Timothy Ravasi

Interferon-γ: an overview of signals, mechanisms and functions

Interferon‐γ (IFN‐γ) coordinates a diverse array of cellular programs through transcriptional regulation of immunologically relevant genes. This article reviews the current understanding of IFN‐γ ligand, receptor, ignal transduction, and cellular effects with a focus on macrophage responses and to a lesser extent, responses from other cell types that influence macrophage function during infection. The current model for IFN‐γ signal transduction is discussed, as well as signal regulation and factors conferring signal specificity. Cellular effects of IFN‐γ are described, including up‐regulation of pathogen recognition, antigen processing and presentation, the antiviral state, inhibition of cellular proliferation and effects on apoptosis, activation of microbicidal effector functions, immunomodulation, and leukocyte trafficking. In addition, integration of signaling and response with other cytokines and pathogen‐associated molecular patterns, such as tumor necrosis factor‐α, interleukin‐4, type I IFNs, and lipopolysaccharide are discussed.

Genome-wide analysis of mammalian promoter architecture and evolution

Mammalian promoters can be separated into two classes, conserved TATA box–enriched promoters, which initiate at a well-defined site, and more plastic, broad and evolvable CpG-rich promoters. We have sequenced tags corresponding to several hundred thousand transcription start sites (TSSs) in the mouse and human genomes, allowing precise analysis of the sequence architecture and evolution of distinct promoter classes. Different tissues and families of genes differentially use distinct types of promoters. Our tagging methods allow quantitative analysis of promoter usage in different tissues and show that differentially regulated alternative TSSs are a common feature in protein-coding genes and commonly generate alternative N termini. Among the TSSs, we identified new start sites associated with the majority of exons and with 3′ UTRs. These data permit genome-scale identification of tissue-specific promoters and analysis of the cis-acting elements associated with them.

The mononuclear phagocyte system revisited

The mononuclear phagocyte system (MPS) was defined as a family of cells comprising bone marrow progenitors, blood monocytes, and tissue macrophages. In this review, we briefly consider markers for cells of this lineage in the mouse, especially the F4/80 surface antigen and the receptor for macrophage colony‐stimulating factor. The concept of the MPS is challenged by evidence that there is a separate embryonic phagocyte lineage, the blurring of the boundaries between macrophages and other cells types arising from phenotypic plasticity and transdifferentiation, and evidence of local renewal of tissue macrophage populations as opposed to monocyte recruitment. Nevertheless, there is a unity to cells of the MPS suggested by their location, morphology, and shared markers. We discuss the origins of macrophage heterogeneity and argue that macrophages and antigen‐representing dendritic cells are closely related and part of the MPS.

LPS regulates proinflammatory gene expression in macrophages by altering histone deacetylase expression

Bacterial LPS triggers dramatic changes in gene expression in macrophages. We show here that LPS regulated several members of the histone deacetylase (HDAC) family at the mRNA level in murine bone marrow‐derived macrophages (BMM). LPS transiently repressed, then induced a number of HDACs (Hdac‐4, 5, 7) in BMM, whereas Hdac‐1 mRNA was induced more rapidly. Treatment of BMM with trichostatin A (TSA), an inhibitor of HDACs, enhanced LPS‐induced expression of the Cox‐2, Cxcl2, and Ifit2 genes. In the case of Cox‐2, this effect was also apparent at the promoter level. Overexpression of Hdac‐8 in RAW264 murine macrophages blocked the ability of LPS to induce Cox‐2 mRNA. Another class of LPS‐inducible genes, which included Ccl2, Ccl7, and Edn1, was suppressed by TSA, an effect most likely mediated by PU.1 degradation. Hence, HDACs act as potent and selective negative regulators of proinflammatory gene expression and act to prevent excessive inflammatory responses in macrophages.—Aung, H. T., Schroder, K., Himes, S. R., Brion, K., van Zuylen, W., Trieu, A., Suzuki, H., Hayashizaki, Y., Hume, D. A., Sweet, M. J., Ravasi, T. LPS regulates proinflammatory gene expression in macrophages by altering histone deacetylase expression. FASEB J. 20, 1315–1327 (2006)

Transcript annotation in FANTOM3: mouse gene catalog based on physical cDNAs.

