Expression of microRNAs miR21, miR146a,and miR155 in tuberous sclerosis complex cortical tubers and their regulation in human astrocytes and SEGA‐derived cell cultures

Tuberous sclerosis complex (TSC) is a genetic disease presenting with multiple neurological symptoms including epilepsy, mental retardation, and autism. Abnormal activation of various inflammatory pathways has been observed in astrocytes in brain lesions associated with TSC. Increasing evidence supports the involvement of microRNAs in the regulation of astrocyte‐mediated inflammatory response. To study the role of inflammation‐related microRNAs in TSC, we employed real‐time PCR and in situ hybridization to characterize the expression of miR21, miR146a, and miR155 in TSC lesions (cortical tubers and subependymal giant cell astrocytomas, SEGAs). We observed an increased expression of miR21, miR146a, and miR155 in TSC tubers compared with control and perituberal brain tissue. Expression was localized in dysmorphic neurons, giant cells, and reactive astrocytes and positively correlated with IL‐1β expression. In addition, cultured human astrocytes and SEGA‐derived cell cultures were used to study the regulation of the expression of these miRNAs in response to the proinflammatory cytokine IL‐1β and to evaluate the effects of overexpression or knockdown of miR21, miR146a, and miR155 on inflammatory signaling. IL‐1β stimulation of cultured glial cells strongly induced intracellular miR21, miR146a, and miR155 expression, as well as miR146a extracellular release. IL‐1β signaling was differentially modulated by overexpression of miR155 or miR146a, which resulted in pro‐ or anti‐inflammatory effects, respectively. This study provides supportive evidence that inflammation‐related microRNAs play a role in TSC. In particular, miR146a and miR155 appear to be key players in the regulation of astrocyte‐mediated inflammatory response, with miR146a as most interesting anti‐inflammatory therapeutic candidate. GLIA 2016;64:1066–1082     

miR147b: A novel key regulator of interleukin 1 beta‐mediated inflammation in human astrocytes

Astrocytes are important mediators of inflammatory processes in the brain and seem to play an important role in several neurological disorders, including epilepsy. Recent studies show that astrocytes produce several microRNAs, which may function as crucial regulators of inflammatory pathways and could be used as therapeutic target. We aim to study which miRNAs are produced by astrocytes during IL‐1β mediated inflammatory conditions in vitro, as well as their functional role and to validate these findings in human epileptogenic brain tissue. Sequencing was used to assess miRNA and mRNA expression in IL‐1β‐stimulated human fetal astrocyte cultures. miRNAs were overexpressed in cell cultures using miRNA mimics. Expression of miRNAs in resected brain tissue from patients with tuberous sclerosis complex or temporal lobe epilepsy with hippocampal sclerosis was examined using in situ hybridization. Two differentially expressed miRNAs were found: miR146a and miR147b, which were associated with increased expression of genes related to the immune/inflammatory response. As previously reported for miR146a, overexpression of miR147b reduced the expression of the pro‐inflammatory mediators IL‐6 and COX‐2 after IL‐1β stimulation in both astrocyte and tuberous sclerosis complex cell cultures. miR146a and miR147b overexpression decreased proliferation of astrocytes and promoted neuronal differentiation of human neural stem cells. Similarly to previous evidence for miR146a, miR147b was increased expressed in astrocytes in epileptogenic brain. Due to their anti‐inflammatory effects, ability to restore aberrant astrocytic proliferation and promote neuronal differentiation, miR146a and miR147b deserve further investigation as potential therapeutic targets in neurological disorders associated with inflammation, such as epilepsy.     

Interleukin‐10 is a critical regulator of white matter lesion containment following viral induced demyelination

Neurotropic coronavirus induces an acute encephalomyelitis accompanied by focal areas of demyelination distributed randomly along the spinal column. The initial areas of demyelination increase only slightly after the control of infection. These circumscribed focal lesions are characterized by axonal sparing, myelin ingestion by macrophage/microglia, and glial scars associated with hypertrophic astrocytes, which proliferate at the lesion border. Accelerated virus control in mice lacking the anti‐inflammatory cytokine IL‐10 was associated with limited initial demyelination, but low viral mRNA persistence similar to WT mice and declining antiviral cellular immunity. Nevertheless, lesions exhibited sustained expansion providing a model of dysregulated white matter injury temporally remote from the acute CNS insult. Expanding lesions in the absence of IL‐10 are characterized by sustained microglial activation and partial loss of macrophage/microglia exhibiting an acquired deactivation phenotype. Furthermore, IL‐10 deficiency impaired astrocyte organization into mesh like structures at the lesion borders, but did not prevent astrocyte hypertrophy. The formation of discrete foci of demyelination in IL‐10 sufficient mice correlated with IL‐10 receptor expression exclusively on astrocytes in areas of demyelination suggesting a critical role for IL‐10 signaling to astrocytes in limiting expansion of initial areas of white matter damage. GLIA 2015;63:2106–2120     

