Overproduction of IL-12 is associated with autoimmune diseases and microbial infections. Newly synthesized leaderless proIL-12 cannot be efficiently secreted from activated monocytes. However, when LPS-primed monocytes are further stimulated with extracellular ATP, they rapidly release large amounts of mature IL-12 and proIL-12. Thus far, the mechanism by which IL-12 is released from human monocytes is not understood. The overall goal of this proposal is to determine how IL-12 is released by investigating the molecular mechanisms by which ?-defensins block the release of IL-12 from human monocytes. Human ?-defensins (HNP-1 and HD-5), a group of antimicrobial peptides produced by neutrophils and epithelial cells, are the only inhibitors that block the release but not the processing of proIL-12 by caspase-1 in human monocytes. This proposal does not investigate the in vivo role of defensins. It only uses defensins as a tool to identify novel molecular targets for IL-12 blockade. Using photo-affinity and confocal microscopy, we have found that defensins bind to cell-membrane-associated proteins in human monocytes. We will test the hypothesis that IL-12 release and the externalization of proIL-12 and secretory lysosomes from human monocytes are regulated by distinct signaling proteins which can be inhibited by ?-defensins via an enzyme- linked receptor. To accomplish that, we have identified two specific aims:
Specific Aim One : Identify the receptor that is responsible for defensin-mediated inhibition of IL-12 release from human monocytes. Using photo-affinity purification and proteomic approaches, we will identify defensin-binding proteins (DBPs) in human monocytes. We will then identify the defensin receptor by determining the effect of siRNA-mediated knockdown of these DBPs on defensin blockade of IL-12 release.
Specific Aim Two : Define the signaling pathway essential for defensin receptor-mediated inhibition of IL- 12 release. We will determine whether protein kinase C, phospholipase C, Ca2????dependent protein kinase, and MAP kinases are involved in defensin blockade of IL-12 release and the externalization of proIL-12 and secretory lysosomes from human monocytes. Successful completion of this project will have broad impacts on both basic and translational medical research. If our hypotheses are correct, it will not only bring much clarity to the field of IL-12 biology, but also provide novel insights to the ER/Golgi-independent secretory pathway used by many other important leaderless proteins, including IL-18, IL-33, MIF, and FGF-2. The proposed studies will also lead to the discovery of novel molecular targets for IL-12 blockade and may lead to the development of new therapeutic approaches to prevent and treat many life-threatening microbial infections and inflammatory diseases.
Defensins are peptides produced by white blood cells and epithelial cells. Using defensins as a tool, we will determine how proinflammatory cytokine IL-12 is released from human monocytes. The proposed studies will lead to the discovery of novel molecular targets for IL-12 blockade and may lead to the development of new therapeutic approaches to prevent and treat many life-threatening microbial infections and inflammatory diseases.
|Galliher-Beckley, A; Pappan, L K; Madera, Rachel et al. (2015) Characterization of a novel oil-in-water emulsion adjuvant for swine influenza virus and Mycoplasma hyopneumoniae vaccines. Vaccine 33:2903-8|
|Galliher-Beckley, Amy; Li, Xiangdong; Bates, John T et al. (2015) Pigs immunized with Chinese highly pathogenic PRRS virus modified live vaccine are protected from challenge with North American PRRSV strain NADC-20. Vaccine 33:3518-25|
|Li, X; Galliher-Beckley, A; Pappan, L et al. (2014) Comparison of host immune responses to homologous and heterologous type II porcine reproductive and respiratory syndrome virus (PRRSV) challenge in vaccinated and unvaccinated pigs. Biomed Res Int 2014:416727|
|Li, Xiangdong; Galliher-Beckley, Amy; Huang, Hongzhou et al. (2013) Peptide nanofiber hydrogel adjuvanted live virus vaccine enhances cross-protective immunity to porcine reproductive and respiratory syndrome virus. Vaccine 31:4508-15|
|Wang, Lei; Liu, Ziyan; Li, Yijing et al. (2012) Pro-inflammatory cytokine interleukin-1? promotes the development of intestinal stem cells. Inflamm Res 61:1085-92|
|Wang, Lei; Liu, Ziyan; Balivada, Sivasai et al. (2012) Interleukin-1? and transforming growth factor-? cooperate to induce neurosphere formation and increase tumorigenicity of adherent LN-229 glioma cells. Stem Cell Res Ther 3:5|
|Li, Yijing; Wang, Lei; Pappan, Loretta et al. (2012) IL-1? promotes stemness and invasiveness of colon cancer cells through Zeb1 activation. Mol Cancer 11:87|