The international FANTOM consortium aims to produce a comprehensive picture of the mammalian transcriptome, based upon an extensive cDNA collection and functional annotation of full-length enriched cDNAs. The previous dataset, FANTOM2, comprised 60,770 full-length enriched cDNAs. Functional annotation revealed that this cDNA dataset contained only about half of the estimated number of mouse protein-coding genes, indicating that a number of cDNAs still remained to be collected and identified. To pursue the complete gene catalog that covers all predicted mouse genes, cloning and sequencing of full-length enriched cDNAs has been continued since FANTOM2. In FANTOM3, 42,031 newly isolated cDNAs were subjected to functional annotation, and the annotation of 4,347 FANTOM2 cDNAs was updated. To accomplish accurate functional annotation, we improved our automated annotation pipeline by introducing new coding sequence prediction programs and developed a Web-based annotation interface for simplifying the annotation procedures to reduce manual annotation errors. Automated coding sequence and function prediction was followed with manual curation and review by expert curators. A total of 102,801 full-length enriched mouse cDNAs were annotated. Out of 102,801 transcripts, 56,722 were functionally annotated as protein coding (including partial or truncated transcripts), providing to our knowledge the greatest current coverage of the mouse proteome by full-length cDNAs. The total number of distinct non-protein-coding transcripts increased to 34,030. The FANTOM3 annotation system, consisting of automated computational prediction, manual curation, and final expert curation, facilitated the comprehensive characterization of the mouse transcriptome, and could be applied to the transcriptomes of other species.

Mouse neutrophilic granulocytes express mRNA encoding the macrophage colony-stimulating factor receptor (CSF-1R) as well as many other macrophage-specific transcripts and can transdifferentiate into macrophages in vitro in response to CSF-1

The differentiation of macrophages from their progenitors is controlled by macrophage colony‐stimulating factor (CSF‐1), which binds to a receptor (CSF‐1R) encoded by the c‐fms proto‐oncogene. We have previously used the promoter region of the CSF‐1R gene to direct expression of an enhanced green fluorescent protein (EGFP) reporter gene to resident macrophage populations in transgenic mice. In this paper, we show that the EGFP reporter is also expressed in all granulocytes detected with the Gr‐1 antibody, which binds to Ly‐6C and Ly‐6G or with a Ly‐6G‐specific antibody. Transgene expression reflects the presence of CSF‐1R mRNA but not CSF‐1R protein. The same pattern is observed with the macrophage‐specific F4/80 marker. Based on these findings, we performed a comparative array profiling of highly purified granulocytes and macrophages. The patterns of mRNA expression differed predominantly through granulocyte‐specific expression of a small subset of transcription factors (Egr1, HoxB7, STAT3), known abundant granulocyte proteins (e.g., S100A8, S100A9, neutrophil elastase), and specific receptors (fMLP, G‐CSF). These findings suggested that appropriate stimuli might mediate rapid interconversion of the major myeloid cell types, for example, in inflammation. In keeping with this hypothesis, we showed that purified Ly‐6G‐positive granulocytes express CSF‐1R after overnight culture and can subsequently differentiate to form F4/80‐positive macrophages in response to CSF‐1.

