Fumaric Acids Directly Influence Gene Expression of Neuroprotective Factors in Rodent Microglia.

Dimethylfumarate (DMF) has been approved the for treatment of relapsing-remitting multiple sclerosis. The mode of action of DMF and its assumed active primary metabolite monomethylfumarate (MMF) is still not fully understood, notably for brain resident cells. Therefore we investigated potential direct effects of DMF and MMF on microglia and indirect effects on oligodendrocytes. Primary rat microglia were differentiated into M1-like, M2-like and M0 phenotypes and treated in vitro with DMF or MMF. The gene expression of pro-inflammatory and anti-inflammatory factors such as growth factors (IGF-1), interleukins (IL-10, IL-1ß), chemokines (CCl3, CXCL-10) as well as cytokines (TGF-1ß, TNFα), iNOS, and the mannose receptor (MRC1) was examined by determining their transcription level with qPCR, and on the protein level by ELISA and FACS analysis. Furthermore, microglia function was determined by phagocytosis assays and indirect effects on oligodendroglial proliferation and differentiation. DMF treatment of M0 and M1-like polarized microglia demonstrated an upregulation of gene expression for IGF-1 and MRC1, but not on the protein level. While the phagocytic activity remained unchanged, DMF and MMF treated microglia supernatants led to an enhanced proliferation of oligodendrocyte precursor cells (OPC). These results suggest that DMF has anti-inflammatory effects on microglia which may result in enhanced proliferation of OPC.

Effectors of Th1 and Th17 cells act on astrocytes and augment their neuroinflammatory properties.

BACKGROUND: Autoreactive Th1 and Th17 cells are believed to mediate the pathology of multiple sclerosis in the central nervous system (CNS). Their interaction with microglia and astrocytes in the CNS is crucial for the regulation of the neuroinflammation. Previously, we have shown that only Th1 but not Th17 effectors activate microglia. However, it is not clear which cells are targets of Th17 effectors in the CNS. METHODS: To understand the effects driven by Th17 cells in the CNS, we induced experimental autoimmune encephalomyelitis in wild-type mice and CD4+ T cell-specific integrin α4-deficient mice where trafficking of Th1 cells into the CNS was affected. We compared microglial and astrocyte response in the brain and spinal cord of these mice. We further treated astrocytes with supernatants from highly pure Th1 and Th17 cultures and assessed the messenger RNA expression of neurotrophic factors, cytokines and chemokines, using real-time PCR. Data obtained was analyzed using the Kruskal-Wallis test. RESULTS: We observed in α4-deficient mice weak microglial activation but comparable astrogliosis to that of wild-type mice in the regions of the brain populated with Th17 infiltrates, suggesting that Th17 cells target astrocytes and not microglia. In vitro, in response to supernatants from Th1 and Th17 cultures, astrocytes showed altered expression of neurotrophic factors, pro-inflammatory cytokines and chemokines. Furthermore, increased expression of chemokines in Th1- and Th17-treated astrocytes enhanced recruitment of microglia and transendothelial migration of Th17 cells in vitro. CONCLUSION: Our results demonstrate the delicate interaction between T cell subsets and glial cells and how they communicate to mediate their effects. Effectors of Th1 act on both microglia and astrocytes whereas Th17 effectors preferentially target astrocytes to promote neuroinflammation.

Upon intranasal vesicular stomatitis virus infection, astrocytes in the olfactory bulb are important interferon Beta producers that protect from lethal encephalitis.

