, thus altering pathogen gene expression programs. Important biological insights can thus be gained through the analysis of gene expression changes in both the host and the pathogen during an infection. Traditionally, research methods have involved the use of qPCR, microarrays and/or RNA sequencing to identify transcriptional changes in either the host or the pathogen. Here we describe the application of RNA sequencing using samples obtained from in vitro infection assays to simultaneously quantify both host and bacterial pathogen gene expression changes, as well as general approaches that can be undertaken to interpret the RNA sequencing data that is generated. These methods can be used to provide insights into host TLR-regulated transcriptional responses to microbial challenge, as well as pathogen subversion mechanisms against such responses.
Sweet, M.J. & Bokil, N.J. 2010, 'The role of monocytes and macrophages in innate immunity: Macrophage anti-microbial pathways' in Marsh, C.B., Tridandapani, S. & Piper, M.G. (eds), Regulation of Innate Immune Function, Research Signpost, India, pp. 1-31.
Sweet, M.J., Schroder, K., Irvine, K.M., Taylor, M., Bokil, N.J., Broomfield, S., Schembri, M.A., Stacey, K.J. & Hume, D.A. 2012, 'Functional significance of evolutionary divergence in Toll-like receptor-regulated gene expression in human versus mouse', IMMUNOLOGY, European Congress of Immunology, WILEY-BLACKWELL, Glasgow, SCOTLAND, pp. 297-297.
1. Prof. Warwick Britton AO Bosch Professor of Medicine and Professor of Immunology Medicine (Immunology & Infectious Diseases), Central Clinical School, Head of Tuberculosis Research Program, Centenary Institute, Research Director, Sydney Local Health District.
2. Morten Thaysen-Andersen, Ph.D, Head, Glycoimmunology Group, Department of Molecular Sciences, Faculty of Science and Engineering, Macquarie University