Final Report Summary - TLR TOLERANCE (Investigation of the Regulation of Toll-like Receptor Mediated Transcription)
Toll-like receptors (TLRs) are key sensors of microbial products and are essential for the development of innate immunity to pathogens. TLR activation induces the expression of hundreds of genes that encode inflammatory cytokines, antimicrobial proteins, and regeneration and metabolic regulators; these molecules in turn mediate inflammation, antimicrobial immunity and tissue regeneration seen in patients with infectious diseases3-5. However, uncontrolled or prolonged activation of TLRs can have devastating consequences, which include the development of septic shock and fatal inflammatory diseases. Fortunately, TLR activation is tightly controlled by two classes of negative regulators: a) signal-specific regulators that inhibit TLR signalling, and b) gene-specific regulators that suppress TLR target gene transcription. These regulators ensure that prolonged or repeated exposure of TLRs to their ligands does not lead to sustained activation of the receptors; instead, it renders them insensitive or hyporesponsive to subsequent ligand stimulation. This phenomenon is referred to as TLR tolerance, or lipopolysaccharide (LPS) tolerance when LPS is the ligand involved. Recent genomic profiling of LPS responses reveals that LPS tolerance is a gene-specific phenomenon, i.e. it selectively targets one set of genes (e.g. inflammatory genes) but not others (e.g. antimicrobial genes); in fact, the expression of antimicrobial genes is further upregulated in LPS tolerised cells. Thus, LPS target genes have been divided into two classes: 1) tolerisable (T) genes or class T genes that are sensitive to LPS tolerance, and 2) non-tolerisable (NT) genes or class NT genes that are not sensitive to LPS tolerance. Because it is the inflammatory genes, not the antimicrobial genes, that cause deleterious inflammatory responses, the selective inactivation of class T (inflammatory) but not class NT (antimicrobial) genes ensures that the host is able to continuously build up its antimicrobial immunity without causing fatal inflammatory diseases even with chronic or prolonged infections 3.
However, the molecular mechanisms through which prolonged LPS exposure activates antimicrobial genes, but paradoxically suppresses inflammatory genes are unknown. I recently discovered that B cell leukaemia (Bcl)-3 mediates LPS tolerance by inhibiting nuclear factor (NF)-B, the major transcription factor activated by LPS. In the absence of Bcl-3, the inhibitory NF-B p50 homodimer is degraded, the inflammatory response exacerbated and LPS tolerance abolished. Additionally, my bioinformatic analysis of hundreds of LPS target genes reveals that the binding sites of NF-B are the only transcription factor sites that are selectively enriched in tolerisable genes, but not non-tolerisable genes (Preliminary Studies). I therefore hypothesize that inflammatory genes but not antimicrobial genes selectively utilise NF-B and that Bcl-3-mediated LPS tolerance targets inflammatory but not antimicrobial genes. These hypotheses will be tested in this project.
This project had three main objectives:
1) To determine the molecular determinants of p50 homodimer ubiquitination
2) The mechanisms by which Bcl-3 inhibits p50 ubiquitination and the testing of Bcl-3 mimetic peptides to inhibit inflammatory gene expression.
3) To test the hypothesis that NF-κB binding sites determine whether a gene is tolerisable or not.
Aims 2 and 3 were completed and significant progress was made on aim 3. A manuscript summarising the role of NF-κB binding sites in tolerisable gene promoters has been published in the Proceedings of the National Academy of Sciences, USA. We identified several critical determinants of p50 ubiquitination including an IKKβ kinase site which triggers p50 homodimer ubiquitination and enhances NF-κB transcriptional activity. In addition we identified the site of p50 homodimer ubiquitination, the mutation of which inhibits p50 ubiquitination. We identified the region of p50 homodimers required for interaction with Bcl-3. The mutation of this region results in the formation of a p50 homodimer that cannot interact with Bcl-3. Cells expressing this form of p50 recapitulate the Bcl-3-/- phenotype and are hyper-responsive to activation of NF-κB. We have also identified regions of Bcl-3 critical for interaction with p50 homodimers. Short cell-permeable peptides containing these sequences inhibited NF-κB induced expression of IL-23 in vitro while peptides mutated at 4 critical residues did not..
The on-going work will further characterise these peptides as modulators of inflammation. The data obtained on the molecular mechanisms of p50 ubiquitination and its inhibition by Bcl-3 will further increase our understanding of the regulation of inflammation at the transcriptional level as well as identify novel therapeutic targets.
However, the molecular mechanisms through which prolonged LPS exposure activates antimicrobial genes, but paradoxically suppresses inflammatory genes are unknown. I recently discovered that B cell leukaemia (Bcl)-3 mediates LPS tolerance by inhibiting nuclear factor (NF)-B, the major transcription factor activated by LPS. In the absence of Bcl-3, the inhibitory NF-B p50 homodimer is degraded, the inflammatory response exacerbated and LPS tolerance abolished. Additionally, my bioinformatic analysis of hundreds of LPS target genes reveals that the binding sites of NF-B are the only transcription factor sites that are selectively enriched in tolerisable genes, but not non-tolerisable genes (Preliminary Studies). I therefore hypothesize that inflammatory genes but not antimicrobial genes selectively utilise NF-B and that Bcl-3-mediated LPS tolerance targets inflammatory but not antimicrobial genes. These hypotheses will be tested in this project.
This project had three main objectives:
1) To determine the molecular determinants of p50 homodimer ubiquitination
2) The mechanisms by which Bcl-3 inhibits p50 ubiquitination and the testing of Bcl-3 mimetic peptides to inhibit inflammatory gene expression.
3) To test the hypothesis that NF-κB binding sites determine whether a gene is tolerisable or not.
Aims 2 and 3 were completed and significant progress was made on aim 3. A manuscript summarising the role of NF-κB binding sites in tolerisable gene promoters has been published in the Proceedings of the National Academy of Sciences, USA. We identified several critical determinants of p50 ubiquitination including an IKKβ kinase site which triggers p50 homodimer ubiquitination and enhances NF-κB transcriptional activity. In addition we identified the site of p50 homodimer ubiquitination, the mutation of which inhibits p50 ubiquitination. We identified the region of p50 homodimers required for interaction with Bcl-3. The mutation of this region results in the formation of a p50 homodimer that cannot interact with Bcl-3. Cells expressing this form of p50 recapitulate the Bcl-3-/- phenotype and are hyper-responsive to activation of NF-κB. We have also identified regions of Bcl-3 critical for interaction with p50 homodimers. Short cell-permeable peptides containing these sequences inhibited NF-κB induced expression of IL-23 in vitro while peptides mutated at 4 critical residues did not..
The on-going work will further characterise these peptides as modulators of inflammation. The data obtained on the molecular mechanisms of p50 ubiquitination and its inhibition by Bcl-3 will further increase our understanding of the regulation of inflammation at the transcriptional level as well as identify novel therapeutic targets.