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Interdisciplinary Biomedical Sciences Program
M.S. 2008, Joint Program at NJIT/Rutgers, Newark
B.S. 1998, University of Mumbai, India
Thesis Advisor: S. J. Leibovich, Ph.D.
Department of Cell Biology and Molecular Medicine
Tuesday, November 22, 2016
12:00 PM, MSB G609b (CBMM conference room)
Our lab has been working to understand the mechanism of an alternative activation pathway of macrophages that differs from the canonical M1 and M2 pathways and that involves an IL4R£-independent activation of macrophages by TLR2, 4, 7 or 9 agonists in the presence of A2AR agonists. This pathway switches macrophages from an M1 phenotype to an ¡§M2-like¡¨ phenotype in an A2AR-dependent manner. Macrophages activated in this manner exhibit low expression of inflammatory cytokines such as TNF£, IL-12 and several small chemokines, and up-regulated expression of IL-10 and VEGF. We have suggested that these macrophages be termed ¡§M2d¡¨. Earlier studies in our lab investigating the signaling pathways involved in the induction of the M2d phenotype found that lipopolysaccharide (LPS) rapidly and specifically suppresses Phospholipase C beta 2 (PLC£]-2) expression in macrophages at the post transcriptional level by destabilizing its mRNA, and that this suppression plays a role in the A2AR -mediated switch of macrophages from an inflammatory to an angiogenic phenotype. My thesis work aimed at elucidating the mechanism of destabilization of PLC£]-2 mRNA upon LPS treatment in murine macrophages. Reporter assays involving the PLC£]-2 3¡¦UTR transfected into RAW 264.7 macrophage-like cells showed decreased luciferase expression upon LPS treatment, suggesting the involvement of the 3¡¦UTR in the PLC£]-2 mRNA destabilization.
Microarray profiling of microRNA (miRNA) expression by macrophages induced by LPS and a combination of LPS and NECA (a non-specific adenosine receptor agonist) indicated that several miRNAs are regulated by these treatments. These include miRNA 155, miRNA 221, miRNA 222, miRNA 146a, miRNA 487b and many others. miRNA155 and miRNA466l were selected for investigation of their roles in PLC-£]2 mRNA destabilization since miRNA 155 was the most strongly modulated miRNA upon LPS treatment of macrophages and miRNA 466l had a conserved binding site in the 3¡¦UTR of the PLC-£]2 mRNA 3¡¦UTR. Treatment of macrophages with miR155 and miR466l mimics alone decreased PLC-£]2 mRNA levels in comparison to negative control mimic treated macrophages, indicating their potential role in regulating PLC-£]2 mRNA stability. Treatment of macrophages with miRNA antagomeres did not cause a significant change in LPS¡Vmediated PLC£]-2 mRNA suppression in the miR155 or the miRNA 466l antagomere treated groups in comparison to the negative control antagomere group, suggesting either an indirect effect of these miRNAs and/or the involvement of additional regulatory factors in the LPS-mediated PLCÒ-2 mRNA destabilization pathway. Mimics of miRNA155 and miRNA466l did not affect LPS-mediated TNF-£ expression and caused a modest decrease in LPS-NECA-mediated VEGF expression, again suggesting an indirect role for these miRNAs in the M1 to M2d macrophage phenotypic switch. Additionally, there was no significant change in LPS-mediated TNF-£ expression or of LPS-NECA mediated VEGF expression upon treatment of RAW 264.7 cells with miRNA155 and miRNA466l antagomeres, further suggesting that these miRNAs are not directly involved in the M1 to M2d switch.
LPS strongly induced the expression of the RNA-binding protein tristetraprolin (TTP) in RAW cells and murine peritoneal macrophages. However, LPS suppressed PLC£]-2 mRNA expression in TTP-/- macrophages to the same degree and with similar kinetics to that observed in wild type macrophages. This clearly indicates that TTP does not play an essential role in the destabilization of PLC£]-2 mRNA by LPS.
The role of specific regions of the PLCÒ-2 mRNA in the LPS-mediated destabilization was studied by systematic deletion of regions in the PLC£]-2 3¡¦UTRusing site-directed mutagenesis. Deletion constructs were cloned into a reporter plasmid to create several mutant 3¡¦UTR plasmids. Transfection of these mutant 3¡¦UTR plasmids into RAW 264.7 cells followed by LPS treatment and reporter assays showed that LPS suppressed luciferase expression from the mutant constructs to the same extent as from the non-mutated 3¡¦UTR construct, strongly suggesting that the destabilization of the mRNA in response to LPS is not mediated by a specific region of 3¡¦UTR alone.