Katharine M. Irvine

The regulated retrotransposon transcriptome of mammalian cells

Although repetitive elements pervade mammalian genomes, their overall contribution to transcriptional activity is poorly defined. Here, as part of the FANTOM4 project, we report that 6–30% of cap-selected mouse and human RNA transcripts initiate within repetitive elements. Analysis of approximately 250,000 retrotransposon-derived transcription start sites shows that the associated transcripts are generally tissue specific, coincide with gene-dense regions and form pronounced clusters when aligned to full-length retrotransposon sequences. Retrotransposons located immediately 5′ of protein-coding loci frequently function as alternative promoters and/or express noncoding RNAs. More than a quarter of RefSeqs possess a retrotransposon in their 3′ UTR, with strong evidence for the reduced expression of these transcripts relative to retrotransposon-free transcripts. Finally, a genome-wide screen identifies 23,000 candidate regulatory regions derived from retrotransposons, in addition to more than 2,000 examples of bidirectional transcription. We conclude that retrotransposon transcription has a key influence upon the transcriptional output of the mammalian genome.

Tiny RNAs associated with transcription start sites in animals

It has been reported that relatively short RNAs of heterogeneous sizes are derived from sequences near the promoters of eukaryotic genes. As part of the FANTOM4 project, we have identified tiny RNAs with a modal length of 18 nt that map within −60 to +120 nt of transcription start sites (TSSs) in human, chicken and Drosophila. These transcription initiation RNAs (tiRNAs) are derived from sequences on the same strand as the TSS and are preferentially associated with G+C-rich promoters. The 5′ ends of tiRNAs show peak density 10–30 nt downstream of TSSs, indicating that they are processed. tiRNAs are generally, although not exclusively, associated with highly expressed transcripts and sites of RNA polymerase II binding. We suggest that tiRNAs may be a general feature of transcription in metazoa and possibly all eukaryotes.

Histone deacetylases as regulators of inflammation and immunity

Histone deacetylases (HDACs) remove an acetyl group from lysine residues of target proteins to regulate cellular processes. Small-molecule inhibitors of HDACs cause cellular growth arrest, differentiation and/or apoptosis, and some are used clinically as anticancer drugs. In animal models, HDAC inhibitors are therapeutic for several inflammatory diseases, but exacerbate atherosclerosis and compromise host defence. Loss of HDAC function has also been linked to chronic lung diseases in humans. These contrasting effects might reflect distinct roles for individual HDACs in immune responses. Here, we review the current understanding of innate and adaptive immune pathways that are regulated by classical HDAC enzymes. The objective is to provide a rationale for targeting (or not targeting) individual HDAC enzymes with inhibitors for future immune-related applications.

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.

Differential effects of selective HDAC inhibitors on macrophage inflammatory responses to the Toll‐like receptor 4 agonist LPS

Broad‐spectrum inhibitors of HDACs are therapeutic in many inflammatory disease models but exacerbated disease in a mouse model of atherosclerosis. HDAC inhibitors have anti‐ and proinflammatory effects on macrophages in vitro. We report here that several broad‐spectrum HDAC inhibitors, including TSA and SAHA, suppressed the LPS‐induced mRNA expression of the proinflammatory mediators Edn‐1, Ccl‐7/MCP‐3, and Il‐12p40 but amplified the expression of the proatherogenic factors Cox‐2 and Pai‐1/serpine1 in primary mouse BMM. Similar effects were also apparent in LPS‐stimulated TEPM and HMDM. The pro‐ and anti‐inflammatory effects of TSA were separable over a concentration range, implying that individual HDACs have differential effects on macrophage inflammatory responses. The HDAC1‐selective inhibitor, MS‐275, retained proinflammatory effects (amplification of LPS‐induced expression of Cox‐2 and Pai‐1 in BMM) but suppressed only some inflammatory responses. In contrast, 17a (a reportedly HDAC6‐selective inhibitor) retained anti‐inflammatory but not proinflammatory properties. Despite this, HDAC6−/− macrophages showed normal LPS‐induced expression of HDAC‐dependent inflammatory genes, arguing that the anti‐inflammatory effects of 17a are not a result of inhibition of HDAC6 alone. Thus, 17a provides a tool to identify individual HDACs with proinflammatory properties.