UNLABELLED: Previously we found that following intranasal (i.n.) infection with neurotropic vesicular stomatitis virus (VSV) type I interferon receptor (IFNAR) triggering of neuroectodermal cells was critically required to constrain intracerebral virus spread. To address whether locally active IFN-ß was induced proximally, we studied spatiotemporal conditions of VSV-mediated IFN-ß induction. To this end, we performed infection studies with IFN-ß reporter mice. One day after intravenous (i.v.) VSV infection, luciferase induction was detected in lymph nodes. Upon i.n. infection, luciferase induction was discovered at similar sites with delayed kinetics, whereas on days 3 and 4 postinfection enhanced luciferase expression additionally was detected in the foreheads of reporter mice. A detailed analysis of cell type-specific IFN-ß reporter mice revealed that within the olfactory bulb IFN-ß was expressed by neuroectodermal cells, primarily by astrocytes and to a lesser extent by neurons. Importantly, locally induced type I IFN triggered distal parts of the brain as indicated by the analysis of ISRE-eGFP mice which after i.n. VSV infection showed enhanced green fluorescent protein (eGFP) expression throughout the brain. Compared to wild-type mice, IFN-ß(-/-) mice showed increased mortality to i.n. VSV infection, whereas upon i.v. infection no such differences were detected highlighting the biological significance of intracerebrally expressed IFN-ß. In conclusion, upon i.n. VSV instillation, IFN-ß responses mounted by astrocytes within the olfactory bulb critically contribute to the antiviral defense by stimulating distal IFN-ß-negative brain areas and thus arresting virus spread. IMPORTANCE: The central nervous system has long been considered an immune privileged site. More recently, it became evident that specialized immune mechanisms are active within the brain to control pathogens. Previously, we showed that virus, which entered the brain via the olfactory route, was arrested within the olfactory bulb by a type I IFN-dependent mechanism. Since peripheral type I IFN would not readily cross the blood-brain barrier and within the brain thus far no abundant type I IFN responses have been detected, here we addressed from where locally active IFN originated from. We found that upon intranasal VSV instillation, primarily astrocytes, and to a lesser extent neurons, were stimulated within the olfactory bulb to mount IFN-ß responses that also activated and protected distal brain areas. Our results are surprising because in other infection models astrocytes have not yet been identified as major type I IFN producers.

Mesenchymal stem cells do not exert direct beneficial effects on CNS remyelination in the absence of the peripheral immune system.

Remyelination is the natural repair mechanism in demyelinating disorders such as multiple sclerosis (MS) and it was proposed that it might protect from axonal loss. For unknown reasons, remyelination is often incomplete or fails in MS lesions and therapeutic treatments to enhance remyelination are not available. Recently, the transplantation of exogenous mesenchymal stem cells (MSC) has emerged as a promising tool to enhance repair processes. This included the animal model experimental autoimmune encephalomyelitis (EAE), a commonly used model for the autoimmune mechanisms of MS. However, in EAE it is not clear if the beneficial effect of MSC derives from a direct influence on brain resident cells or if this is an indirect phenomenon via modulation of the peripheral immune system. The aim of this study was to determine potential regenerative functions of MSC in the toxic cuprizone model of demyelination that allows studying direct effects on de- and remyelination without the influence of the peripheral immune system. MSC from three different species (human, murine, canine) were transplanted either intraventricularly into the cerebrospinal fluid or directly into the lesion of the corpus callosum at two time points: at the onset of oligodendrocyte progenitor cell (OPC) proliferation or the peak of OPC proliferation during cuprizone induced demyelination. Our results show that MSC did not exert any regenerative effects after cuprizone induced demyelination and oligodendrocyte loss. During remyelination, MSC did not influence the dynamics of OPC proliferation and myelin formation. In conclusion, MSC did not exert direct regenerative functions in a mouse model where peripheral immune cells and especially T lymphocytes do not play a role. We thus suggest that the peripheral immune system is required for MSC to exert their effects and this is independent from a direct influence of the central nervous system.

Long-term impact of neonatal inflammation on demyelination and remyelination in the central nervous system.

