The role of lipids in lysosome dysfunction and the pathogenesis of neurodegenerative diseases

Lizosomi su primarni katabolički odjeljci eukariotskih stanica. Za funkciju lizosoma neophodne su dvije skupine proteina: kisele hidrolaze i integralni lizosomalni membranski proteini. Lizosomi su uključeni u različite fiziološke procese kao što su homeostaza kolesterola, popravak plazma membrane, obrana od patogena, stanična smrt i stanična signalizacija. Uzrok lizosomalnih bolesti nakupljanja lipida je u većini slučajeva neispravna aktivnost lizosomalnih proteina što rezultira akumuliranjem nerazgrađenih metabolita unutar lizosoma. Postoji sve više dokaza da su lizosomi uključeni i u proces patogeneze različitih neurodegenerativnih bolesti uključujući Alzheimerovu bolest, Parkinsonovu bolest te Huntingtonovu bolest. Promijenjen metabolizam lipida uzrokuje disfunkciju endosomalnog/lizosomalnog puta. Pretpostavlja se da je nefunkcionalna razgradnja proteina inducirana disfunkcijom endosomalno/lizosomalnog sustava primarni uzrok nakupljanja proteina te nastanka neurodegenerativnih bolesti. U ovom sam seminarskom radu dala kratak osvrt na strukturu i funkciju lizosoma, promjenu metabolizma lipida u lizosomalnim bolestima nakupljanja lipida te na disfunkciju endosomalno/lizosomalnog sustava u neurodegenerativnim bolestima.Lysosomes are the primary catabolic compartments of eukaryotic cells. Two classes of proteins are essential for the lysosome function: lysosomal hydrolases and integral lysosomal membrane proteins. Lysosomes are involved in various physiological processes, such as cholesterol homeostasis, plasma membrane repair, pathogen defence, cell death and cell signalling. Lysosomal storage disorders (LSD) are mainly caused by the defective activity of lysosomal proteins, which results in the intra-lysosomal accumulation of undegraded metabolites. There is increasing evidence that lysosomes are also involved in the pathogenesis of a variety of neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease and Huntington’s disease. Endosomal/lysosomal dysfunction is caused by altered lipid metabolism. It is hypothesized that abnormal protein degradation and deposition caused by endosomal/lysosomal dysfunction may be the primary trigger of age-related neurodegeneration. In this review, the structure and function of lysosomes, the role of abnormal lipid metabolism in relation to aberrant endosomal/lysosomal function and the relationship between lysosome dysfunction and various neurodegenerative diseases is described

The role of lipids in lysosome dysfunction and the pathogenesis of neurodegenerative diseases

Lizosomi su primarni katabolički odjeljci eukariotskih stanica. Za funkciju lizosoma neophodne su dvije skupine proteina: kisele hidrolaze i integralni lizosomalni membranski proteini. Lizosomi su uključeni u različite fiziološke procese kao što su homeostaza kolesterola, popravak plazma membrane, obrana od patogena, stanična smrt i stanična signalizacija. Uzrok lizosomalnih bolesti nakupljanja lipida je u većini slučajeva neispravna aktivnost lizosomalnih proteina što rezultira akumuliranjem nerazgrađenih metabolita unutar lizosoma. Postoji sve više dokaza da su lizosomi uključeni i u proces patogeneze različitih neurodegenerativnih bolesti uključujući Alzheimerovu bolest, Parkinsonovu bolest te Huntingtonovu bolest. Promijenjen metabolizam lipida uzrokuje disfunkciju endosomalnog/lizosomalnog puta. Pretpostavlja se da je nefunkcionalna razgradnja proteina inducirana disfunkcijom endosomalno/lizosomalnog sustava primarni uzrok nakupljanja proteina te nastanka neurodegenerativnih bolesti. U ovom sam seminarskom radu dala kratak osvrt na strukturu i funkciju lizosoma, promjenu metabolizma lipida u lizosomalnim bolestima nakupljanja lipida te na disfunkciju endosomalno/lizosomalnog sustava u neurodegenerativnim bolestima.Lysosomes are the primary catabolic compartments of eukaryotic cells. Two classes of proteins are essential for the lysosome function: lysosomal hydrolases and integral lysosomal membrane proteins. Lysosomes are involved in various physiological processes, such as cholesterol homeostasis, plasma membrane repair, pathogen defence, cell death and cell signalling. Lysosomal storage disorders (LSD) are mainly caused by the defective activity of lysosomal proteins, which results in the intra-lysosomal accumulation of undegraded metabolites. There is increasing evidence that lysosomes are also involved in the pathogenesis of a variety of neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease and Huntington’s disease. Endosomal/lysosomal dysfunction is caused by altered lipid metabolism. It is hypothesized that abnormal protein degradation and deposition caused by endosomal/lysosomal dysfunction may be the primary trigger of age-related neurodegeneration. In this review, the structure and function of lysosomes, the role of abnormal lipid metabolism in relation to aberrant endosomal/lysosomal function and the relationship between lysosome dysfunction and various neurodegenerative diseases is described

