The IL-12 family of heterodimeric cytokines, comprising IL-12, IL-23 and IL-27, is an integral component of the inflammatory response. The IL-12 p40 subunit, which is part of both IL-12 and IL-23, is expressed specifically in macrophages and dendritic cells in response to microbial stimuli which signal through toll-like receptors (TLRs) and nucleotide-oligomerization-binding domain (NOD) proteins. Dysregulated expression of IL-12 p40 could lead to prolonged, unresolved inflammation manifesting into chronic inflammatory disorders such as inflammatory bowel disease (IBD). Understandably, IL-12 p40 expression is tightly regulated. We have demonstrated the requirement of a complex comprising nuclear factor of activated T cells (NFAT) and interferon regulatory factor 8 (IRF8) in IL-12 p40 gene transcriptional regulation. Subsequently, IRF8 was shown to be a target for an important anti-inflammatory pathway activated by carbon monoxide (CO) and heme oxygenase-1 (HO-1) in murine IBD. This dissertation explored two molecular mechanisms of IL-12 p40 inhibition targeting the transcription factor NFAT using a novel cell-permeable inhibitory peptide and phosphoinositide 3-kinase (PI3K) mediated inhibition of IL-12. Firstly, VIVIT peptide which prevents the nuclear translocation of NFAT was employed to examine the role of NFAT in macrophage gene expression. We observed that NFAT inhibition attenuated the expression of inflammatory cytokines including IL-12, IL-23 and TNF. Secondly, we studied PI3K mediated inhibition of IL-12 and characterized immune responses and macrophage defects in PI3K p110δ mutant mice which spontaneously develop IBD. We observed that the PI3K mutant mice recapitulate certain aspects of human IBD, including a profound increase in several proinflammatory cytokines in the intestinal mucosa and in macrophages, such as IL-12, IL-23, IL-17, TNF and IFN-γ. Furthermore, macrophages from PI3K p110δ mutant mice were defective in IL-10 and C5a mediated inhibition of IL-12 p40.
Thus, the expression of proinflammatory cytokines is coordinately regulated by transcription factors (such as NFAT and IRF8) and signaling molecules (such as PI3K), and mechanisms limiting inflammation are crucial for maintenance of immune homeostasis. Manipulation of such inhibitory pathways is a potential therapeutic approach for treating chronic inflammatory disorders.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-08152006-171506 |
| Date | 17 August 2006 |
| Creators | Rao, Kavitha N |
| Contributors | Binfeng Lu, Lawrence Kane, Sidney Morris, Anuradha Ray, Scott Plevy |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-08152006-171506/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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