Perinatal inflammation causes immediate changes of the blood-brain barrier (BBB) and thus may have different consequences in adult life including an impact on neurological diseases such as demyelinating disorders. In order to determine if such a perinatal insult affects the course of demyelination in adulthood as "second hit," we simulated perinatal bacterial inflammation by systemic administration of lipopolysaccharide (LPS) to either pregnant mice or newborn animals. Demyelination was later induced in adult animals by cuprizone [bis(cyclohexylidenehydrazide)], which causes oligodendrocyte death with subsequent demyelination accompanied by strong microgliosis and astrogliosis. A single LPS injection at embryonic day 13.5 did not have an impact on demyelination in adulthood. In contrast, serial postnatal LPS injections (P0-P8) caused an early delay of myelin removal in the corpus callosum, which was paralleled by reduced numbers of activated microglia. During remyelination, postnatal LPS treatment enhanced early remyelination with a concomitant increase of mature oligodendrocytes. Furthermore, the postnatal LPS challenge impacts the phenotype of microglia since an elevated mRNA expression of microglia related genes such as TREM 2, CD11b, TNF-α, TGF-ß1, HGF, FGF-2, and IGF-1 was found in these preconditioned mice during early demyelination. These data demonstrate that postnatal inflammation has long-lasting effects on microglia functions and modifies the course of demyelination and remyelination in adulthood.

Limited role of regulatory T cells during acute Theiler virus-induced encephalitis in resistant C57BL/6 mice.

BACKGROUND: Theiler's murine encephalomyelitis virus (TMEV) infection represents a commonly used infectious animal model to study various aspects of the pathogenesis of multiple sclerosis (MS). In susceptible SJL mice, dominant activity of Foxp3(+) CD4(+) regulatory T cells (Tregs) in the CNS partly contributes to viral persistence and progressive demyelination. On the other hand, resistant C57BL/6 mice rapidly clear the virus by mounting a strong antiviral immune response. However, very little is known about the role of Tregs in regulating antiviral responses during acute encephalitis in resistant mouse strains. METHODS: In this study, we used DEREG mice that express the diphtheria toxin (DT) receptor under control of the foxp3 locus to selectively deplete Foxp3(+) Tregs by injection of DT prior to infection and studied the effect of Treg depletion on the course of acute Theiler's murine encephalomyelitis (TME). RESULTS: As expected, DEREG mice that are on a C57BL/6 background were resistant to TMEV infection and cleared the virus within days of infection, regardless of the presence or absence of Tregs. Nevertheless, in the absence of Tregs we observed priming of stronger effector T cell responses in the periphery, which subsequently resulted in a transient increase in the frequency of IFNγ-producing T cells in the brain at an early stage of infection. Histological and flow cytometric analysis revealed that this transiently increased frequency of brain-infiltrating IFNγ-producing T cells in Treg-depleted mice neither led to an augmented antiviral response nor enhanced inflammation-mediated tissue damage. Intriguingly, Treg depletion did not change the expression of IL-10 in the infected brain, which might play a role for dampening the inflammatory damage caused by the increased number of effector T cells. CONCLUSION: We therefore propose that unlike susceptible mice strains, interfering with the Treg compartment of resistant mice only has negligible effects on virus-induced pathologies in the CNS. Furthermore, in the absence of Tregs, local anti-inflammatory mechanisms might limit the extent of damage caused by strong anti-viral response in the CNS.

Effector molecules released by Th1 but not Th17 cells drive an M1 response in microglia.

Microglia act as sensors of inflammation in the central nervous system (CNS) and respond to many stimuli. Other key players in neuroinflammatory diseases are CD4+ T helper cell (Th) subsets that characteristically secrete IFN-γ (Th1) or IL-17 (Th17). However, the potential of a distinct cytokine milieu generated by these effector T cell subsets to modulate microglial phenotype and function is poorly understood. We therefore investigated the ability of factors secreted by Th1 and Th17 cells to induce microglial activation. In vitro experiments wherein microglia were cultured in the presence of supernatants derived from polarized Th1 or Th17 cultures, revealed that Th1-associated factors could directly activate and trigger a proinflammatory M1-type gene expression profile in microglia that was cell-cell contact independent, whereas Th17 cells or its associated factors did not have any direct influence on microglia. To assess the effects of the key Th17 effector cytokine IL-17A in vivo we used transgenic mice in which IL-17A is specifically expressed in astrocytes. Flow cytometric and histological analysis revealed only subtle changes in the phenotype of microglia suggesting only minimal effects of constitutively produced IL-17A on microglia in vivo. Neither IL-23 signaling nor addition of GM-CSF, a recently described effector molecule of Th17 cells, changed the incapacity of Th17 cells to activate microglia. These findings demonstrate a potent effect of Th1 cells on microglia, however, the mechanism of how Th17 cells achieve their effect in CNS inflammation remains unclear.