Genetic control of the innate immune response

BackgroundSusceptibility to infectious diseases is directed, in part, by the interaction between the invading pathogen and host macrophages. This study examines the influence of genetic background on host-pathogen interactions, by assessing the transcriptional responses of macrophages from five inbred mouse strains to lipopolysaccharide (LPS), a major determinant of responses to gram-negative microorganisms.ResultsThe mouse strains examined varied greatly in the number, amplitude and rate of induction of genes expressed in response to LPS. The response was attenuated in the C3H/HeJlpsdstrain, which has a mutation in the LPS receptor Toll-like receptor 4 (TLR4). Variation between mouse strains allowed clustering into early (C57Bl/6J and DBA/2J) and delayed (BALB/c and C3H/ARC) transcriptional phenotypes. There was no clear correlation between gene induction patterns and variation at the Bcg locus (Slc11A1) or propensity to bias Th1 versus Th2 T cell activation responses.ConclusionMacrophages from each strain responded to LPS with unique gene expression profiles. The variation apparent between genetic backgrounds provides insights into the breadth of possible inflammatory responses, and paradoxically, this divergence was used to identify a common transcriptional program that responds to TLR4 signalling, irrespective of genetic background. Our data indicates that many additional genetic loci control the nature and the extent of transcriptional responses promoted by a single pathogen-associated molecular pattern (PAMP), such as LPS.

Histone deacetylase inhibitors decrease toll-like receptor-mediated activation of proinflammatory gene expression by impairing transcription factor recruitment

Post‐translational modifications of histone proteins are major mechanisms that modify chromatin structure and regulate gene expression in eukaryotes. Activation of histone acetyltransferases or inhibition of histone deacetylases (HDACs) is generally believed to allow chromatin to assume a more open state, permitting transcriptional activity. We report here the surprising observation that treatment of murine dendritic cells with the HDAC inhibitors trichostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA) in non‐apoptotic concentrations strongly inhibited induction of both interleukin‐12 protein p40 (IL‐12p40) mRNA and protein upon stimulation of Toll‐like receptors (TLRs). Moreover, TLR‐mediated up‐regulation of costimulatory molecules was also inhibited. Up‐regulation of tumour necrosis factor‐α mRNA and protein in response to TLR agonists was only affected upon prolonged exposure to HDAC inhibitors and regulation of IL‐1β was not affected. Similar effects were apparent in murine and human macrophages. Regarding the mode of action, HDAC inhibition increased the acetylation status at the IL‐12p40 locus. Nevertheless, IL‐12p40 chromatin remodelling, binding of Rel‐A and IRF1 to the IL‐12p40 promoter and transcriptional activation were abrogated. In contrast, HDAC inhibitors had no effects on upstream nuclear factor‐κB and mitogen‐activated protein kinase activation. Thus HDACs positively regulate the expression of a subset of cytokine genes by enabling transcription factor recruitment.

Pirfenidone Is Renoprotective in Diabetic Kidney Disease

Although several interventions slow the progression of diabetic nephropathy, current therapies do not halt progression completely. Recent preclinical studies suggested that pirfenidone (PFD) prevents fibrosis in various diseases, but the mechanisms underlying its antifibrotic action are incompletely understood. Here, we evaluated the role of PFD in regulation of the extracellular matrix. In mouse mesangial cells, PFD decreased TGF-beta promoter activity, reduced TGF-beta protein secretion, and inhibited TGF-beta-induced Smad2-phosphorylation, 3TP-lux promoter activity, and generation of reactive oxygen species. To explore the therapeutic potential of PFD, we administered PFD to 17-wk-old db/db mice for 4 wk. PFD treatment significantly reduced mesangial matrix expansion and expression of renal matrix genes but did not affect albuminuria. Using liquid chromatography with subsequent electrospray ionization tandem mass spectrometry, we identified 21 proteins unique to PFD-treated diabetic kidneys. Analysis of gene ontology and protein-protein interactions of these proteins suggested that PFD may regulate RNA processing. Immunoblotting demonstrated that PFD promotes dosage-dependent dephosphorylation of eukaryotic initiation factor, potentially inhibiting translation of mRNA. In conclusion, PFD is renoprotective in diabetic kidney disease and may exert its antifibrotic effects, in part, via inhibiting RNA processing.

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.