Innate immunity consists of two systems; humoral and cellular systems. The humoral system includes anti-microbial peptides and opsonins while the cellular system involves specialized cells including phagocytes. The major functions of phagocytes are scavenging toxic compounds and producing inflammatory mediators to destroy infectious organisms and to transfer signals to other immune cells. Dysregulation of the phagocytic system by various conditions including the defect of immune cells and insufficient nutrition can lead to inflammatory diseases. Therefore, understanding the characteristics of phagocytes and the molecular mechanism in response to extracellular stimulations is critical for the development of therapeutics for inflammatory diseases. The focus of my thesis was to evaluate cellular signaling and its pathophysiological relationship in one type of phagocytes, macrophages. All-trans retinoic acid (RA) and its derivatives have been proved as potent therapeutics for inflammatory diseases, but the molecular mechanism of RA action in macrophages was not well established. In order to shed light on the functional role of RA in macrophages, I first found that topical application of RA significantly improved wound healing and the co-stimulation with RA and IL-4 synergistically activated Arginase-1 (Arg1), a critical gene for tissue repair, in macrophages. This involves feed forward regulation of Raldh2, a rate-limiting enzyme for RA biosynthesis, and requires Med25 to remodel the +1 nucleosome of Arg1 for transcription initiation and to facilitate transcriptional initiation-elongation coupling by recruiting elongation factor TFIIS. This study demonstrated RA’s modulatory activity in IL-4-induced anti-inflammatory macrophages, which involves synergistic activation of Arg1 by RA and IL-4 and a functional role of Med25 in chromatin remodeling of this gene promoter. In macrophage activation, there are two well-established phenotypes; classically activated (M1, pro-inflammatory) and alternatively activated (M2, anti-inflammatory) macrophages. The switch from M1 to M2 is critical for the control of inflammatory responses including wound repair. Previously, it has been found that Receptor-interacting Protein 140 (RIP140) is an enhancer of M1 by acting as a co-activator for NF-κB. Related to this, my research has uncovered that RIP140 delays the wound healing process by suppressing the macrophage phenotype switch from M1 to M2. With regards to mechanism, IL-4 treatment stimulates RIP140 export from the nucleus to the cytoplasm to form a complex with calpain regulatory subunit (CAPNS1) to activate calpain1/2 that enhances the activity of PTP1B, a negative regulator for STAT6 in M2 macrophages. Together, these results have established a new modulatory role of RIP140 in macrophage phenotype switch during wound healing by regulating both M1 and M2 activations (enhancing M1 and suppressing M2 activation). Another new finding I discovered about RIP140 was its repressive effect on osteoclast (OC) differentiation. OCs are derived from monocyte/macrophage lineage of hematopoietic cells and are responsible for bone resorptive activity. OCs maintain a balance in bone remodeling with osteoblasts (OB) that are involved in bone formation. RIP140 forms a complex with orphan nuclear receptor TR4 in pre-osteoclastic cells to suppress OC differentiation. Receptor Activator of Nuclear factor Kappa-B Ligand (RANKL) induces RIP140 protein degradation and represses TR4 mRNA level, which terminates the repressive activity of TR4/RIP140 complex in OC differentiation. In vivo micro CT analysis of macrophage/monocyte-specific RIP140 KD (mϕRIP140KD) mice showed an osteopenia phenotype with reduced OB function and increased OC activity, indicating uncoupling between OC and OB. This study demonstrated RIP140’s additional role in OC differentiation and bone diseases such as osteoporosis. Taken all together, these studies have established fine-tuning molecular mechanisms in macrophages, including their phenotypic switch and polarization/maturation. Specifically, we uncovered additional pathways of signal inputs and stimuli that regulate these processes such as RA, IL-4 and RANKL, and determined their physiological relevance in wound healing, inflammation and osteoclastogenesis. Differential activation of macrophages by these biological cues further confirms the plastic nature of macrophages. These findings contribute to our understanding of signaling mechanisms in macrophage polarization and their impact on diseases.
University of Minnesota Ph.D. dissertation.August 2016. Major: Pharmacology. Advisor: Li-Na Wei. 1 computer file (PDF); ix, 126 pages.
The functional impact of Retinoic acid and RIP140 in macrophages: from chromatin to physiology.
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