Transcriptional profiling and targeted proteomics reveals common molecular changes associated with cigarette smoke-induced lung emphysema development in five susceptible mouse strains.

BACKGROUND: Mouse models are useful for studying cigarette smoke (CS)-induced chronic pulmonary pathologies such as lung emphysema. To enhance translation of large-scale omics data from mechanistic studies into pathophysiological changes, we have developed computational tools based on reverse causal reasoning (RCR). OBJECTIVE: In the present study we applied a systems biology approach leveraging RCR to identify molecular mechanistic explanations of pathophysiological changes associated with CS-induced lung emphysema in susceptible mice. METHODS: The lung transcriptomes of five mouse models (C57BL/6, ApoE (-/-) , A/J, CD1, and Nrf2 (-/-) ) were analyzed following 5-7 months of CS exposure. RESULTS: We predicted 39 molecular changes mostly related to inflammatory processes including known key emphysema drivers such as NF-κB and TLR4 signaling, and increased levels of TNF-α, CSF2, and several interleukins. More importantly, RCR predicted potential molecular mechanisms that are less well-established, including increased transcriptional activity of PU.1, STAT1, C/EBP, FOXM1, YY1, and N-COR, and reduced protein abundance of ITGB6 and CFTR. We corroborated several predictions using targeted proteomic approaches, demonstrating increased abundance of CSF2, C/EBPα, C/EBPß, PU.1, BRCA1, and STAT1. CONCLUSION: These systems biology-derived candidate mechanisms common to susceptible mouse models may enhance understanding of CS-induced molecular processes underlying emphysema development in mice and their relevancy for human chronic obstructive pulmonary disease.

Identification of IRF-8 and IRF-1 target genes in activated macrophages.

Interferon regulatory factor 1 (IRF-1) and IRF-8, also known as interferon consensus sequence binding protein (ICSBP), are important regulators of macrophage differentiation and function. These factors exert their activities through the formation of heterocomplexes. As such, they are coactivators of various interferon-inducible genes in macrophages. To gain better insights into the involvement of these two transcription factors in the onset of the innate immune response and to identify their regulatory network in activated macrophages, DNA microarray was employed. Changes in the expression profile were analyzed in peritoneal macrophages from wild type mice and compared to IRF-1 and IRF-8 null mice, before and following 4 h exposure to IFN-gamma and LPS. The expression pattern of 265 genes was significantly changed (up/down) in peritoneal macrophages extracted from wild type mice following treatment with IFN-gamma and LPS, while no changes in the expression levels of these genes were observed in samples of the same cell-type from both IRF-1 and IRF-8 null mice. Among these putative target genes, numerous genes are involved in macrophage activity during inflammation. The expression profile of 10 of them was further examined by quantitative RT-PCR. In addition, the promoter regions of three of the identified genes were analyzed by reporter gene assay for the ability to respond to IRF-1 and IRF-8. Together, our results suggest that both IRF-1 and IRF-8 are involved in the transcriptional regulation of these genes. We therefore suggest a broader role for IRF-1 and IRF-8 in macrophages differentiation and maturation, being important inflammatory mediators.

Interferon regulatory factor-8 is indispensable for the expression of promyelocytic leukemia and the formation of nuclear bodies in myeloid cells.

Interferon (IFN) regulatory factor-8 (IRF-8), previously known as ICSBP, is a myeloid cell essential transcription factor. Mice with null mutation in IRF-8 are defective in the ability of myeloid progenitor cells to mature toward macrophage lineage. Accordingly, these mice develop chronic myelogenous leukemia (CML). We demonstrate here that IRF-8 is an obligatory regulator of the promyelocytic leukemia (PML) gene in activated macrophages, leading to the expression of the PML-I isoform. This regulation is most effective together with two other transcription factors, IRF-1 and PU.1. PML is a tumor suppressor gene that serves as a scaffold protein for nuclear bodies. IRF-8 is not only essential for the IFN-gamma-induced expression of PML in activated macrophages but also for the formation of nuclear bodies. Reduced IRF-8 transcript levels were reported in CML patients, and a recovery to normal levels was observed in patients in remission following treatment with IFN-alpha. We demonstrate a significant correlation between the levels of IRF-8 and PML in these CML patients. Together, our results indicate that some of the myeloleukemia suppressor activities of IRF-8 are mediated through the regulation of PML. When IRF-8 levels are compromised, the reduced PML expression may lead to genome instability and eventually to the leukemic phenotype.

Nramp1-mediated innate resistance to intraphagosomal pathogens is regulated by IRF-8, PU.1, and Miz-1.

Natural resistance-associated macrophage protein 1 (Nramp1) is a proton/divalent cation antiporter exclusively expressed in monocyte/macrophage cells with a unique role in innate resistance to intraphagosomal pathogens. In humans, it is linked to several infectious diseases, including leprosy, pulmonary tuberculosis, visceral leishmaniasis, meningococcal meningitis, and human immunodeficiency virus as well as to autoimmune diseases such as rheumatoid arthritis and Crohn's disease. Here we demonstrate that the restricted expression of Nramp1 is mediated by the macrophage-specific transcription factor IRF-8. This factor exerts its activity via protein-protein interaction, which facilitates its binding to target DNA. Using yeast two-hybrid screen we identified Myc Interacting Zinc finger protein 1 (Miz-1) as new interacting partner. This interaction is restricted to immune cells and takes place on the promoter Nramp1 in association with PU.1, a transcription factor essential for myelopoiesis. Consistent with these data, IRF-8 knockout mice are sensitive to a repertoire of intracellular pathogens. Accordingly, IRF-8-/- mice express low levels of Nramp1 that can not be induced any further. Thus, our results explain in molecular terms the role of IRF-8 in conferring innate resistance to intracellular pathogens and point to its possible involvement in autoimmune diseases.