Immune correlates of protection of the four-segmented Rift Valley fever virus candidate vaccine in mice.

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease caused by RVF virus (RVFV). RVFV infections in humans are usually asymptomatic or associated with mild febrile illness, although more severe cases of haemorrhagic disease and encephalitis with high mortality also occur. Currently, there are no licensed human vaccines available. The safety and efficacy of a genetically engineered four-segmented RVFV variant (hRVFV-4s) as a potential live-attenuated human vaccine has been tested successfully in mice, ruminants, and marmosets though the correlates of protection of this vaccine are still largely unknown. In the present study, we have assessed hRVFV-4s-induced humoral and cellular immunity in a mouse model of RVFV infection. Our results confirm that a single dose of hRVFV-4s is highly efficient in protecting naïve mice from developing severe disease following intraperitoneal challenge with a highly virulent RVFV strain and data show that virus neutralizing (VN) serum antibody titres in a prime-boost regimen are significantly higher compared to the single dose. Subsequently, VN antibodies from prime-boost-vaccinated recipients were shown to be protective when transferred to naïve mice. In addition, hRVFV-4s vaccination induced a significant virus-specific T cell response as shown by IFN-γ ELISpot assay, though these T cells did not provide significant protection upon passive transfer to naïve recipient mice. Collectively, this study highlights hRVFV-4s-induced VN antibodies as a major correlate of protection against lethal RVFV infection.

Leishmania-infected macrophages are targets of NK cell-derived cytokines but not of NK cell cytotoxicity.

Natural killer (NK) cells are important components of a protective immune response against intracellular pathogens such as Leishmania parasites, which reside within myeloid cells. Previous in vivo studies in murine cutaneous or visceral leishmaniasis showed that NK cells are activated by conventional dendritic cells in a Toll-like receptor 9-, interleukin-12 (IL-12)-, and IL-18-dependent manner during the early phase of infection and help to restrict the tissue parasite burden by unknown mechanisms. Here, we tested whether NK cells contribute to the control of Leishmania infections by lysing or by activating infected host cells. Coculture experiments revealed that activated NK cells from poly(I:C)-treated mice readily killed tumor target cells, whereas Leishmania infantum- or L. major-infected macrophages or dendritic cells remained viable. Infection with Leishmania did not significantly alter the expression of NK cell-activating molecules (retinoic acid early transcript alpha [Rae-1α], mouse UL16-binding protein-like transcript 1 [MULT-1], CD48) or inhibitory molecules (major histocompatibility complex [MHC] class I, nonclassical MHC class 1b molecule Qa-1) on the surface of myeloid cells, which offers an explanation for their protection from NK cell cytotoxicity. Consistent with these in vitro data, in vivo cytotoxicity assays revealed poor cytolytic activity of NK cells against adoptively transferred infected wild-type macrophages, whereas MHC class I-deficient macrophages were efficiently eliminated. NK cells activated by IL-12 and IL-18 stimulated macrophages to kill intracellular Leishmania in a cell contact-independent but gamma interferon-, tumor necrosis factor-, and inducible nitric oxide synthase-dependent manner. We conclude that Leishmania parasites, unlike viruses, do not render infected myeloid cells susceptible to the cytotoxicity of NK cells. Instead, soluble products of NK cells trigger the leishmanicidal activity of macrophages.

