Role of TLR3 in response to herpes simplex virus encephalitis (HSE) in the central nervous system

            Published in Nature Immunology, Sato et al. (2018) research deconstructed the role of toll-like receptor (TLR) 3 in the central nervous system (CNS) in response to herpes simplex virus (HSV) encephalitis. HSV-1 is one of eight human herpes viruses, which is a large, double-stranded DNA virus that gains access to the CNS and affects sensory neurons (Bradshaw & Venkatesan, 2016). The authors demonstrated that cells in the CNS combat HSV-1 by activating the TLR3-mTORC2 pathway which led to the production to inflammatory chemokine (CCL5) and cytokine (IFN-beta) (Sato et al. 2018). TLRs are transmembrane pattern recognition receptor (PRR) proteins located on the cell surface and on endosomes (Takagi, 2011). TLRs are main components of the innate immune response by recognizing exogeneous pathogen-associated molecular pattern (PAMP) and endogenous damage-associated molecular pattern (DAMP) and initiate immune response accordingly (Takagi, 2011). There are 10 identified TLRs in humans and they recognize a number of different ligands which leads to the production of various cytokines and chemokines via the activation of adapter proteins (Mak et al. 2014). These chemical signals recruit and activate different immune cells resulting in the destruction of the pathogen. 

(Liu et al. 2008)

Using agonists to stimulate TLRs, a recent study demonstrated that activation of TLR3 and TLR4 leads to the production of proinflammatory cytokines in brain endothelial cells (Johnson et al. 2018). Proinflammatory cytokines are important for the recruitment of immune cells to the site of infection to fight off pathogens such as the HSV-1. Previous research has also demonstrated that besides TLRs, another pathway is involved in the innate defense against viral infection - the cyclic GMP-AMP synthase (cGAS) and its downstream adaptor protein, STING (Gao et al. 2013). Sato et al. focused their research on TLR3 in the CNS as the main activator of the innate immune response against HSV-1in neurons and investigate the role of cGAS-STING in other glial cells [astrocytes and microglia]. Here, they focused on the pathways and mechanisms through which cells of the CNS combat HSV-1. 

            The authors first assessed the role of TLR3 in neuronal response to HSV-1 by creating a genetic line of mice where the TLR3 gene was knocked out (Tlr3^-/-). The mice lacking TLR3 gene had a higher viral load three days after being transfected with HSV-1, which was correlated with lower mRNA expressions of Cxcl5 and Ifnb1, genes associated with CCL5 and IFN-beta. Mice lacking TLR3 did not survive past day 12 because they were unable to fight off the HSV-1 infection. In comparison, the wild type (WT) mice expressing TLR3 gene had higher levels of Cxcl5 and Ifnb1 mRNA expression and were able to survive the HSV-1 infection. To further elucidate the role of TLR3 in neurons, the authors cultured embryonic brain cells from mice lacking the Tlr3 gene and WT mice. Using flow cytometry - a technique that allow cells to be analyzed based on specific cellular markers - they found no TLR3 positive cells in Tlr3^-/-cell culture. Adding TLR3 agonist or HSV-1 lead to an increase in the expression of Ccl5 in WT but no change in gene expression in Tlr3 deficient culture. The authors also demonstrated that stimulating cGAS-STING did not increase the production of Ccl5 or Ifnb1 which suggested that this pathway was not active in neurons following HSV-1 infection. From these results, the authors were able to conclude that TLR3 was the primary receptor activated in neurons during HSV-1 infection. 

            Next, they investigated whether TLR3 had similar role in glial response to HSV-1. Analyzing neonatal brain cells, Sato et al. found that TLR3 expression was greater in astrocytes and microglia compared to neurons. Activating the TLR3 and cGAS pathway lead to the production of CCL5, demonstrating that TLR3 was not the only pathway activated in astrocytes. However, it was found that activation of both pathways was necessary for the production of IFN-beta because in astrocytes that were deficient in STING, the downstream adaptor protein of cGAS, IFN-beta production was significantly reduced. On the other hand, microglia were able to produce CCL5 and IFN-beta even when lacking Tlr3 gene. However, in STING deficient microglia, the production of CCL5 was abolished, suggesting that unlike the neuron and astrocyte, cGAS-STING pathway as the main activator in response to HSV-1 in microglia. 