Anti‐inflammatory effects of ADAMTS‐4 in a mouse model of ischemic stroke

ADAMTS‐4 (a disintegrin and metalloproteinase with thrombospondin motifs type 4) is a metalloprotease capable to degrade chondroitin sulfate proteoglycans leading to cartilage destruction during arthritis or to neuroplasticity during spinal cord injury (SCI). Although ADAMTS‐4 is an inflammatory‐regulated enzyme, its role during inflammation has never been investigated. The aim of this study was to investigate the role of ADAMTS‐4 in neuroinflammation. First, we evidenced an increase of ADAMTS‐4 expression in the ischemic brain hemisphere of mouse and human patients suffering from ischemic stroke. Then, we described that ADAMTS‐4 has predominantly an anti‐inflammatory effect in the CNS. Treatment of primary microglia or astrocyte cultures with low doses of a human recombinant ADAMTS‐4 prior to LPS exposure decreased NO production and the synthesis/release of pro‐inflammatory cytokines including NOS2, CCL2, TNF‐α, IL‐1β and MMP‐9. Accordingly, when cell cultures were transfected with silencing siRNA targeting ADAMTS‐4 prior to LPS exposure, the production of NO and the synthesis/release of pro‐inflammatory cytokines were increased. Finally, the feasibility of ADAMTS‐4 to modulate neuroinflammation was investigated in vivo after permanent middle cerebral artery occlusion in mice. Although ADAMTS‐4 treatment did not influence the lesion volume, it decreased astrogliosis and macrophage infiltration, and increased the number of microglia expressing arginase‐1, a marker of alternatively activated cells with inflammation inhibiting functions. Additionally, ADAMTS‐4 increased the production of IL‐10 and IL‐6 in the peri‐ischemic area. By having anti‐inflammatory and neuroregenerative roles, ADAMTS‐4 may represent an interesting target to treat acute CNS injuries, such as ischemic stroke, SCI or traumatic brain injury. GLIA 2016;64:1492–1507     

Increased levels of proinflammatory cytokines in the aged rat brain attenuate injury‐induced cytokine response after excitotoxic damage

In order to evaluate proinflammatory cytokine levels and their producing cell types in the control aged rat brain and after acute excitotoxic damage, both adult and aged male Wistar rats were injected with N‐methyl‐D ‐aspartate in the striatum. At different survival times between 6 hr and 7 days after lesioning, interleukin‐1 beta (IL‐1β), interleukin‐6 (IL‐6), and tumor necrosis factor alpha (TNF‐α) were analyzed by enzyme‐linked immunosorbent assay and by double immunofluorescence of cryostat sections by using cell‐specific markers. Basal cytokine expression was attributed to astrocytes and was increased in the normal aged brain showing region specificity: TNF‐α and IL‐6 displayed age‐dependent higher levels in the aged cortex, and IL‐1β and IL‐6 in the aged striatum. After excitotoxic striatal damage, notable age‐dependent differences in cytokine induction in the aged vs. the adult were seen. The adult injured striatum exhibited a rapid induction of all cytokines analyzed, but the aged injured striatum showed a weak induction of cytokine expression: IL‐1β showed no injury‐induced changes at any time, TNF‐α presented a late induction at 5 days after lesioning, and IL‐6 was only induced at 6 hr after lesioning. At both ages, in the lesion core, all cytokines were early expressed by neurons and astrocytes, and by microglia/macrophages later on. However, in the adjacent lesion border, cytokines were found in reactive astrocytes. This study highlights the particular inflammatory response of the aged brain and suggests an important role of increased basal levels of proinflammatory cytokines in the reduced ability to induce their expression after damage. © 2009 Wiley‐Liss, Inc.     

