No abstract

No abstract

TH17 cells are recognized as a unique subset of T helper cells that have critical roles in the pathogenesis of autoimmunity and tissue inflammation. Although RORγt is necessary for the generation of TH17 cells, the molecular mechanisms underlying the functional diversity of TH17 cells are not fully understood. Here we show that a member of interferon regulatory factor (IRF) family of transcription factors, IRF8, has a critical role in silencing TH17-cell differentiation. Mice with a conventional knockout, as well as a T cell-specific deletion, of the Irf8 gene exhibited more efficient TH17 cells. Indeed, studies of an experimental model of colitis showed that IRF8 deficiency resulted in more severe inflammation with an enhanced TH17 phenotype. IRF8 was induced steadily and inhibited TH17-cell differentiation during TH17 lineage commitment at least in part through its physical interaction with RORγt. These findings define IRF8 as a novel intrinsic transcriptional inhibitor of TH17-cell differentiation.

Abasic sites and deamination of cytosine to uracil are probably the most common types of endogenous DNA damage. The effects of such lesions on DNA topoisomerase I (top1) activity were examined in oligonucleotides containing a unique top1 cleavage site. The presence of uracils and abasic sites within the first 4 bases immediately 5 to the cleavage site suppressed normal top1 cleavage and induced new top1 cleavage sites. Uracils immediately 3 to the cleavage site increased cleavage and produced a camptothecin mimicking effect. A mismatch with a bulge or abasic sites immediately 3 to the top1 cleavage site irreversibly trapped top1 cleavable complexes in the absence of camptothecin and produced a suicide cleavage complex. These results demonstrate that top1 activity is sensitive to physiological, environmental, and pharmacological DNA modifications and that top1 can act as a specific mismatch-and abasic site-nicking enzyme.

Nitric oxide derived from iNOS in activated T cells negatively regulates Th17 cell differentiation.

DNA-PK is a nuclear, serine/threonine protein kinase required for repairing DNA double-strand breaks and for V(D)J recombination. To determine the distribution of DNA-PK in human tissues, we assayed paraffin-embedded sections of normal and cancerous tissues for DNA-PKcs and Ku80 by immunohistochemistry. We also assayed for Brca2, a human tumor suppressor gene that is implicated in the repair of DNA strand-breaks. Brca2 was strongly expressed in epithelial cells of the breast, endometrium, and thymus, in tingible body macrophages of follicular germinal centers of lymphoid tissue, and in reticuloendothelial cells in the spleen. DNA-PKcs and Ku80 expression was usually parallel, but both were expressed in a highly cell- and tissue-specific manner. The highest levels were observed in spermatogenic cells (but not in spermatozoa), and in neurons and glial cells of the central and autonomic nervous system. Neither protein was consistently expressed in liver nor in resting mammary epithelium, but lactating breast epithelium was strongly positive for DNA-PKcs and Ku80. In contrast to established human cell cultures, expression between cells in the same tissue was highly selective in the epidermis, exocrine pancreas, renal glomeruli, the red pulp of the spleen, and within cellular compartments of tonsils, lymph nodes, and thymus. Most cancerous tissues were consistently positive for DNA-PKcs and Ku80, except invasive carcinoma of the breast. DNA-PKcs, Ku80, and Ku70 mRNAs were expressed in all normal tissues with relatively little variation in levels. Our results suggest that the apparent absence of DNA-PKcs and Ku80 from some cells or tissues is a consequence of post-transcriptional mechanisms that regulate protein levels.

Autophagy, activated by many stresses, plays a critical role in innate immune responses. Here we show that Interferon Regulatory Factor 8 (IRF8) is required for expression of autophagy-related genes in dendritic cells. Furthermore in macrophages, IRF8 is induced by multiple autophagy-inducing stresses, including IFNγ and toll like receptor stimulation, bacterial infection, starvation and by macrophage colony-stimulating factor. IRF8 directly activates many genes involved in various steps of autophagy, promoting autophagosome formation and lysosomal fusion. Consequently, Irf8-/- macrophages are deficient in autophagic activity, and excessively accumulate SQSTM1 and ubiquitin-bound proteins. We show that clearance of Listeria monocytogenes in macrophages requires IRF8-dependent activation of autophagy genes and subsequent autophagic capturing and degradation of Listeria antigens. These processes are defective in Irf8-/- macrophages where uninhibited bacterial growth ensues. Together, these data suggest that IRF8 is a major autophagy regulator in macrophages, essential for macrophage maturation, survival and innate immune responses.

The effects of 3'-azido-3'-deoxythymidine (AZT) and three of its intracellular metabolites, azido-thymidine mono-, di-, and triphosphates, on the human immunodeficiency virus type 1 Integrase have been determined. AZT mono-, di-, and triphosphate have an IC50 for integration between 110 and 150 FM, whereas AZT does not inhibit the integrase. The inhibition by AZT monophosphate can be partially reversed by coincubation with either thymidine monophosphate or 2',3'-ddeoxythymldine monophosphate, suggesting that either of these monophosphates can bind to the integrase but that the azido group at the 3' position could be responsible for the inhibition. Integrase inhibition is associated with reduced enzyme-DNA binding but does not appear to be competitive with respect to the DNA substrate. Inhibition of an integrase deletion mutant containing only amino acids 50-212 suggests that these nucleotides bind in the catalytic core. Concentrations up to 1 mM AZT monophosphate can accumulate in vivo, indicating that integrase inhibition may contribute to the antiviral effects of AZT. The increasing incidence of AZT-resistant virus strains may, therefore, be associated with mutations not only in the reverse transcriptase but also in the human immundeficiency virus integrase. Finally, these observations suggest that additional strategies for antiviral drug development could be based upon nucleotide analogs as inhibitors of human immunodeficiency virus integrase.The first clinically approved drug in the treatment ofAIDS was 3'-azido-3'-deoxythymidine (AZT). This thymidine analog is converted to AZT monophosphate (AZTMP), diphosphate (AZTDP), and triphosphate (AZTTP) by thymidine, thymidylate, and nucleoside diphosphate kinases, respectively (1). The antiretroviral effect of AZT is attributed to AZTTP, which interferes with viral DNA replication by two mechanisms (2). (i) It can competitively inhibit the human immunodeficiency virus (HIV) reverse transcriptase against normal dTTP for DNA polymerization; and (ii) it can act as a chain terminator of the nascent viral DNA chain. The major limitation to AZT therapy is bone marrow suppression, leading to anemia or neutropenia (3). The most likely mechanism for AZT-induced toxicity could be incorporation ofAZT into newly synthesized host (i.e., nonviral) DNA and inhibition of strand elongation due to chain termination (4).Studies on the cellular metabolism ofAZT have shown that AZTMP accumulates in vivo because it is a competitive substrate inhibitor of thymidylate kinase that converts AZ-