            As the authors developed an understanding of the role of TLR3 in neurons, astrocytes and microglia, they next investigated the molecular mechanisms that led to the production of CCL5 and IFNb. In investigating IFNbproduction, Sato et al. focused on the mTOR complexes [mTORC1 and mTORC2]. Mammalian target of rapamycin, or mTOR, is a downstream signaling molecule that becomes activated by TLR3/4 activation and facilitate the translation of IFN-beta genes (Bodur et al. 2018). In order to determine the roles of mTORC1 and mTORC2, the authors used rapamycin to selectively inhibit mTORC1 and Torin 1 which inhibited both mTORC1 and mTORC2. By manipulating the mTOR pathways, they were able demonstrate that mTORC2 was necessary for the production of CCL5 in neurons, while IFN-beta production was abolished with both inhibitors. Similar observations were made in astrocytes. However, both mTORC1 and mTORC2 pathways were involved in the production of CCL5 in microglia. The role of mTORC2 dependent production of CCL5 via TLR3 activation was corroborated with experiments using mouse embryonic fibroblasts (MEFs). 

(Bodur et al. 2018)

            MEFs were further studied to understand the role of mTORC1 and mTORC2 in the production of IFN-beta. Using a line of MEFs that express TLR3 along with rapamycin and Torin 1, the researchers demonstrate that both mTORC1 and mTORC2 was necessary for the production of IFN-beta in HSV-1 infected fibroblasts. When TLR3 expressing MEFs were stimulated, the mTOR complexes’ physical proximity to TLR3 was enhanced; immunoprecipitation analysis also revealed the colocalization of TLR3 signaling molecules such as RICTOR and TRAF6 [RAPTOR and TRAF3 also colocalized after HSV-1 infection]. When mTORC2 was inhibited, the colocalization of these molecules and mTORC2 to TLR3 was not observed. These secondary signaling molecules, identified as identified as type 1 interferon signaling molecules, are essential for the production of IFN-beta upon infection with HSV-1. The trafficking of type 1 IFN signaling molecules to TLR3, and subsequent colocalization, was dependent on the mTORC2 phosphorylation of kinases [protein kinase C] which lead to the polymerization of actin and microtubules. Inhibiting mTORC2 and PKC prevented the polymerization of actin and microtubules. These cytoskeletal changes resulted in the trafficking of TLR3 from the perinuclear region of fibroblasts to the cell periphery – this trafficking of TLR3 was found to require the hydrolysis of GTP to GDP by Rab7a. 

            The authors then proposed that it is during the trafficking of TLR3 form the perinuclear region to the cell periphery that TLR3 interacts with signaling molecules to induce IFN-beta production. When they restricted TLR3 trafficking via inhibition of PKC or deficiency in Rab7a, HSV-1-dependent production of IFN-beta was impaired. Additionally, when transfected with HSV-1, there was a high colocalization of TLR3 and type IFN signaling molecules such as TRAF3. This lead the authors to conclude that HSV-1 enable the induction of IFN-beta during TLR3 trafficking by allowing TLR3 get close to type 1 IFN signaling molecules. 

            To translate these findings back to the CNS, they demonstrated that the response and trafficking of TLR3 in neurons transfected with HSV-1 is dependent on mTOR. Torin 1 inhibited the expression of Ccl5and Ifnb1 mRNA while the use of an mTOR activator increased their mRNA expressions. They also conducted in vivo experiments showing that intracranial administration of Torin 1 to inhibit mTOR pathways significantly increase the viral yield in mice after three days and significantly decreased the expression of Ccl5 and Ifnb1 mRNA; no effect on mice that lacked the Tlr3 gene was observed. Furthermore, they used a monoclonal antibody to demonstrate that potentiating the TLR3 response to HSV-1 infection lead to an increase survival of WT mice and decrease the viral yield in the CNS after three days. The decrease in viral yield was associated with increased expression of Ccl5 and Ifnb1 mRNA. Together these results demonstrate the importance of the TLR3-mTORC2 pathway in combating viral infection of HSV-1 in the CNS. The use of monoclonal antibody to TLR3 to enhance the immune response provides a novel therapeutic approach for patients with HSV-1 infections.       

