Allergies to nickel (Ni(2+)) are the most frequent cause of contact hypersensitivity (CHS) in industrialized countries. The efficient development of CHS requires both a T lymphocyte-specific signal and a proinflammatory signal. Here we show that Ni(2+) triggered an inflammatory response by directly activating human Toll-like receptor 4 (TLR4). Ni(2+)-induced TLR4 activation was species-specific, as mouse TLR4 could not generate this response. Studies with mutant TLR4 proteins revealed that the non-conserved histidines 456 and 458 of human TLR4 are required for activation by Ni(2+) but not by the natural ligand lipopolysaccharide. Accordingly, transgenic expression of human TLR4 in TLR4-deficient mice allowed efficient sensitization to Ni(2+) and elicitation of CHS. Our data implicate site-specific human TLR4 inhibition as a potential strategy for therapeutic intervention in CHS that would not affect vital immune responses.

PRDI-BF1, the human ortholog of mouse Blimp-1, is a DNA-binding protein involved in postinduction repression of interferon-beta gene transcription in response to viral infection. PRDI-BF1 also has an essential function in driving terminal differentiation of B lymphocytes and therein silences multiple genes. Here we show PRDI-BF1 assembles silent chromatin over the interferon-beta promoter in the osteosarcoma cell line U2OS through recruitment of the histone H3 lysine methyltransferase G9a. G9a is recruited only when in a complex with PRDI-BF1. G9a catalytic activity is required for the accumulation of methylated histone H3 and transcriptional silencing mediated by PRDI-BF1 in vivo. This establishes a mechanism for the recruitment of G9a, the main mammalian euchromatic methyltransferase, and defines nonembryonic targets of G9a.

Post-translational modifications of histones, in general, and acetylation/deacetylation, in particular, can dramatically alter gene expression in eukaryotic cells. In humans, four highly homologous class I HDAC enzymes (HDAC1, HDAC2, HDAC3, and HDAC8) have been identified to date. Although HDAC3 shares some structural and functional similarities with other class I HDACs, it exists in multisubunit complexes separate and different from other known HDAC complexes, implying that individual HDACs might function in a distinct manner. In this current study, to understand further the cellular function of HDAC3 and to uncover possible unique roles this protein may have in gene regulation, we performed a detailed analysis of HDAC3 using deletion mutations. Surprisingly, we found that the non-conserved C-terminal region of HDAC3 is required for both deacetylase and transcriptional repression activity. In addition, we discovered that the central portion of the HDAC3 protein possesses a nuclear export signal, whereas the C-terminal part of HDAC3 contributes to the protein's localization in the nucleus. Finally, we found that HDAC3 forms oligomers in vitro and in vivo and that the N-terminal portion of HDAC3 is necessary for this property. These data indicate that HDAC3 comprises separate, non-overlapping domains that contribute to the unique properties and function of this protein.Post-translational modification of nucleosomal histones can convert regions of chromosomes into transcriptionally active or inactive chromatin. The best understood post-translational modification of histones is the acetylation of ⑀-amino groups on conserved lysine residues in the histones' N-terminal tail domains. In addition to its effect on transcription, acetylation of core histones has been correlated with many important cellular processes, including chromatin assembly, DNA repair, and recombination (1-7).In general, the status of histone acetylation in a cell is regulated by histone acetyltransferases and histone deacetylases (HDACs).1 In humans, HDACs are divided into three categories (8 -11): the class I RPD3-like proteins (HDAC1, HDAC2, HDAC3, and HDAC8); the class II HDA1-like proteins (HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10); and the class III SIR2-like proteins. The recent availability of the human genome sequence revealed the possibility of additional HDACs whose class assignment has yet to be determined (12). Because class I HDACs are ubiquitously expressed, whereas most class II HDACs are tissue-specific, it has been proposed that the class I enzymes may be important in the regulation of housekeeping genes (13,14). By far, the most thoroughly studied human HDACs are the two founding members of the class I enzymes, HDAC1 and the closely related HDAC2 protein. HDAC1 and HDAC2 exist together in at least three distinct multiprotein complexes called the Sin3, the NuRD/NRD/Mi2, and the CoREST complexes (15-22). In addition, many transcription factors interact directly with HDAC1/2 and thus may target HDAC1/2 to specific promoters (8,...

In humans and mice, CD8α+ conventional dendritic cells are the primary source of interferon-λ released in response to the adjuvant and Toll-like receptor 3 agonist poly IC.