Microglia are less pro‐inflammatory than myeloid infiltrates in the hippocampus of mice exposed to status epilepticus

Activated microglia, astrogliosis, expression of pro‐inflammatory cytokines, blood brain barrier (BBB) leakage and peripheral immune cell infiltration are features of mesial temporal lobe epilepsy. Numerous studies correlated the expression of pro‐inflammatory cytokines with the activated morphology of microglia, attributing them a pro‐epileptogenic role. However, microglia and myeloid cells such as macrophages have always been difficult to distinguish due to an overlap in expressed cell surface molecules. Thus, the detrimental role in epilepsy that is attributed to microglia might be shared with myeloid infiltrates. Here, we used a FACS‐based approach to discriminate between microglia and myeloid infiltrates isolated from the hippocampus 24 h and 96 h after status epilepticus (SE) in pilocarpine‐treated CD1 mice. We observed that microglia do not express MHCII whereas myeloid infiltrates express high levels of MHCII and CD40 96 h after SE. This antigen‐presenting cell phenotype correlated with the presence of CD4^pos T cells. Moreover, microglia only expressed TNFα 24 h after SE while myeloid infiltrates expressed high levels of IL‐1β and TNFα. Immunofluorescence showed that astrocytes but not microglia expressed IL‐1β. Myeloid infiltrates also expressed matrix metalloproteinase (MMP)?9 and 12 while microglia only expressed MMP‐12, suggesting the involvement of both cell types in the BBB leakage that follows SE. Finally, both cell types expressed the phagocytosis receptor Axl, pointing to phagocytosis of apoptotic cells as one of the main functions of microglia. Our data suggests that, during early epileptogenesis, microglia from the hippocampus remain rather immune supressed whereas myeloid infiltrates display a strong inflammatory profile. GLIA 2016 GLIA 2016;64:1350–1362     

Astrocytic transforming growth factor‐beta signaling reduces subacute neuroinflammation after stroke in mice

Astrocytes limit inflammation after CNS injury, at least partially by physically containing it within an astrocytic scar at the injury border. We report here that astrocytic transforming growth factor‐beta (TGFβ) signaling is a second, distinct mechanism that astrocytes utilize to limit neuroinflammation. TGFβs are anti‐inflammatory and neuroprotective cytokines that are upregulated subacutely after stroke, during a clinically accessible time window. We have previously demonstrated that TGFβs signal to astrocytes, neurons and microglia in the stroke border days after stroke. To investigate whether TGFβ affects astrocyte immunoregulatory functions, we engineered “Ast‐Tbr2DN” mice where TGFβ signaling is inhibited specifically in astrocytes. Despite having a similar infarct size to wildtype controls, Ast‐Tbr2DN mice exhibited significantly more neuroinflammation during the subacute period after distal middle cerebral occlusion (dMCAO) stroke. The peri‐infarct cortex of Ast‐Tbr2DN mice contained over 60% more activated CD11b⁺ monocytic cells and twice as much immunostaining for the activated microglia and macrophage marker CD68 than controls. Astrocytic scarring was not altered in Ast‐Tbr2DN mice. However, Ast‐Tbr2DN mice were unable to upregulate TGF‐β1 and its activator thrombospondin‐1 2 days after dMCAO. As a result, the normal upregulation of peri‐infarct TGFβ signaling was blunted in Ast‐Tbr2DN mice. In this setting of lower TGFβ signaling and excessive neuroinflammation, we observed worse motor outcomes and late infarct expansion after photothrombotic motor cortex stroke. Taken together, these data demonstrate that TGFβ signaling is a molecular mechanism by which astrocytes limit neuroinflammation, activate TGFβ in the peri‐infarct cortex and preserve brain function during the subacute period after stroke. GLIA 2014;62:1227–1240     

Expression of proinflammatory cytokines and its relationship with virus infection in the brain of macaques inoculated with macrophage‐tropic simian immunodeficiency virus

The pathogenesis of acquired immunodeficiency syndrome dementia complex (ADC) is still poorly understood. Many studies suggest that proinflammatory cytokines such as IL‐1β and TNF‐α released by microglia/macrophages or astrocytes play a role in CNS injury. A microscopic finding of a microglial nodule with multinucleated giant cells (MNGCs) is a histopathologic hallmark of ADC and named HIV encephalitis. However, in vivo expression of these cytokines in this microenvironment of HIV encephalitis is not yet clarified. One of the main reasons is complexities of brain pathology in patients who have died from terminal AIDS. In this study, we infected two macaques with macrophage‐tropic Simian immunodeficiency virus SIV239env/MERT and examined expression of TNF‐α and IL‐1β in inflammatory lesions with MNGCs and its relation to virus‐infected cells using immunohistochemistry. One macaque showed typical inflammatory lesions with MNGCs in the frontal white matter. Small microglial nodules were also detected in the basal ganglia and the spinal cord. SIVenv positive cells were detected mainly in inflammatory lesions, and seemed to be microglia/macrophages and MNGCs based on their morphology. Expression of IL‐1β and TNF‐α were detected in the inflammatory lesions with MNGCs, and these positive cells were found to be negative for SIVenv by double‐labeling immunohistochemistry or immunohistochemistry of serial sections. There were a few TNF‐α positive cells and almost no IL‐1β positive cells in the area other than inflammatory lesions. Another macaque showed scattered CD3+ cells and CD68+ cells in the perivascular regions of the white matter. SIVenv and TNF‐α was demonstrated in a few perivascular macrophages. These findings indicate that virus‐infected microglia/macrophages do not always express IL‐1β and TNF‐α, which suggests an indirect role of HIV‐1‐infected cells in cytokine‐mediated pathogenesis of ADC. Our macaque model for human ADC may be useful for better understanding of its pathogenesis.     