Innate lymphoid cells in neuroinflammation

Innate lymphoid cells (ILCs) are largely tissue-resident cells that participate in the maintenance of tissue homeostasis and react early to inflammatory events. Mature ILCs are divided into three major groups based on the transcription factors required for their development and function. Under physiological conditions, ILCs are present within the choroid plexus and meninges while the CNS parenchyma is almost devoid of these cells. However, pathological conditions such as autoimmune neuroinflammation and viral infections of the CNS result in the infiltration of ILCs into parenchyma. In this article, we provide an overview of the involvement and function of the ILCs within the CNS during physiological conditions and in infections, autoimmune diseases, neurodegeneration, and injury

Murine cytomegalovirus infection induces susceptibility to eae in resistant BalB/c Mice

In contrast to C57BL/6 mice, BALB/c mice are relatively resistant to the induction of experimental autoimmune encephalomyelitis (EAE) after challenge with MOG35–55 peptide. Here, we provide the first evidence that infection with murine cytomegalovirus (MCMV) in adulthood abrogates this resistance. Infected BALB/c mice developed clinical and histological signs similar to those seen in susceptible C57BL/6 mice. In addition to CD4+ cells, large proportion of cells in the infiltrate of diseased BALB/c mice was CD8+, similar with findings in multiple sclerosis. CD8+ cells that responded to ex vivo restimulation with MOG35–55 were not specific for viral epitopes pp89 and m164. MCMV infection favors proinflammatory type of dendritic cells (CD86+CD40+CD11c+) in the peripheral lymph organs, M1 type of microglia in central nervous system, and increases development of Th1/Th17 encephalitogenic cells. This study indicates that MCMV may enhance autoimmune neuropathology and abrogate inherent resistance to EAE in mouse strain by enhancing proinflammatory phenotype of antigen-presenting cells, Th1/Th17, and CD8 response to MOG35–55

Inflammatory monocytes and NK cells play a crucial role in DNAM-1-dependent control of cytomegalovirus infection

The poliovirus receptor (PVR) is a ubiquitously expressed glycoprotein involved in cellular adhesion and immune response. It engages the activating receptor DNAX accessory molecule (DNAM)-1, the inhibitory receptor TIG IT, and the CD96 receptor with both activating and inhibitory functions. Human cytomegalovirus (HCMV) down-regulates PVR expression, but the significance of this viral function in vivo remains unknown. Here, we demonstrate that mouse CMV (MCMV) also down-regulates the surface PVR. The m20.1 protein of MCMV retains PVR in the endoplasmic reticulum and promotes its degradation. A MCMV mutant lacking the PVR inhibitor was attenuated in normal mice but not in mice lacking DNAM-1. This attenuation was partially reversed by NK cell depletion, whereas the simultaneous depletion of mononuclear phagocytes abolished the virus control. This effect was associated with the increased expression of DNAM-1, whereas TIG IT and CD96 were absent on these cells. An increased level of proinflammatory cytokines in sera of mice infected with the virus lacking the m20.1 and an increased production of iNOS by inflammatory monocytes was observed. Blocking of CCL2 or the inhibition of iNOS significantly increased titer of the virus lacking m20.1. In this study, we have demonstrated that inflammatory monocytes, together with NK cells, are essential in the early control of CMV through the DNAM-1–PVR pathwa

