Nilesh J. Bokil

Conservation and divergence in Toll-like receptor 4-regulated gene expression in primary human versus mouse macrophages

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-7Rα) 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.

Molecular genetics of long QT syndrome

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.

Metal ions in macrophage antimicrobial pathways: emerging roles for zinc and copper

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.

Intramacrophage survival of uropathogenic Escherichia coli: Differences between diverse clinical isolates and between mouse and human macrophages

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.

The co‐transcriptome of uropathogenic Escherichia coli‐infected mouse macrophages reveals new insights into host–pathogen interactions

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.

Innate immune perturbations, accumulating DAMPs and inflammasome dysregulation: A ticking time bomb in ageing.

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 (Aβ), 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-1β, 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.

Experimental and bioinformatic characterisation of the promoter region of the Marfan syndrome gene, FBN1

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.

Senescent human hepatocytes express a unique secretory phenotype and promote macrophage migration

AIM To develop a model of stress-induced senescence to study the hepatocyte senescence associated secretory phenotype (SASP). METHODS 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 1β, and senescence-associated-β-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. RESULTS Senescent hepatocytes induced classical markers of senescence (p21, heterochromatin protein 1β, and senescence-associated-β-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. CONCLUSION Our novel model of hepatocyte senescence provides insights into mechanisms by which senescent hepatocytes may promote chronic liver disease pathogenesis.

Salmonella employs multiple mechanisms to subvert the TLR-inducible zinc-mediated antimicrobial response of human macrophages

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. FASEB J. 30, 1901–1912 (2016). www.fasebj.org

TLR9-independent effects of inhibitory oligonucleotides on macrophage responses to S. typhimurium.

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‐κB‐dependent reporter gene expression and interleukin‐12p40 secretion) to not only CpG DNA, but also the TLR2 ligand Pam3Cys, 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.