|
|
|
Regulation of Cytokine Production Among the primary cytokines produced in response to TLR activation, TNF, IL-12 and the type I interferons are of key importance for the induction of further innate immune processes and also for activation of adaptive immunity. The TLR signal is propagated by serine kinase IRAK4, and if LPS is the inciting stimulus, also by way of the TRAM-TRIF-TRAF6 and TRAM-TRIF-TBK1-IRF3 pathways. TRAF6 coordinates the activation of still other protein kinases, which collectively lead to the activation of numerous transcription factors. Activated NF-kB, AP-1 and IRF3 are responsible for most of the hundred to thousands of transcriptional changes that ensue in the immediate aftermath of cell activation. Transcriptional Regulation Cytokines are regulated by different families of transcription factors such as NF-қB, AP-1, c-myc and NFAT1. These transcription factors, for example, are essential for the expression of IL-2, GM-CSF, and IFN-γ. In addition to control by NFκB, specific transcription factors, such as NFIL-1 and NFIL-6, also regulate cytokine production. Three transcription factors have been implicated in the development of a Th2 response: STAT6, GATA3 and c-maf. c-Maf was identified in a genetic screen of NF-AT1-associated factors. It is a distant cousin of the Ap-1 fmaily of transcription factors and is selectively expressed in Th2 but not Th1 cells. It appears to have an important and direct role in IL-4 transcription. C-maf-deficient T cells have significantly diminished IL_4 expression but produce normal levels of other Th2 type cytokines like IL-13. Cmaf plays distinct and nonoverlapping roles in Th2 differentation and cytokine production with GATA-3. c-Maf is a highly selective transactivator of the Il4 promoter but does not affect expression of IL-5 and IL-13 as with GATA-3. In addition, c-Maf-deficient T cells do not differ from wild-type cells in their ability to remodel the Th2 locus. C-maf binds to a consensus MARE binding site located in the proximal IL-4 promtoer, adjacent to the P0 NFAT binding site. Txk is a transcription factor that activates IFN-y gene directly. It is a member of the Tec family of nonreceptor tyrosine kinases. Many members of the Tec family function as signal mediators upon stimulation by an antigen via TCR. Txk is expressed only in Th1 and Th0 cells and acts as a Th1 specific transcrption factor. However, there is no evidence yet that T-bet and STAT-4 activate the gene for IFN-y directly. Suppressor of cytokine signalling (SOCS) are a family of molecules that act as negative regulators of cytokine signalling by interfering with the binding of cytokine receptors and intracellular molecules that act downstream, such as Janus Kinases (JAKs) SOCS-5 is expressed preferentially in Th1 cells and inhibits IL-4 receptor-mediated signal. The expression of SOCS-5 can result in a reduction in the extent of Th2 differentiation. SOCs-3 is expressed exclusively in Th2 cells and its pattern of expression is the mirror image of that of SOCS-5. It inhibits signal transduction vial the IL-12 receptor and its expression can result in a reduction in the extent of Th1 differentation. Post Transcription Regulation One of the features of ctyokine genes is the TTATTTAT sequence, which makes the mRNA prone for degradation. It has been analyzed if this sequence in the 3'UTR has any influence on regulation of the TNFα gene. Various CAT constructs with different 3'UTRs are transfected and CAT activity measured. As a general rule, those clones containing longer mRNA molecules express CAT mRNA and protein at a lower level then those encoding shorter 3'UTRs. All mRNAs derived from constructs containing the TTATTTAT element are poorly translated, indicating a posttranscriptional regulation. In addition the TTATTTAT element, just as it is imiportant for translational suppression, is important for the resposne to endotoxin. Modifications in Chromatin Structure See also Transcriptional Regulation Acetylation of the core histone N-terminal tails at lysine residues regulates transcription in several ways. First, it disrupts the ability of nucleosomal arrays to form higher order folded structures, thus enhancing accessibility of the underlying genome to DNA-binding proteins. Second, it counteracts the inhibitory effect exerted by histone tails on transcription factor access to both nucleosomal and internucleosomal DNA. Finally, acetylation favors recruitment to chromatin and/or activation of proteins implicated in transcriptional regulation, such as coactivators and chromatin remodeling enzymes. For example, the promoters of IL-6, IL-8, IL-12p40 and MCP-1 were found in one study to undergo H3 phosphorylation. Conversely, no H3 phsophorylation occured on the promoters of other LPS induced genes such as MIP1alpha, TNF-alpha im primary human monocyte-derived DCs. The histone acetylation profile at a locus can be inherited through mitosis, thus contributing to the maintenance of specific states of gene activity from one cell generation to the next. The ability of Th1 and Th2 restricted transcription factors to induce remodeling events at the corresponding loci suggests that modifications in chromatin structure underlie the acquisition of heritable competence to transcribe the Th1 and Th2 cytokines. DNase I-hypersensitive sites: Changes in chromatin structure have classically been assessed by changes in accessibility to restriction enzymes. The identification of specific sites on DNA that are cleaved by very low concentrations of DNase I is usually indicative of the presence of regulatory elements such as promoters, enhancers, LCRs, silencers, insulators or matrix attachment regions. For example, in the IL-4 locus, two such hypersensitive sites HSS3 and HS-IV are present in all T helper cell types on mouse chromosome 11.
|
