THE ROLE OF INNATE IMMUNE CELLS IN THE PATHOGENESIS OF CONGENITAL CYTOMEGALOVIRUS INFECTION IN THE CENTRAL NERVOUS SYSTEM

Abstract

Cilj istraživanja: Infekcija MCMV-om kod novookoćenih miševa inducira snažan upalni odgovor u mozgu koji dovodi do aktivacije mikroglije i infiltacije urođenih imunosnih stanica. Imunosne stanice luče proupalne citokine, poput TNFα, koji uzrokuje promjene u razvoju malog mozga. Tretman inficiranih miševa glukokortikoidima ili neutralizacija TNFα smanjuje upalu i korigira parametre postnatalnog razvoja malog mozga, što ukazuje da je upalni odgovori domaćina na infekciju MCMV-om, a ne citopatski učinak virusa odgovoran za opažene promjene u razvoju malog mozga. Naši preliminarni rezultati ukazuju na ulogu stanica NK u neuroinflamaciji nakon prirođene infekcije MCMV-om. Stoga je cilj ovog istraživanja odrediti ulogu stanica NK u aktivaciji mikroglije i promjenama u razvoju malog mozga. Materijali i metode: Kako bismo odredili utjecaj infekcije MCMV-om na mikrogliju, proveli smo RNA-seq analizu te analizu fenotipa navedenih stanica protočnom citometrijom. Dodatno, proveli smo imunohistokemijsku analizu kako bismo utvrdili može li MCMV inficirati mikrogliju. Kako bismo odredili ulogu stanica NK u neuroinflamaciji, prvo smo odredili kinetiku infiltracije stanica NK u mozak i izvršili analizu fenotipa navedenih stanica protočnom citometrijom te odredili njihovu tkivnu lokalizaciju imunohistokemijom. Također, utvrdili smo čimbenike odgovorne za privlačenje stanica NK u mozak. Kako bismo odredili ulogu stanica NK i IFN-γ u promijenjenom razvoju malog mozga, uklonili smo stanice NK ili smo neutralizirali IFN-γ in vivo ili koristili miševe kojima nedostaje receptor za IFN-γ na rezidentnim stanicama mozga i mjerili debljinu EGL-a te izražaj gena u signalnom putu SHH. Dodatno, koristili smo test virusnih čistina kako bismo utvrdili ulogu stanica NK i IFN-γ u kontroli infekcije MCMV-om u mozgu. Rezultati: Pokazali smo da aktivirane stanice NK/ILC1 ne sudjeluju u kontroli virusne replikacije u mozgu, već posreduju imunopatologiju. Imunopatologiji prethodi aktivacija mikroglije i proizvodnja CXCL9 i CXCL10, kemokina koji privlače stanice NK/ILC1 u mozak miševa na način ovisan o CXCR3. IFN-γ kojeg izlučuju aktivirane stanice NK/ILC1 glavni je čimbenik koji doprinosi promijenjenom razvoju malog mozga. Zaključak: Ovo istraživanje pokazalo je da nezrele stanice NK/ILC1 posreduju imunopatologiju u mozgu nakon prirođene infekcije MCMV-om. Stoga ovo istraživanje daje važan doprinos razumijevanju patogeneze prirođene infekcije HCMV-om, te se može iskoristiti za dizajniranje novih terapijskih ciljeva.Objectives: MCMV infection in newborn mice induces a strong inflammatory response which leads to the activation of microglia and infiltation of innate immune cells. These immune cells can further produce proinflammatory cytokines, such as TNFα which can then exacerbate cerebellar developmental problems. This is underscored by the finding that the treatment of infected animals with glucocorticoids or blocking of TNFα attenuates neuroinflammation and limits deficits in cerebellar development indicating that host inflammatory responses to MCMV infection, rather than the cytopathic effect of virus on infected cells, are important drivers of deficits in cerebellar development. The exact mechanisms and critical components involved are, however, largely unknown. Our preliminary results indicate the role of NK cells in neuroinflammation following congenital MCMV infection. Therefore, the aim of this study is to determine the role of NK cells in activation of microglia and altered cerebellar development. Material and methods: To gain more insight into the impact of MCMV infection on the microglia we performed RNA-seq and phenotype analysis of these cells by flow cytometry. In addition, we performed immunohistochemical analysis to determine whether microglia can be infected with MCMV. To determine the role of NK cells in neuroinflammation we first determined the kinetics of NK cell infiltration in the brain and performed phenotype analysis of these cells by flow cytometry and their tissue localization by immunohistochemistry. We also determined factors responsible for recruitment of NK cells to the brain. To determine the role of NK cells and IFN-γ in altered cerebellar development we depleted NK cells or neutralize IFN-γ in vivo or use mice that lack IFN-γ receptor on brain resident cells and measure EGL thickness and expression of genes in SHH pathway. In addition, we used the plaque assay to determine the role of NK cells and IFN-γ on the control of MCMV infection in the brain. Results: Here, we show that activated newborn NK and ILC1 cells mediate immunopathology instead of controlling the infection and limiting tissue damage. Immunopathology is preceded by activation of microglia and production of CXCL9 and CXCL10, chemokines that recruit NK/ILC1 cells to the brain of MCMV-infected newborns in a CXCR3-dependent manner. IFNγ released by highly activated, brain infiltrating NK/ILC1 cells is a major contributing factor to the altered cerebellar development. Conclusions: This study is the first to demonstrate the immune-pathogenic action of immature newborn NK/ILC1 cells in the brain following congnital MCMV infection. Thus, this study provides an important contribution to understanding the pathogenesis of cHCMV infection, which can be harnessed to design novel therapeutic targets