Innate immune responses are dictated by a panel of pathogen recognition receptors, downstream signaling from the receptors and the stimulated activities of various effector molecules. IRF8 is known as an interferon (IFN)-responsive transcription factor that plays critical roles in regulating the development of myeloid and dendritic cells and the activity of a number of genes, such as IL-12 and iNOS, involved in innate responses. Much of the activity of IRF8 in vitro was previously shown to require its ability to heterodimerize with PU.1 and possibly other transcription factors to mediate transcriptional activation or repression. An in vivo test of this model was provided by studies of BXH2 mice that identified a point mutation in IRF8 in the domain required for heterodimerization. It was shown that mice bearing this mutation were very similar to those bearing a null mutation of the gene, but that the null and point-mutant mice differed in their patterns of dendritic cell maturation. This indicated that most, but not all in vivo activities of IRF8 are dependent on its ability to dimerize with other transcription factors. Previous studies demonstrated that IRF8 is expressed to varying extents in subsets of dendritic cells (DC) of bone marrow origin. The status of IRF8 expression in follicular dendritic cells (FDC) that are not of bone marrow origin was not known. Studies of human and mouse FDC demonstrated that they expressed little or no IRF8. In addition, it was found also that IRF8 is expressed a much lower levels in tingible body macrophages of germinal centers than in other macrophage subsets. Studies of IRF8 knockout mice showed that the distribution and number of marginal zone macrophages was greatly altered while metallophilic macrophages on the other side of the marginal sinus were unaffected. These findings indicated that IRF8 was differentially expressed and functional in subsets of DC and macrophages. The function of IRF8 was previously shown by us and our collaborators to be affected by partnering with TRIM21, a member of the tripartite family and genes and and E3 ubiquitin ligase. To examine the role(s) played by TRIM21 in biology we generated a TRIM21 knockout mouse with an EGFP reporter inserted into the gene. Remarkably, the mouse exhibited essentially not abnormalities in innate immunity mediated by hematopoietic cells but was shown to negatively regulate NF-KB dependent proinflammatory responses in fibroblasts. We published a review on the contributions of the entire TRIM family to innate and acquired immunity including responses to HIV. We have previously identified TREX1 as a novel target of IRF8 in GC B cells. TREX1 is the major 3 ->5 DNA exonuclease in mammalian cells. Mutations identified within the single coding exon of the gene have been associated with a variety of human familial syndromes associated with inflammation and autoimmunity including systemic lupus erythematosus, Sjogrens syndrome, Aicardi-Goutieres syndrome (AGS) and retinal vasculopathy with cerebral leukodystrophy (RVCL). Loss of function mutation causes either mislocalization of the protein within intracellular compartments or a complete loss of enzymetic activity. Upon DNA damage induction, mutant forms of TREX1 fail to clear double and single stranded DNA species leading to expression of type I IFN. To understand the mechanisms of TREX1 in this type of innate immune responses, we have started to develop an allelic series of mice with humanized alleles of Trex1 that will conditionally express the normal or mutant alleles of this gene. Constructs designed to introduce the normal, one SLE-associated and one RVCL-associated allele have been generated and the first mice with the normal allele should be available soon. The mouse studies are being coordinated with the laboratory of Dr. John Atkinson, Chief of Rheumatology (Washington University, St. Louis), who is following several families with RVCL. In collaboration with Dr. Steve Holland (NIAID), individuals carrying the RVCL mutation but currently healthy are being admitted to the NIH for baseline studies and serial follow up. In addition, we have carried out studies on several human cell lines derived from patients with RVCL. By using a powerful Re-PCA assay and mass spectrometry analysis, we have identified 17 potential partner molecules of TREX1. Among these targets, PARP1 (poly(ADP-ribose)polymerase 1) has particularly drawn our attention in that this molecule is involved in DNA repair and has been investigated extensively as a therapeutic target for the treatment of cancer. We found that TREX1 physically associates with PARP1 and TREX1 mutation affects degradation of PARP1. Further characterization of TREX1 and PARP1 in human cells will help us to understand the molecular pathways that lead to activation of type I IFN and help to develop new strategies to reduce IFN effects in TREX1-related diseases. Additional studies are directer at understanding the role of IRF8 in T cells, where it was shown to inhibit expression of IL17 by Th17 CD4-positive T cells, and, unexpectedly, in some non-hematopoietic cells.
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