The synthetic TLR2 agonist BPPcysMPEG leads to efficient cross-priming against co-administered and linked antigens.

The property of DC to generate effective CTL responses is influenced by TLR signaling. TLR ligands contain molecular signatures associated with pathogens, have an impact on the antigen processing and presentation by DC, and are being exploited as potential adjuvants. We hypothesized that the TLR2/6 heterodimer agonist S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxyl polyethylene glycol (BPP), a synthetic derivative of the Mycoplasma macrophage activating lipopeptide-2, is a potent adjuvant for cross-priming against cellular antigens. Systemic administration of BPP-induced maturation of CD8alpha+ DC and CD8alpha- DC in the spleen and resulted in enhanced cross-presentation of intravenously co-administered antigen in mice. In addition, administration of BPP and cell-associated OVA generated an effective CTL response against OVA in vivo in a CD4+ T helper cell-dependent manner, but independent of IFN-alpha. Delivering antigenic peptides directly linked to BPP led to superior CTL immunity as compared to giving antigens and adjuvants admixed. In contrast to other TLR ligands, such as CpG, systemic activation of DC with BPP did not result in shut-down of antigen presentation by splenic DC subsets, although cross-priming against subsequently encountered antigens was reduced. Together, our data provide evidence that BPP is a potent stimulus to generate CTL via cross-priming.

Polarized microglia do not influence oligodendrocyte lineage cells via astrocytes.

Microglia can adopt different activation patterns, ranging from a pro-inflammatory M1- to an anti-inflammatory M2-like phenotype in which they play crucial roles in various neuroinflammatory diseases. M2-like microglia are described to drive remyelination, whereas detrimental effects have been attributed to M1-like microglia. How polarized microglia might act on oligodendrocyte lineage cells indirectly by influencing astrocytes has not been studied in detail. In this study, conditioned media from polarized murine microglia were used to treat astrocytes and astrocytic gene expression was analyzed by microarray for genes known to influence oligodendrocyte lineage cells. Supernatants of astrocytes previously stimulated with soluble effectors from polarized microglia were used to investigate effects on oligodendrocyte precursor cells (OPC). Growth factors known to induce OPC proliferation, differentiation, and survival were upregulated in astrocytes treated with supernatants from M1-like microglia while M0- and M2-like microglia only had negligible effects on the expression of these factors in astrocytes. Despite the upregulation of these factors in M1 stimulated astrocytes there were no significant effects on OPC in vitro. All astrocyte supernatants induced proliferation of A2B5+ OPC and inhibited differentiation of OPC into mature oligodendrocytes. A trend toward enhanced migration of OPC was induced by M1 stimulated astrocytes. Our data suggest that M1-like microglia may potentially influence OPC and remyelination indirectly via astrocytes by inducing the expression of respective growth factors, however, this has no significant effect in addition to the already strong effects of unstimulated astrocytes on OPC. Nevertheless, the observed effect may be of relevance in other pathophysiological scenarios.

Fumaric Acids Do Not Directly Influence Gene Expression of Neuroprotective Factors in Highly Purified Rodent Astrocytes.