Allergic contact hypersensitivity (CHS) is a T cell–mediated inflammatory skin disease. Interleukin (IL)-12 is considered to be important in the generation of the allergen-specific T cell response. Loss of IL-12 function in IL-12Rβ2–deficient mice, however, did not ameliorate the allergic immune response, suggesting alternate IL-12–independent pathways in the induction of CHS. Because exposure to contact allergens always takes place in the presence of microbial skin flora, we investigated the potential role of Toll-like receptors (TLRs) in the induction of CHS. Using mice deficient in TLR4, the receptor for bacterial lipopolysaccharide (LPS), IL-12 receptor (R) β2, or both, we show that the concomitant absence of TLR4 and IL-12Rβ2, but not the absence of TLR4 or IL-12Rβ2 alone, prevented DC-mediated sensitization, generation of effector T cells, and the subsequent CHS response to 2,4,6-trinitro-1-chlorobenzene (TNCB), oxazolone, and fluorescein isothiocyanate. Introduction of the TLR4 transgene into the TLR4/IL-12Rβ2 mutant restored the CHS inducibility, showing a requirement for TLR4 in IL-12–independent CHS induction. Furthermore, the concomitant absence of TLR2 and TLR4 prevented the induction of CHS to TNCB in IL-12–competent mice. Finally, CHS was inducible in germ-free wild-type and IL-12Rβ2–deficient mice, but not in germ-free TLR4/IL-12Rβ2 double deficient mice, suggesting that the necessary TLR activation may proceed via endogenous ligands.

Methylation of specific residues within the N-terminal histone tails plays a critical role in regulating eukaryotic gene expression. Although great advances have been made toward identifying histone methyltransferases (HMTs) and elucidating the consequences of histone methylation, little is known about the recruitment of HMTs to regulatory regions of chromatin. Here we report that the sequence-specific DNA-binding transcription factor Yin Yang 1 (YY1) binds to and recruits the histone H4 (Arg 3)-specific methyltransferase, PRMT1, to a YY1-activated promoter. Our data confirm that histone methylation does not occur randomly but rather is a targeted event and provides one mechanism by which HMTs can be recruited to chromatin to activate gene expression.

Significance Macrophages—cells crucially involved in defense against infections—exhibit, depending on their anatomical location, distinct biological properties. Studies of the underlying mechanisms are of scientific and clinical interest, but are hampered by the difficulty of obtaining primary tissue macrophages in sufficient numbers and purity. Here, we report the generation of nontransformed murine macrophages, which are similar to alveolar macrophages and can be grown continuously without change of phenotype and in unlimited amounts. Such macrophages helped us to recognize several innate immune properties of alveolar macrophages that are involved in the pathogenesis of infectious lung inflammation.

The early systemic production of interferon (IFN)-αβ is an essential component of the antiviral host defense mechanisms, but is also thought to contribute to the toxic side effects accompanying gene therapy with adenoviral vectors. Here we investigated the IFN-αβ response to human adenoviruses (Ads) in mice. By comparing the responses of normal, myeloid (m)DC- and plasmacytoid (p)DC-depleted mice and by measuring IFN-αβ mRNA expression in different organs and cells types, we show that in vivo, Ads elicit strong and rapid IFN-αβ production, almost exclusively in splenic mDCs. Using knockout mice, various strains of Ads (wild type, mutant and UV-inactivated) and MAP kinase inhibitors, we demonstrate that the Ad-induced IFN-αβ response does not require Toll-like receptors (TLR), known cytosolic sensors of RNA (RIG-I/MDA-5) and DNA (DAI) recognition and interferon regulatory factor (IRF)-3, but is dependent on viral endosomal escape, signaling via the MAP kinase SAPK/JNK and IRF-7. Furthermore, we show that Ads induce IFN-αβ and IL-6 in vivo by distinct pathways and confirm that IFN-αβ positively regulates the IL-6 response. Finally, by measuring TNF-α responses to LPS in Ad-infected wild type and IFN-αβR−/− mice, we show that IFN-αβ is the key mediator of Ad-induced hypersensitivity to LPS. These findings indicate that, like endosomal TLR signaling in pDCs, TLR-independent virus recognition in splenic mDCs can also produce a robust early IFN-αβ response, which is responsible for the bulk of IFN-αβ production induced by adenovirus in vivo. The signaling requirements are different from known TLR-dependent or cytosolic IFN-αβ induction mechanisms and suggest a novel cytosolic viral induction pathway. The hypersensitivity to components of the microbial flora and invading pathogens may in part explain the toxic side effects of adenoviral gene therapy and contribute to the pathogenesis of adenoviral disease.