Induction of interleukin‐1β by activated microglia is a prerequisite for immunologically induced fatigue

We previously reported that an intraperitoneal (i.p.) injection of synthetic double‐stranded RNA, polyriboinosinic:polyribocytidylic acid (poly‐I:C), produced prolonged fatigue in rats, which might serve as a model for chronic fatigue syndrome. The poly‐I:C‐induced fatigue was associated with serotonin transporter (5‐HTT) overexpression in the prefrontal cortex (PFC), a brain region that has been suggested to be critical for fatigue sensation. In the present study, we demonstrated that microglial activation in the PFC was important for poly‐I:C‐induced fatigue in rats, as pretreatment with minocycline, an inhibitor of microglial activation, prevented the decrease in running wheel activity. Poly‐I:C injection increased the microglial interleukin (IL)‐1β expression in the PFC. An intracerebroventricular (i.c.v.) injection of IL‐1β neutralising antibody limited the poly‐I:C‐induced decrease in activity, whereas IL‐1β (i.c.v.) reduced the activity in a dose‐dependent manner. 5‐HTT expression was enhanced by IL‐1β in primary cultured astrocytes but not in microglia. Poly‐I:C injection (i.p.) caused an increase in 5‐HTT expression in astrocytes in the PFC of the rat, which was inhibited by pretreatment with minocycline (i.p.) and rat recombinant IL‐1 receptor antagonist (i.c.v.). Poly‐I:C injection (i.p.) led to a breakdown of the blood–brain barrier and enhanced Toll‐like receptor 3 signaling in the brain. Furthermore, direct application of poly‐I:C enhanced IL‐1β expression in primary microglia. We therefore propose that poly‐I:C‐induced microglial activation, which may be at least partly caused by a direct action of poly‐I:C, enhances IL‐1β expression. Then, IL‐1β induces 5‐HTT expression in astrocytes, resulting in the immunologically induced fatigue.     

ATP‐induced IL‐1β secretion is selectively impaired in microglia as compared to hematopoietic macrophages

Under stressful conditions nucleotides are released from dying cells into the extracellular space, where they can bind to purinergic P2X and P2Y receptors. High concentrations of extracellular ATP in particular induce P2X7‐mediated signaling, which leads to inflammasome activation. This in turn leads to the processing and secretion of pro‐inflammatory cytokines, like interleukin (IL)−1β. During neurodegenerative diseases, innate immune responses are shaped by microglia and we have previously identified microglia‐specific features of inflammasome‐mediated responses. Here, we compared ATP‐induced IL‐1β secretion in primary rhesus macaque microglia and bone marrow‐derived macrophages (BMDM). We assessed the full expression profile of P2 receptors and characterized the induction and modulation of IL‐1β secretion by extracellular nucleotides. Microglia secreted significantly lower levels of IL‐1β in response to ATP when compared to BMDM. We demonstrate that this is not due to differences in sensitivity, kinetics or expression of ATP‐processing enzymes, but rather to differences in purinergic receptor expression levels and usage. Using a combined approach of purinergic receptor agonists and antagonists, we demonstrate that ATP‐induced IL‐1β secretion in BMDM was fully dependent on P2X7 signaling, whereas in microglia multiple purinergic receptors were involved, including P2X7 and P2X4. These cell type‐specific features of conserved innate immune responses may reflect adaptations to the vulnerable CNS microenvironment. GLIA 2016;64:2231–2246     

CD38 regulation in activated astrocytes: Implications for neuroinflammation and HIV‐1 brain infection