Gpnmb is induced in macrophages by IFN-gamma and lipopolysaccharide and acts as a feedback regulator of proinflammatory responses

The process of inflammation requires the selective expression of a suite of genes in cells of the macrophage lineage. To identify candidate regulators of inflammation, we used cDNA microarrays to compare the transcriptome of inflammatory macrophages (thioglycolate-elicited peritoneal macrophages), bone marrow-derived macrophages, nonadherent spleen cells, and fibroblasts. We identified genes that were macrophage restricted and further elevated in inflammatory macrophages, and characterized the function of one such gene, gpnmb. Gpnmb mRNA expression was enriched in myelomonocytic cell lines and macrophage-related tissues and strongly up-regulated during macrophage differentiation. Epitope-tagged GPNMB expressed in RAW264.7 cells exhibited a perinuclear distribution and colocalized with the Golgi marker coat protein β. Upon activation of macrophages with IFN-γ and LPS, GPNMB translocated from the Golgi apparatus to vesicular compartments scattered toward the periphery. Gpnmb overexpression in RAW264.7 cells caused a 2-fold reduction in the production of the cytokines IL-6 and IL-12p40 and the inflammatory mediator NO in response to LPS. DBA mice, which have an inactivating point mutation in the gpnmb gene, exhibited reduced numbers of myeloid cells, elevated numbers of thioglycolate-elicited peritoneal macrophages, and higher levels of proinflammatory cytokines in response to LPS. Thus, GPNMB acts as a negative regulator of macrophage inflammatory responses.

The portal inflammatory infiltrate and ductular reaction in human nonalcoholic fatty liver disease

Although nonalcoholic fatty liver disease (NAFLD) is conventionally assessed histologically for lobular features of inflammation, development of portal fibrosis appears to be associated with disease progression. We investigated the composition of the portal inflammatory infiltrate and its relationship to the ductular reaction (DR), a second portal phenomenon implicated in fibrogenesis. The portal inflammatory infiltrate may contribute directly to fibrogenesis as well as influence the fate of the DR hepatic progenitor cells (HPCs), regulating the balance between liver repair and fibrosis. The presence of portal inflammation in NAFLD was strongly correlated with disease severity (fibrosis stage) and the DR. The portal infiltrate was characterized by immunostaining NAFLD liver biopsy sections (n = 33) for broad leukocyte subset markers (CD68, CD3, CD8, CD4, CD20, and neutrophil elastase) and selected inflammatory markers (matrix metalloproteinase 9 and interleukin [IL]‐17). Cells expressing all markers examined were identified throughout the liver lobules and in portal tracts, although portal tracts were more densely populated (P < 0.01), and dominated by CD68+ macrophages and CD8+ lymphocytes, at all stages of disease. An increase in portal macrophages in NAFLD patients with steatosis alone (P < 0.01) was the earliest change detected, even before elevated expression of the proinflammatory cytokines, IL1B and TNF, in patients with early NASH (P < 0.05). Portal and periductal accumulation of all other cell types examined occurred in progressed NASH (all P < 0.05). Conclusion: Knowledge of the complex cellular composition of the portal inflammatory infiltrate and HPC/DR niche in NAFLD will shape future functional studies to elucidate the contribution of portal inflammation to HPC differentiation and NAFLD pathogenesis. (Hepatology 2014;59:1393‐1405)

Data-driven normalization strategies for high-throughput quantitative RT-PCR

BackgroundHigh-throughput real-time quantitative reverse transcriptase polymerase chain reaction (qPCR) is a widely used technique in experiments where expression patterns of genes are to be profiled. Current stage technology allows the acquisition of profiles for a moderate number of genes (50 to a few thousand), and this number continues to grow. The use of appropriate normalization algorithms for qPCR-based data is therefore a highly important aspect of the data preprocessing pipeline.ResultsWe present and evaluate two data-driven normalization methods that directly correct for technical variation and represent robust alternatives to standard housekeeping gene-based approaches. We evaluated the performance of these methods against a single gene housekeeping gene method and our results suggest that quantile normalization performs best. These methods are implemented in freely-available software as an R package qpcrNorm distributed through the Bioconductor project.ConclusionThe utility of the approaches that we describe can be demonstrated most clearly in situations where standard housekeeping genes are regulated by some experimental condition. For large qPCR-based data sets, our approaches represent robust, data-driven strategies for normalization.

