Identification of IRF-8 and IRF-1 target genes in activated macrophages
Introduction
Interferon (IFN) regulatory factor-8 (IRF-8), also known as interferon consensus sequence binding protein (ICSBP), and IRF-1 are members of the IRF family of transcription factors, which include nine cellular members (Mamane et al., 1999). In general, these factors are important immunomodulators and are essential components of the IFN signaling cascade. Using in vitro assays, these two transcription factors were identified in association on the promoter region of numerous macrophage essential genes (Marecki and Fenton, 2000, Xiong et al., 2003). IRF-8 is a key element for the differentiation of myeloid progenitor cells towards macrophages and for mature macrophage activity. Accordingly, IRF-8−/− mice exhibit clinical manifestation that resembles the human chronic myelogenous leukemia (CML). Comparable to CML patients, these mice experience a rapid and systemic expansion of granulocytes that eventually results in a fatal blast crisis (Holtschke et al., 1996). Consistent with this, lack of IRF-8 was reported in human myeloid leukemias, implicating its pivotal role as a tumor suppressor gene (Schmidt et al., 1998). Thus, IRF-8 drives bipotential myeloid progenitor cells towards mature macrophages while inhibiting the differentiation pathway towards granulocytes (Tamura et al., 2000). In addition, it was shown that IRF-8 is an essential factor for proper functioning of mature macrophages. For example, IRF-8 null mice fail to mount Th1-mediated immune response, since they fail to produce the IL-12 p40 subunit (Wang et al., 2000). Similarly, IRF-1−/− are also devoid of Th1 responses due to deficiency of the same IL-12 subunit (Taki et al., 1997, Lohoff et al., 1997). Further, clinical studies have assigned important role for IRF-1 in the onset of myelodysplastic syndromes, which are clonal myeloid disorders (Willman et al., 1993). These studies indicate that IRF-1, like IRF-8, is a tumor suppressor gene with essential functions in the maturation of myeloid cells to macrophages.
Unlike other IRF members, IRF-8 is capable of binding to its target DNA sequence only following association with either IRF-1 and IRF-2 or non-IRF transcription factors, such as PU.1, an essential factor for hematopoiesis (Sharf et al., 1997, Wang et al., 2000, Dahl and Simon, 2003). The domain essential for these protein–protein associations, termed IRF association domain (IAD), is conserved among all other IRF members excluding IRF-1 and IRF-2. The association modules of IRF-1 and IRF-2 with IRF-8 were identified as PEST domains, enriched with proline, glutamic acid, serine, and threonine, originally shown in PU.1 (Levi et al., 2002). The specific partner that interacts with IRF-8 dictates not only the DNA binding site but also the transcriptional activity e.g. activation or repression. The stoichiometry between the interacting partners was not determined and it is possible that several PEST domains interact with an IAD, as observed on promoters of several macrophage specific genes, such as IL-12, IL-1β, and ISG15 (Meraro et al., 2002, Marecki et al., 2001).
As mentioned above, IRF-1 is one of the partners associated with IRF-8. Together, they play critical role in the regulation of several pro-inflammatory genes that function during macrophage activation; These two factors synergistically regulate the transcription of iNOS (Xiong et al., 2003), the two components of the phagocyte respiratory burst oxidase: p67 and gp91 (Eklund and Kakar, 1999), the transcription of IL-12 (Masumi et al., 2002), IL-18 (Kim et al., 1999), IL-1β (Marecki et al., 2001), and ISG15 (Meraro et al., 2002). Taken together, IRF-8 and IRF-1 are essential components of macrophage functioning and innate immune response.
To get better insight into the role of these transcription factors in the onset of the innate immune response and to identify their regulatory network in macrophages, we performed DNA microarray analysis. We have used mRNA retrieved from macrophages extracted from IRF-8 knockout (KO) mice, IRF-1 KO mice, and C57BL/6J, the counterpart wild type (WT) strain. Changes in the expression profiles were analyzed before and after 4 h of exposure to both IFN-γ and LPS. The levels of mRNA transcripts from treated and untreated macrophages were compiled and compared. The expression of 265 genes was significantly altered (fold of change ≥2.0, p < 0.001) in WT mice while no changes were observed in the transcripts of these genes in both KO mice. Thus, our results suggest that both IRF-1 and IRF-8 are involved in the transcriptional regulation of these differentially expressed genes. Among these putative target genes, numerous are involved in macrophage activity during inflammation pointing to the pivotal regulatory role of both IRF-1 and IRF-8 in the inflammatory process.
Section snippets
Animals
The mouse strains; C57BL/6J (Harlan Biotech, Israel), IRF-1 deficient (kindly obtained from Dr. Rubinstein, The Weizmann Institute, Israel, originally from The Jackson Laboratory), and IRF-8 deficient (Holtschke et al., 1996), were maintained in microisolator cages in a viral pathogen-free facility.
Isolation of peritoneal macrophages
Four days prior to experiment, mice were injected i.p. with 3 ml 3% sterile thioglycolate medium. Mice were sacrificed by CO2 inhalation, 10 ml sterile PBS was injected to the peritoneal cavity,
Stimulation of peritoneal macrophage with IFN-γ and LPS leads to differential expression of genes involved in various aspects of macrophage activity
To look for changes in peritoneal macrophages expression profile following stimulation, mice were injected intraperitoneally with thioglycolate and cells were extracted by peritoneal lavage 4 days later. The isolated macrophages were either treated or not treated with IFN-γ and LPS to mimic bacterial infection. Four hours later, total RNA was extracted and utilized for DNA microarray analysis. We focused on early response transcripts whose expression level changed significantly following
Discussion
Both IRF-1 and IRF-8 have important roles in the maturation and the function of macrophages. Accordingly, mice with null mutations for these two genes exhibit severe defects in both innate and adaptive immunity. These mice manifest abnormalities in the early phases of myelopoiesis, are unable to mount Th1 mediated immune response, and are sensitive to various pathogens (Tamura and Ozato, 2002, Taniguchi et al., 1901, Testa et al., 1995). These two IRF members associate through characterized
Acknowledgments
We are grateful to the Arison family donation to the center of DNA Chips, Pediatric Oncology, Chaim Sheba Medical Center and to Prof. Rubinstein from the Weizmann Institute for the generous gift of IRF-1−/− mice. We thank Dr. Naama Rave-Harel from our laboratory for critical reading of the manuscript, and Dr. Shifra Ben-Dor from the Weizmann Institute for valuable help in data analysis. GR holds the Djerassi Chair in Oncology at the Sackler School of Medicine, and B.Z. Levi is an incumbent of
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