Reactive astrogliosis is a key pathological aspect of neuroinflammatory disorders including human immunodeficiency virus type 1 (HIV‐1)‐associated neurological disease. On the basis of previous data that showedastrocytes activated with interleukin (IL)‐1β induce neuronal injury, we analyzed global gene changes in IL‐1β‐activated human astrocytes by gene microarray. Among the up‐regulated genes, CD38, a 45‐kDa type II single chain transmembrane glycoprotein, was a top candidate, with a 17.24‐fold change that was validated by real‐time polymerase chain reaction. Key functions of CD38 include enzymatic activities and involvement in adhesion and cell signaling. Importantly, CD38⁺CD8⁺ T‐cell expression is a clinical correlate for progression of HIV‐1 infection and biological marker for immune activation. Thus, CD38 expression in HIV‐1 and/or IL‐1β‐stimulated human astrocytes and human brain tissues was analyzed. IL‐1β and HIV‐1 activation of astrocytes enhanced CD38 mRNA levels. Both CD38 immunoreactivity and adenosine 5′‐diphosphate (ADP)‐ribosyl cyclase activity were up‐regulated in IL‐1β‐activated astrocytes. CD38 knockdown using specific siRNAs significantly reduced astrocyte proinflammatory cytokine and chemokine production. However, CD38 mRNA levels were unchanged in IL‐1β knockdown conditions, suggesting that IL‐1β autocrine loop is not implicated in this process. Quantitative immunohistochemical analysis of HIV‐seropositive without encephalitis and HIV‐1 encephalitis brain tissues showed significant up‐regulation of CD38, which colocalized with glial fibrillary acidic protein–positive cells in areas of inflammation. These results suggest an important role of CD38 in the regulation of astrocyte dysfunction during the neuroinflammatory processes involved in neurodegenerative/neuroinflammatory disorders such as HIV‐1 encephalitis. © 2009 Wiley‐Liss, Inc.     

Selective estrogen receptor modulators decrease the production of interleukin‐6 and interferon‐γ‐inducible protein‐10 by astrocytes exposed to inflammatory challenge in vitro

Expression of proinflammatory molecules by glial cells is involved in the pathophysiological changes associated with chronic neurological diseases. Under pathological conditions, astrocytes release a number of proinflammatory molecules, such as interleukin‐6 (IL‐6) and interferon‐γ‐inducible protein‐10 (IP‐10). The ovarian hormone estradiol exerts protective effects in the central nervous system that, at least in part, may be mediated by a reduction of local inflammation. This study was designed to assess whether estradiol affects the production of IL‐6 and IP‐10 by primary cultures of newborn mice astrocytes exposed to lipopolysaccharide (LPS), a bacterial endotoxin known to cause neuroinflammation. In addition, the possible anti‐inflammatory effect of several selective estrogen receptor modulators (SERMs) was also assessed. LPS induced an increase in the expression of IL‐6 and IP‐10 mRNA levels in astrocytes and an increase in IL‐6 and IP‐10 protein levels in the culture medium. These effects of LPS were impaired by estradiol and by the four SERMs tested in our study: tamoxifen, raloxifene, ospemifene, and bazedoxifene. All SERMs tested showed a similar effect on IL‐6 and IP‐10 mRNA levels, but raloxifene and ospemifene were more effective than tamoxifen and bazedoxifene in reducing protein levels in LPS‐treated cultures. Finally, we report that news SERMs, ospemifene and bazedoxifene, exert anti‐inflammatory actions by a mechanism involving classical estrogen receptors and by the inhibition of LPS‐induced NFκB p65 transactivation. The results suggest that estrogenic compounds may be candidates to counteract brain inflammation under neurodegenerative conditions by targeting the production and release of proinflammatory molecules by astrocytes. © 2009 Wiley‐Liss, Inc.     

Microglial activation induced by the alarmin S100B is regulated by poly(ADP‐ribose) polymerase‐1

Brain injury resulting from stroke or trauma can be exacerbated by the release of proinflammatory cytokines, proteases, and reactive oxygen species by activated microglia. The microglial activation resulting from brain injury is mediated in part by alarmins, which are signaling molecules released from damaged cells. The nuclear enzyme poly(ADP‐ribose) polymerase‐1 (PARP‐1) has been shown to regulate microglial activation after brain injury, and here we show that signaling effects of the alarmin S100B are regulated by PARP‐1. S100B is a protein localized predominantly to astrocytes. Exogenous S100B added to primary microglial cultures induced a rapid change in microglial morphology, upregulation of IL‐1β, TNFα, and iNOS gene expression, and release of matrix metalloproteinase 9 and nitric oxide. Most, though not all of these effects were attenuated in PARP‐1^‐/‐ microglia and in wild‐type microglia treated with the PARP inhibitor, veliparib. Microglial activation and gene expression changes induced by S100B injected directly into brain were likewise attenuated by PARP‐1 inhibition. The anti‐inflammatory effects of PARP‐1 inhibitors in acutely injured brain may thus be mediated in part through effects on S100B signaling pathways. GLIA 2016;64:1869–1878