GSBS Dissertations and Theses

Approval Date

4-29-2013

Document Type

Doctoral Dissertation

Academic Program

MD/PhD

Department

Medicine

First Thesis Advisor

Katherine A. Fitzgerald, PhD

Keywords

Innate Immunity, Pattern Recognition Receptors, Signal Transduction, Virus Diseases, Virus Physiological Phenomena, Viruses, Oligodeoxyribonucleotides, Inflammasomes

Subjects

Dissertations, UMMS; Immunity, Innate; Receptors, Pattern Recognition; Signal Transduction; Virus Diseases; Virus Physiological Phenomena; Viruses; Oligodeoxyribonucleotides; Inflammasomes

Abstract

The innate immune system provides an essential first line of defense against infection. Innate immune cells detect pathogens through several classes of Pattern Recognition Receptors (PRR) allowing rapid response to a broad spectrum of infectious agents. Activated receptors initiate signaling cascades that lead to the production of cytokines, chemokines and type I interferons all of which are vital for controlling pathogen load and coordinating the adaptive immune response. Detection of nucleic acids by the innate immune system has emerged as a mechanism by which infection is recognized. Recognition of DNA is complex, influenced by sequence, structure, covalent modification and subcellular localization.

Interestingly certain synthetic oligodeoxynucleotides comprised of the TTAGGG motif inhibit proinflammatory responses in a variety of disease models. These suppressive oligodeoxynucleotides (sup ODN) have been shown to directly block TLR9 signaling as well as prevent STAT1 and STAT4 phosphorylation. Recently AIM2 has been shown to engage ASC and assemble an inflammasome complex leading to the caspase-1-dependent maturation of IL-1β and IL-18. The AIM2 inflammasome is activated in response to cytosolic dsDNA and plays an important role in controlling replication of murine cytomegalovirus (MCMV). In the second chapter of this thesis, a novel role for the sup ODN A151 in inhibiting cytosolic nucleic acid sensing pathways is described. Treatment of dendritic cells and macrophages with the A151 abrogated type I IFN, TNF-α and ISG induction in response to cytosolic dsDNA. A151 also reduced INF-β and TNF-α induction in BMDC and BMDM responding to the herpesviruses HSV-1 and MCMV but had no effect on the responses to LPS or Sendai virus. In addition, A151 abrogated caspase-1-dependent IL-1β and IL-18 maturation in dendritic cells stimulated with dsDNA and MCMV. Although inhibition of interferon-inducing pathways and inflammasome assembly was dependent on backbone composition, sequence differentially affected these pathways. While A151 more potently suppressed the AIM2 inflammasome, a related construct C151, proved to be a more potent inhibitor of interferon induction. A151 suppressed inflammasome signaling by binding to AIM2 and competing with immune-stimulatory DNA. The interaction of A151 and AIM2 prevented recruitment of the adapter ASC and assembly of the macromolecular inflammasome complex. Collectively, these findings reveal a new route by which suppressive ODNs modulate the immune system and unveil novel applications for suppressive ODNs in the treatment of infectious and autoimmune diseases.

The innate immune response to HSV-1 infection is critical for controlling early viral replication and coordinating the adaptive immune response. The cytokines IL-1β and IL-18 are important effector molecules in the innate response to HSV-1 in vivo. However, the PRRs responsible for the production and maturation of these cytokines have not been fully defined. In the third chapter of this thesis, The TLR2-MyD88 pathway is shown to be essential for the induction of pro-IL-1β transcription in dendritic cells and macrophages responding to HSV-1. The HSV-1 immediate-early protein ICP0 has previously been shown to block TLR2 responses and in keeping with this finding, ICP0 blocked pro-IL-1β expression. Following translation, pro-IL-1β exists as an inactive precursor that must be proteolytically cleaved by a multiprotein complex known as the inflammasome to yield its active form. Inflammasomes are composed of cytoplasmic receptors such as NLRP3 or AIM2, the adapter molecule ASC, and pro-caspase-1. In the present study we found that the NLRP3 inflammasome is important for maturation of IL-1β in macrophages and dendritic cells responding to HSV-1. In contrast the related NLRP12 protein controls IL-1β production in neutrophils. These data indicate that sensing of HSV-1 by TLR2 drives pro-IL-1β transcription and infection activates the inflammasome to mature this cytokine. Moreover, these studies reveal cell type-specific roles for NLRP3 and NLRP12 in inflammasome assembly.

DOI

10.13028/M24C7T

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Copyright is held by the author, with all rights reserved.

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