Controlling immune responses in blood cells
Abstract
A future goal is to control immune responses to rapidly allow hosts to respond to infection while preventing over reactions that generate undesired side effects. Infection and cellular maintenance of synthetic endosymbionts requires the immune system to be dampened but this needs to be controlled as it still is essential to fight undesirable infectious agents. This application focuses on developing a greater understanding of natural methods used by blood cells of the innate immune system to dampen immune responses. A natural dampening immune protein is IRAK3 (interleukin 1 receptor associated kinase 3), which has the hallmarks of an excellent target to be manipulated for controlling immune responses especially in blood cells. IRAK3 acts as a negative regulator of pro-inflammatory cytokine production. IRAK3 production in blood monocytes and endothelial cells is therapeutically induced by glucocorticoids but it remains uncertain how to decrease IRAK3 production which is necessary to prevent serious disease following secondary infections. Inside the immune cells, IRAK3 interacts with components of the myddosome that forms part of the signalling complex to activate the transcription factor nuclear factor .B (NF.B) following bacterial infection. A complete understanding of the mechanisms of action of IRAK3 remains obscure but we previously identified a cryptic catalytic centre within IRAK3 that generates cGMP which appears integral to IRAK3Õs ability to abrogate NF.B activity and thus inflammation. We hypothesize that the catalytic center within IRAK3 generates cGMP that modifies the immediate nano-environment surrounding the protein thereby facilitating inhibition of TLR-mediated signal cascades. We further propose that disruption of IRAK3 catalytic activity may lead to resumption of the innate inflammation cascade that could be advantageous for control of synthetic endosymbionts, and establishment or suppression, as desired, of bacterial presence. In this study, we will define the spatiotemporal interaction of IRAK3 with myddosome components and how this is influenced by its catalytic product, cGMP. New molecular imaging tools now render questions regarding cellular distribution and complex formation addressable at the single molecule level and we have built a bespoke state-of-the-art super resolution microscope where we are based at La Trobe University Bendigo. Therefore, we will specifically interrogate the spatiotemporal interaction of IRAK3 with the myddosome using the super resolution microscope to follow IRAK3 in cells. We will undertake mass spectrometry experiments to identify key components necessary for the interaction using model cell lines and confirm these interactions by immunoprecipitation and FRET analyses including visualizing FRET interactions. To gain insight to the role of the cryptic enzyme function of IRAK3, we will manipulate cellular cGMP levels to understand its contribution to the immunosuppressive effect of IRAK3. The planned experiments will resolve spatiotemporal distribution of IRAK3 interactions with the myddosome and other proteins and how this is influenced by the guanylate cyclase activity of IRAK3. Interrogating IRAK3 mechanisms at the single molecular level will thus inform understanding of its actions in dampening immune responses and is essential to manipulate the innate immune system and todevelop a framework to inform design of IRAK3 specific ligands of significant relevance in the control of innate immunity.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Aug 06, 2019
- Source ID
- W911NF1910435
Entities
People
- Helen R. Irving
Organizations
- Army Contracting Command
- La Trobe University
- United States Army