Macrophage Activation and Differentiation Signals Regulate Schlafen-4 Gene Expression: Evidence for Schlafen-4 as a Modulator of Myelopoiesis

Background The ten mouse and six human members of the Schlafen (Slfn) gene family all contain an AAA domain. Little is known of their function, but previous studies suggest roles in immune cell development. In this report, we assessed Slfn regulation and function in macrophages, which are key cellular regulators of innate immunity. Methodology/Principal Findings Multiple members of the Slfn family were up-regulated in mouse bone marrow-derived macrophages (BMM) by the Toll-like Receptor (TLR)4 agonist lipopolysaccharide (LPS), the TLR3 agonist Poly(I∶C), and in disease-affected joints in the collagen-induced model of rheumatoid arthritis. Of these, the most inducible was Slfn4. TLR agonists that signal exclusively through the MyD88 adaptor protein had more modest effects on Slfn4 mRNA levels, thus implicating MyD88-independent signalling and autocrine interferon (IFN)-β in inducible expression. This was supported by the substantial reduction in basal and LPS-induced Slfn4 mRNA expression in IFNAR-1−/− BMM. LPS causes growth arrest in macrophages, and other Slfn family genes have been implicated in growth control. Slfn4 mRNA levels were repressed during macrophage colony-stimulating factor (CSF-1)-mediated differentiation of bone marrow progenitors into BMM. To determine the role of Slfn4 in vivo, we over-expressed the gene specifically in macrophages in mice using a csf1r promoter-driven binary expression system. Transgenic over-expression of Slfn4 in myeloid cells did not alter macrophage colony formation or proliferation in vitro. Monocyte numbers, as well as inflammatory macrophages recruited to the peritoneal cavity, were reduced in transgenic mice that specifically over-expressed Slfn4, while macrophage numbers and hematopoietic activity were increased in the livers and spleens. Conclusions Slfn4 mRNA levels were up-regulated during macrophage activation but down-regulated during differentiation. Constitutive Slfn4 expression in the myeloid lineage in vivo perturbs myelopoiesis. We hypothesise that the down-regulation of Slfn4 gene expression during macrophage differentiation is a necessary step in development of this lineage.

A CSF-1 receptor kinase inhibitor targets effector functions and inhibits pro-inflammatory cytokine production from murine macrophage populations

CSF‐1 regulates macrophage differentiation, survival, and function, and is an attractive therapeutic target for chronic inflammation and malignant diseases. Here we describe the effects of a potent and selective inhibitor of CSF‐1R—CYC10268—on CSF‐1R‐dependent signaling. In in vitro kinase assays, CYC10268 was active in the low nanomolar range and showed selectivity over other kinases such as Abl and Kit. CYC10268 blocked survival mediated by CSF‐1R in primary murine bone marrow‐derived macrophages (BMM) and in the factor‐dependent cell line Ba/F3, in which the CSF‐1R was ectopically expressed. CYC10268 also inhibited CSF‐1 regulated signaling (Akt, ERK‐1/2), gene expression (urokinase plasminogen activator, toll‐like receptor 9, and apolipoprotein E), and priming of LPS‐inducible cytokine production in BMM. In thioglycollate‐elicited peritoneal macrophages (TEPM), which survive in the absence of exogenous CSF‐1, CYC10268 impaired LPS‐induced cytokine production and regulated expression of known CSF‐1 target genes. These observations support the conclusion that TEPM are CSF‐1 autocrine and that CSF‐1 plays a central role in macrophage effector functions during inflammation. CSF‐1R inhibitors such as CYC10268 provide a powerful tool to dissect the role of the CSF‐1/CSF‐1R signaling system in a range of biological systems and have potential for a number of therapeutic applications.—Irvine, K. M., Burns, C. J., Wilks, A. F., Su, S., Hume, D. A., Sweet, M. J. A CSF‐1 receptor kinase inhibitor targets effector functions and inhibits pro‐inflammatory cytokine production from murine macrophage populations. FASEB J. 20, 1315–1326 (2006)