(1) Background: Dimethylfumarate (DMF) has been approved for the treatment of relapsing remitting multiple sclerosis. However, the mode of action of DMF and its assumed active primary metabolite monomethylfumarate (MMF) is still not fully understood. Former reports suggest a neuroprotective effect of DMF mediated via astrocytes by reducing pro-inflammatory activation of these glial cells. We investigated potential direct effects of DMF and MMF on neuroprotective factors like neurotrophic factors and growth factors in astrocytes to elucidate further possible mechanisms of the mode of action of fumaric acids; (2) Methods: highly purified cultures of primary rat astrocytes were pre-treated in vitro with DMF or MMF and incubated with lipopolysaccharides (LPS) or a mixture of interferon gamma (IFN-γ) plus interleukin 1 beta (IL-1ß) in order to simulate an inflammatory environment. The gene expression of neuroprotective factors such as neurotrophic factors (nuclear factor E2-related factor 2 (NGF), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF)) and growth factors (fibroblast growth factor 2 (FGF2), platelet-derived growth factor subunit A (PDGFa), ciliary neurotrophic factor (CNTF)) as well as cytokines (tumor necrosis factor alpha (TNFα), interleukin 6 (IL-6), IL-1ß, inducible nitric oxide synthase (iNOS)) was examined by determining the transcription level with real-time quantitative polymerase chain reaction (qPCR); (3) Results: The stimulation of highly purified astrocytes with either LPS or cytokines changed the expression profile of growth factors and pro- inflammatory factors. However, the expression was not altered by either DMF nor MMF in unstimulated or stimulated astrocytes; (4) Conclusions: There was no direct influence of fumaric acids on neuroprotective factors in highly purified primary rat astrocytes. This suggests that the proposed potential neuroprotective effect of fumaric acid is not mediated by direct stimulation of neurotrophic factors in astrocytes but is rather mediated by other pathways or indirect mechanisms via other glial cells like microglia as previously demonstrated.

IFN-γ Producing Th1 Cells Induce Different Transcriptional Profiles in Microglia and Astrocytes.

Autoreactive T cells that infiltrate into the central nervous system (CNS) are believed to have a significant role in mediating the pathology of neuroinflammatory diseases like multiple sclerosis. Their interaction with microglia and astrocytes in the CNS is crucial for the regulation of neuroinflammatory processes. Our previous work demonstrated that effectors secreted by Th1 and Th17 cells have different capacities to influence the phenotype and function of glial cells. We have shown that Th1-derived effectors altered the phenotype and function of both microglia and astrocytes whereas Th17-derived effectors induced direct effects only on astrocytes but not on microglia. Here we investigated if effector molecules associated with IFN-γ producing Th1 cells induced different gene expression profiles in microglia and astrocytes. We performed a microarray analysis of RNA isolated from microglia and astrocytes treated with medium and Th-derived culture supernatants and compared the gene expression data. By using the criteria of 2-fold change and a false discovery rate of 0.01 (corrected p < 0.01), we demonstrated that a total of 2,106 and 1,594 genes were differentially regulated in microglia and astrocytes, respectively, in response to Th1-derived factors. We observed that Th1-derived effectors induce distinct transcriptional changes in microglia and astrocytes in addition to commonly regulated transcripts. These distinct transcriptional changes regulate peculiar physiological functions, and this knowledge can help to better understand T cell mediated neuropathologies.

Type I Interferon Receptor Signaling of Neurons and Astrocytes Regulates Microglia Activation during Viral Encephalitis.

In sterile neuroinflammation, a pathological role is proposed for microglia, whereas in viral encephalitis, their function is not entirely clear. Many viruses exploit the odorant system and enter the CNS via the olfactory bulb (OB). Upon intranasal vesicular stomatitis virus instillation, we show an accumulation of activated microglia and monocytes in the OB. Depletion of microglia during encephalitis results in enhanced virus spread and increased lethality. Activation, proliferation, and accumulation of microglia are regulated by type I IFN receptor signaling of neurons and astrocytes, but not of microglia. Morphological analysis of myeloid cells shows that type I IFN receptor signaling of neurons has a stronger impact on the activation of myeloid cells than of astrocytes. Thus, in the infected CNS, the cross talk among neurons, astrocytes, and microglia is critical for full microglia activation and protection from lethal encephalitis.

Astrocytes Enhance Streptococcus suis-Glial Cell Interaction in Primary Astrocyte-Microglial Cell Co-Cultures.

