Date of Completion
Graduate School of Biomedical Sciences, Interdisciplinary Graduate Program
Dissertations, UMMS; Adaptive Immunity; Herpes Simplex; Immune System; Interferon Type I; Ligands; Receptors, Immunologic; Receptors, Pattern Recognition
The innate immune system provides the first line of defense against infection. Pathogens are detected though a variety of Pattern Recognition Receptors (PRRs), which activate downstream signaling cascades. Effector molecules such as cytokines and chemokines are released upon activation and aid in cell recruitment, control of pathogen replication, and coordination of the adaptive immune response. Nucleic acids that are released into the cytosol during viral and bacterial infection are recognized through a special class of PRRs, coined cytosolic nucleic acid sensors. Upon recognition, these receptors induce the production of type I interferons and other cytokines to aid in pathogen clearance. Although many cytosolic nucleic acid sensors have been discovered, it is unclear how they work in concert to mediate these responses.
The Interferon Gamma Inducible protein (IFI)16 and its proposed mouse orthologue IFI204 are cytosolic DNA sensors that have been linked to the detection of cytosolic DNA during infection with Herpes Simplex Virus (HSV-1). IFI16 binds dsDNA that has been released into the cytosol during viral infection and engages the adaptor molecule Stimulator of Interferon Genes (STING) leading to TANK binding kinase-1 (TBK1) dependent phosphorylation of interferon regulatory factor 3 (IRF3) and transcription of type I interferons and interferon stimulated genes. In addition to its role as a sensor, in chapter two of this thesis we describe a broader role for IFI16 in the regulation of the type I IFN response to RNA and DNA viruses in anti-viral immunity. In an effort to better understand the role of IFI16 in coordinating type I IFN gene regulation, we generated cell lines with stable knockdown of IFI16 and examined responses to DNA and RNA viruses as well as other inducers of IFN such as cyclic-dinucleotides. As expected, stable knockdown of IFI16 led to a severely attenuated type I IFN response to cytosolic DNA ligands and DNA viruses. In contrast, expression of the NF-κB regulated cytokines such as IL-6 and IL-1β were unaffected in IFI16 knockdown cells, suggesting that the role of IFI16 in sensing these triggers was unique to the type I IFN pathway. Surprisingly, we also found that knockdown of IFI16 led to a severe attenuation of expression of IFN-α and IFN stimulated genes such as RIG-I in response to cyclic GMP-AMP (cGAMP), a second messenger produced in response to cGAS, as well as RNA ligands and viruses. Analysis of IFI16 knockdown cells revealed compromised occupancy of RNA polymerase II on the IFN-α promoter in IFI16 knockdown cells suggesting that transcription of ISGs is dependent on IFI16. Since IFI16 knockdown compromised not only DNA virus driven pathways, we propose additional regulatory roles outside of DNA sensing. Collectively, these results indicate that IFI16 plays a role in the regulation of type I IFN gene transcription and production in response to both RNA and DNA viruses.
The role of IFI16/IFI204 has been studied extensively in vitro, however the role of the receptors in vivo has yet to be determined. In chapter three of this thesis, we developed a mouse deficient in IFI204 to explore the role of IFI204 in in vivo immune responses to viruses. We investigated the ability of IFI204 deficient cells to induce type I interferons and other cytokines in response to a panel of DNA and RNA ligands in vitro. IFI204 deficient BMDMs displayed a partial defect in type I interferon induction in response to both DNA and RNA ligands and viruses as compared to WT mice. We also observed that this phenotype is time dependent, since there was no change in type I interferon induction after 12 hours post infection as compared to earlier time points. In contrast to these results, expression of the NF-κB regulated cytokines IL-6 and IL-1β were unaffected in IFI16 knockdown cells. These results suggest that IFI204 plays a partial role in the induction of type I interferons in response to both DNA and RNA ligands. Additionally, IFI204 may work in tandem with other receptors in a sequential manner to amplify the type I interferon response. We also studied the involvement of IFI204 in an in vivo model of HSV-1 infection. IFI204 knockout mice produce less brain and serum IFN-β, IL-6, and IL-1β 72 hours post intraperitoneal infection with HSV-1. Furthermore, IFI204 -/- mice are more susceptible to HSV-1 infection as compared to WT mice. These data indicate that IFI204 mediates the response to HSV-1 in vivo by inducing the production of cytokines that are necessary for the control of viral infection.
Thompson, Mikayla R., "Dissecting the Role of Cytosolic Nucleic Acid Sensors in the Type I Interferon Response to Herpes Simplex Virus-1 and other Ligands: A Dissertation" (2014). University of Massachusetts Medical School. GSBS Dissertations and Theses. Paper 708.
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