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

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

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

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

The effect of perinatal treatment with 5HT agonists on monoamine transporters` gene expression in adult rats

Serotonin (5HT) je biološki aktivan amin koji se kod sisavaca nalazi u mozgu i perifernim tkivima. U mozgu regulira razvoj 5HT neurona i ciljnih tkiva tijekom neurogeneze, dok kasnije ima funkciju neurotransmitera. Unutarstaničnu i izvanstaničnu koncentraciju 5HT reguliraju dvije vrste transportera. U mozgu, serotoninski transporter (5HTt) odgovoran je za prijenos 5HT iz sinaptičke pukotine u presinaptički neuron dok je vezikularni monoaminski transporter 2 (VMAT2) odgovoran za prijenos 5HT iz citoplazme presinaptičkog neurona u sinaptičke vezikule. U ovom radu istražila sam utjecaj perinatalne primjene 5-hidroksitriptofana ili tranilcipromina na ekspresiju gena za 5HTt i VMAT2 u odraslih štakora. Tretman 5-hidroksitriptofanom uzrokovao je značajno povećanje ekspresije gena za VMAT2 u jezgrama rafe. Tretman tranilciprominom uzrokovao je značajno povećanje ekspresije gena za VMAT2 u jezgrama rafe i gena za 5HTt u frontalnoj kori. Rezultati pokazuju da je tretman agonistima serotonina uzrokovao (dugo)trajne kompenzatorne promjene u ekspresiji gena koji reguliraju homeostazu serotonina u središnjem odjeljku.Serotonin (5HT) is a biologically active amine present in mammals in the brain and the peripheral tissues. In the brain, it regulates the development of 5HT neurons and target tissues during neurogenesis, while later it assumes the function of neurotransmitter. Intracellular and extracellular concentration of 5HT is controlled by two types of transporters. Serotonin transporter (5HTt) in brain is responsible for the transport of serotonin from the synaptic cleft into the presynaptic neurons, while vesicular monoamine transporter 2 (VMAT2) is responsible for the transport of monoamines from the cytoplasm of presynaptic neuron into the synaptic vesicles. In this study I examined the impact of the perinatal treatment with 5- hydroxytryptophan or tranylcypromine on the expression of genes for 5HTt and VMAT2 in adult rats. Treatment with 5-hydroxytryptophan induced a significant increase in VMAT2 gene expression in raphe nuclei. Treatment with tranylcypromine induced a significant increase in VMAT2 gene expression in raphe nuclei and significant increase in 5HTt gene expression in frontal cortex. The results suggest that treatment with serotonin agonists have induced long-lasting compensatory changes in the expression of genes involved in central serotonin homeostasis

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

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

Immunobiology of congenital cytomegalovirus infection of the central nervous system—the murine cytomegalovirus model

Congenital human cytomegalovirus infection is a leading infectious cause of long-term neurodevelopmental sequelae, including mental retardation and hearing defects. Strict species specificity of cytomegaloviruses has restricted the scope of studies of cytomegalovirus infection in animal models. To investigate the pathogenesis of congenital human cytomegalovirus infection, we developed a mouse cytomegalovirus model that recapitulates the major characteristics of central nervous system infection in human infants, including the route of neuroinvasion and neuropathological findings. Following intraperitoneal inoculation of newborn animals with mouse cytomegalovirus, the virus disseminates to the central nervous system during high-level viremia and replicates in the brain parenchyma, resulting in a focal but widespread, non-necrotizing encephalitis. Central nervous system infection is coupled with the recruitment of resident and peripheral immune cells as well as the expression of a large number of pro-inflammatory cytokines. Although infiltration of cellular constituents of the innate immune response characterizes the early immune response in the central nervous system, resolution of productive infection requires virus-specific CD8(+) T cells. Perinatal mouse cytomegalovirus infection results in profoundly altered postnatal development of the mouse central nervous system and long-term motor and sensory disabilities. Based on an enhanced understanding of the pathogenesis of this infection, prospects for novel intervention strategies aimed to improve the outcome of congenital human cytomegalovirus infection are proposed