     

This research provided a very thorough understanding of the role of TLR3 and mTORC2 in response to viral infection from HSV-1. To summarize, the researchers demonstrate that the activation of TLR3 by viral PAMP activates the downstream mTORC2 pathway leading to the polymerization of microtubules via PKC which enables the trafficking of TLR3 from the perinuclear region to the periphery of the cell. The trafficking of TLR3 subsequently induce the production of IFNbby interaction with type 1 interferon signaling molecules. The authors can build on their research by further investigating the pathways and mechanisms of CCL5 and IFNbin initiating an immune response in the CNS. Inflammatory response in the brain can be dangerous and can potentially lead to neurodegenerative disorders such as Alzheimer’s disease and multiple sclerosis (Sochocka et al. 2017; Hemmer et al. 2015). Further research can be focused on the mechanisms that prevent further damage in the CNS by the presence of immune cells. Additionally, research can also be directed towards the mechanisms involved in cytokine and chemokine signaling that allows immune cells to cross the blood-brain barrier and migrate to the site of infection. These investigation can give a more holistic view from the activation of TLR3 to the targeting and destruction of pathogens in the CNS. 

References: 

Bodur, C., Kazyken, D., Huang, K., Ustunel, B. E., Siroky, K. A., Tooley, A. S., ... & Steinl, G. K. (2018). The IKK‐related kinase TBK1 activates mTORC1 directly in response to growth factors and innate immune agonists. The EMBO journal, 37(1), 19-38.

Bradshaw, M. J., & Venkatesan, A. (2016). Herpes simplex virus-1 encephalitis in adults: pathophysiology, diagnosis, and management. Neurotherapeutics, 13(3), 493-508.

Gao, D., Wu, J., Wu, Y. T., Du, F., Aroh, C., Yan, N., ... & Chen, Z. J. (2013). Cyclic GMP-AMP synthase is an innate immune sensor of HIV and other retroviruses. Science, 341(6148), 903-906.

Johnson, R. H., Kho, D. T., O’Carroll, S. J., Angel, C. E., & Graham, E. S. (2018). The functional and inflammatory response of brain endothelial cells to toll-like receptor agonists. Scientific reports, 8.

Hemmer, B., Kerschensteiner, M., & Korn, T. (2015). Role of the innate and adaptive immune responses in the course of multiple sclerosis. The Lancet Neurology, 14(4), 406-419.

Liu, L., Botos, I., Wang, Y., Leonard, J. N., Shiloach, J., Segal, D. M., & Davies, D. R. (2008). Structural basis of toll-like receptor 3 signaling with double-stranded RNA. Science, 320(5874), 379-381.

Mak, T. W., Saunders, M. E., & Jett, B. D. (2014). Primer to the immune response. Newnes.

Takagi, M. (2011). Toll-like receptor. Journal of Clinical and Experimental Hematopathology, 51(2), 77-92.

Sochocka, M., Diniz, B. S., & Leszek, J. (2017). Inflammatory response in the CNS: friend or foe?. Molecular neurobiology, 54(10), 8071-8089.

Sato, R., Kato, A., Chimura, T., Saitoh, S. I., Shibata, T., Murakami, Y., ... & Sun-Wada, G. H. (2018). Combating herpesvirus encephalitis by potentiating a TLR3–mTORC2 axis. Nature immunology, 19(10), 1071.

M. (2017, February 02). HSV Encephalitis - Pathogenesis, Clinical Presentation, and Diagnosis. Retrieved October 24, 2018, from https://www.youtube.com/watch?v=oD26ys9P1Ks