Streptococcus (S.) suis infections are the most common cause of meningitis in pigs. Moreover, S. suis is a zoonotic pathogen, which can lead to meningitis in humans, mainly in adults. We assume that glial cells may play a crucial role in host-pathogen interactions during S. suis infection of the central nervous system. Glial cells are considered to possess important functions during inflammation and injury of the brain in bacterial meningitis. In the present study, we established primary astrocyte-microglial cell co-cultures to investigate interactions of S. suis with glial cells. For this purpose, microglial cells and astrocytes were isolated from new-born mouse brains and characterized by flow cytometry, followed by the establishment of astrocyte and microglial cell mono-cultures as well as astrocyte-microglial cell co-cultures. In addition, we prepared microglial cell mono-cultures co-incubated with uninfected astrocyte mono-culture supernatants and astrocyte mono-cultures co-incubated with uninfected microglial cell mono-culture supernatants. After infection of the different cell cultures with S. suis, bacteria-cell association was mainly observed with microglial cells and most prominently with a non-encapsulated mutant of S. suis. A time-dependent induction of NO release was found only in the co-cultures and after co-incubation of microglial cells with uninfected supernatants of astrocyte mono-cultures mainly after infection with the capsular mutant. Only moderate cytotoxic effects were found in co-cultured glial cells after infection with S. suis. Taken together, astrocytes and astrocyte supernatants increased interaction of microglial cells with S. suis. Astrocyte-microglial cell co-cultures are suitable to study S. suis infections and bacteria-cell association as well as NO release by microglial cells was enhanced in the presence of astrocytes.

The antiviral drug ganciclovir does not inhibit microglial proliferation and activation.

Ganciclovir is effective in the treatment of human infections with viruses of the Herpesviridae family. Beside antiviral properties, recently ganciclovir was described to inhibit microglial proliferation and disease severity of experimental autoimmune encephalomyelitis, an inflammatory model of multiple sclerosis. Microglial activation and proliferation are main characteristics of neuroinflammatory CNS diseases and inhibition of microglial functions might be beneficial in autoimmune diseases, or detrimental in infectious diseases. The objective of this study was to determine potential inhibitory effects of ganciclovir in three different murine animal models of CNS neuroinflammation in which microglia play an important role: Theiler´s murine encephalomyelitis, the cuprizone model of de- and remyelination, and the vesicular stomatitis virus encephalitis model. In addition, in vitro experiments with microglial cultures were performed to test the hypothesis that ganciclovir inhibits microglial proliferation. In all three animal models, neither microglial proliferation or recruitment nor disease activity was changed by ganciclovir. In vitro experiments confirmed that microglial proliferation was not affected by ganciclovir. In conclusion, our results show that the antiviral drug ganciclovir does not inhibit microglial activation and proliferation in the murine CNS.

A pivotal role of nonmuscle myosin II during microglial activation.

Microglia are resident macrophages in the central nervous system (CNS) and the primary cells that contribute to CNS inflammation in many pathological conditions. Upon any signs of brain damage, microglia become activated and undergo tremendous cellular reorganization to adopt appropriate phenotypes. They migrate to lesion areas, accumulate, phagocytose cells or cellular debris, and produce a large array of inflammatory mediators like cytokines, chemokines, reactive oxygen species, and other mediators. To cope with the extreme cellular rearrangements during activation, microglia have to be highly dynamic. One major component of the cytoskeleton in nonmuscle cells is nonmuscle myosin II (NM II). This study was aimed to examine the functional role of NM II in resting and activated microglia. Using immunohistochemistry, we demonstrate strong expression of NM II isoform B (NM IIB) in microglia during cuprizone-induced demyelination as well as in cultured microglia. Treatment with the NM II inhibitor blebbistatin prevented the morphological shaping of microglial cells, led to functional deficits during chemokine-directed migration and phagocytosis, induced NM IIB redistribution, and affected actin microfilament patterning. In addition, inhibition of NM II led to an attenuated release of nitric oxide (NO), while TNFα secretion was not altered. In conclusion, we propose a pivotal role of NM II in cytoskeleton organization during microglial activation. This is of great importance to understand the mechanisms of microglial action in inflammatory CNS diseases.