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Tick-borne encephalitis Targeted by Human Active NK cells

Periodic Reporting for period 1 - Tick ThaNK (Tick-borne encephalitis Targeted by Human Active NK cells)

Reporting period: 2019-01-01 to 2020-12-31

Natural killer (NK) cells are important players during the immune response against viral infections. However, their role in tick borne encephalitis (TBE) and other viral infections in the central nervous system (CNS) has been understudied. The aim of this project was to understand how NK cells fight the infection produced by the tick-borne encephalitis virus (TBEV) that may cause brain inflammation and long-term neurological sequelae. We also studied what NK cells do in the CNS and what are the mechanisms of their migration into the brain during TBEV infection. We think that a better understanding of the mechanisms responsible for NK cell activation and recruitment to the site of infection in this particular disease can give us a glimpse of the possible function of NK cells during other viral infections that affect the CNS.

The European TBEV causes a mortality rate of 1-2% and deaths occur in average one week after the onset of neurological signs and symptoms. The Far-Eastern TBEV strain is more severe, and the mortality rate rises up to 5-20%.TBE may cause a long recovery period, leaving long-term or even permanent neurologic sequelae in 30 to 60% of patients with the disease.

TBE is a disease occurring in different areas within Europe and Asia and is transmitted by the bite of an infected tick. The incubation period takes in average seven days after the bite of an infected tick. Most of the TBEV infections are asymptomatic, however those who develop symptoms show a clinical debut with a biphasic pattern. Unspecific symptoms such as fever, headache, myalgia and nausea appear during the first phase that lasts from 2-10 days. An asymptomatic period follows and subsequently, a second phase with neurological involvement occurs producing meningitis, encephalitis or meningoencephalitis. Since there is no cure or specific treatment for TBE, hospitalization, supportive care, use of anti-inflammatory drugs such as corticosteroids, intubation and/or ventilator support may be considered based on disease severity.

NK cells constitute a first line of defense against viral infections and tumors. According to the expression of the CD56 molecule on their surface, NK cells are classified into two main subsets, both with specific functions and locations within the body. The CD56dim NK cell population is predominant in peripheral blood and has a cytotoxic function, whereas the CD56bright immunoregulatory subset is mainly found in peripheral and lymphoid tissues.
Recently, the field of tissue resident NK cells (trNK) in solid organs and their function in tissue development and remodeling have gained increasing interest. However, in the brain tissue, knowledge has been limited with respect to the existence of tissue resident lymphocytic populations including NK cells. Conventionally, the CNS was known as an immune privileged site with low penetrability of immune cells. However, recent studies have demonstrated the existence of an important inflammatory response to infection in the CNS. Studies of NK cell recruitment into the brain in pathological conditions such as multiple sclerosis and viral encephalitis have led to important but limited information to understand the role of NK cells in such compartment. The aim of the Tick ThaNK project is to generate knowledge that will provide new insights into innate immunity responses executed by NK cells that could lead to novel strategies to improve treatments for infectious diseases.
We studied the migratory and functional characteristics of different immune cells, mainly NK cells, using an in vitro model of the blood brain barrier (BBB) and complex multiparameter flow cytometry. Different immune cells can be added into the model and it is possible to study the immune phenotype, capability to transmigrate, cytotoxicity and other functions of the cells that are capable to cross the barrier. Thus, it is possible to determine possible mechanisms that allow cells to migrate. Furthermore, our study of the immune cell compartment in blood and in cerebrospinal fluid of patients with TBE has been possible due to the collection of a small cohort of such patients that we have analysed. Additionally, we are currently studying the effect of different neurotropic viruses on the integrity of the BBB as well as on the activation of immune cells.

Here, we report that among the lymphocytic cell population, the CD56bright, a subset of NK cells, possess a high migratory capability in the presence of brain endothelial cells and the expression of cell adhesion molecules such as CD11a, CD11b and CD29 are important in the total lymphocytic migration process. The cells with higher migratory capability also seem to possess a higher cytotoxic capability as determined by the high specific target cell lysis and the association with a higher activation status determined by high expression of CD69, Granzyme A, Granzyme B and perforin.
About the study of patient samples with TBE, we are still acquiring and processing data. However, we have observed that the CD56bright NK cell population is highly increased in the CFS of TBE patients in comparison to peripheral blood, and the cells also seem to have a similar pattern of activation. Our results in vitro partially resemble the observations we have obtained in vivo and thus, the collection of more TBE patients is crucial to complete a broad picture of the immunological features during a viral infection in CNS. Furthermore, our current studies on the effect of neurotropic viruses on the BBB integrity and on immune cell activation will help us understand more the interplay between different immune components in mounting an immune response towards a viral infection in the CNS. Altogether, we have acquired insights on the migratory capabilities of different cell populations, which may add value in understanding the complex immune mobilization process between blood and tissues taking place to keep a homeostatic balance.
The characterization of the immune phenotype and function of cells that are able to migrate through a biological membrane, as well as the characteristics of the immune compartment in biological samples of TBE patients allow us to understand more the immunopathogenesis of a viral disease affecting the CNS. The mechanisms found in TBE can be potentially extrapolated to other pathological processes. Currently, we have extended the project to the study of other neurological conditions that might have an impact on the BBB. Among other, we are studying the effect of neurotropic viruses such as influenza, TBEV, West Nile virus and SarsCoV2 on cell recruitment and cell activation using the BBB in vitro model. Viral infections that induce neurological alterations have important socio-economic implications due to the long-term, and in some cases, permanent sequelae. A clear example is the accumulation of persistent neurologic symptoms and cognitive dysfunction in patients who had Covid19, the so-called “long haulers” experiencing PASC or post-acute sequelae of Covid19. Further studies are crucial to understand the immunopathogenesis of such diseases and thus, develop therapies to target the immune system, or tissue specific immune responses, as a way to fight viral infections.
NK cell mediated immune response in the different clinical phases of TBE