Manon Blain

TLR Signaling Tailors Innate Immune Responses in Human Microglia and Astrocytes

The specific signals mediating the activation of microglia and astrocytes as a prelude to, or consequence of, CNS inflammation continue to be defined. We investigated TLRs as novel receptors mediating innate immune responses in human glial cells. We find that microglia express mRNA for TLRs 1–9, whereas astrocytes express robust TLR3, low-level TLR 1, 4, 5, and 9, and rare-to-undetectable TLR 2, 6, 7, 8, and 10 mRNA (quantitative real-time PCR). We focused on TLRs 3 and 4, which can signal through both the MyD88-dependent and -independent pathways, and on the MyD88-restricted TLR2. By flow cytometry, we established that microglia strongly express cell surface TLR2; TLR3 is expressed at higher levels intracellularly. Astrocytes express both cell surface and intracellular TLR3. All three TLRs trigger microglial activation upon ligation. TLR3 signaling induces the strongest proinflammatory polarizing response, characterized by secretion of high levels of IL-12, TNF-α, IL-6, CXCL-10, and IL-10, and the expression of IFN-β. CXCL-10 and IL-10 secretion following TLR4 ligation are comparable to that of TLR3; however, other responses were lower or absent. TLR2-mediated responses are dominated by IL-6 and IL-10 secretion. Astrocytes respond to TLR3 ligation, producing IL-6, CXCL-10, and IFN-β, implicating these cells as contributors to proinflammatory responses. Initial TLR-mediated glial activation also regulates consequent TLR expression; while TLR2 and TLR3 are subject to positive feedback, TLR4 is down-regulated in microglia. Astrocytes up-regulate all three TLRs following TLR3 ligation. Our data indicate that activation of innate immune responses in the CNS is not homogeneous but rather tailored according to cell type and environmental signal.

Type I interferons and microbial metabolites of tryptophan modulate astrocyte activity and central nervous system inflammation via the aryl hydrocarbon receptor

Astrocytes have important roles in the central nervous system (CNS) during health and disease. Through genome-wide analyses we detected a transcriptional response to type I interferons (IFN-Is) in astrocytes during experimental CNS autoimmunity and also in CNS lesions from patients with multiple sclerosis (MS). IFN-I signaling in astrocytes reduces inflammation and experimental autoimmune encephalomyelitis (EAE) disease scores via the ligand-activated transcription factor aryl hydrocarbon receptor (AHR) and the suppressor of cytokine signaling 2 (SOCS2). The anti-inflammatory effects of nasally administered interferon (IFN)-β are partly mediated by AHR. Dietary tryptophan is metabolized by the gut microbiota into AHR agonists that have an effect on astrocytes to limit CNS inflammation. EAE scores were increased following ampicillin treatment during the recovery phase, and CNS inflammation was reduced in antibiotic-treated mice by supplementation with the tryptophan metabolites indole, indoxyl-3-sulfate, indole-3-propionic acid and indole-3-aldehyde, or the bacterial enzyme tryptophanase. In individuals with MS, the circulating levels of AHR agonists were decreased. These findings suggest that IFN-Is produced in the CNS function in combination with metabolites derived from dietary tryptophan by the gut flora to activate AHR signaling in astrocytes and suppress CNS inflammation.

Regulation of astrocyte activation by glycolipids drives chronic CNS inflammation

Astrocytes have complex roles in health and disease, thus it is important to study the pathways that regulate their function. Here we report that lactosylceramide (LacCer) synthesized by β-1,4-galactosyltransferase 6 (B4GALT6) is upregulated in the central nervous system (CNS) of mice during chronic experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). LacCer acts in an autocrine manner to control astrocyte transcriptional programs that promote neurodegeneration. In addition, LacCer in astrocytes controls the recruitment and activation of microglia and CNS-infiltrating monocytes in a non–cell autonomous manner by regulating production of the chemokine CCL2 and granulocyte-macrophage colony–stimulating factor (GM-CSF), respectively. We also detected high B4GALT6 gene expression and LacCer concentrations in CNS MS lesions. Inhibition of LacCer synthesis in mice suppressed local CNS innate immunity and neurodegeneration in EAE and interfered with the activation of human astrocytes in vitro. Thus, B4GALT6 regulates astrocyte activation and is a potential therapeutic target for MS and other neuroinflammatory disorders.

NKG2D-Mediated Cytotoxicity toward Oligodendrocytes Suggests a Mechanism for Tissue Injury in Multiple Sclerosis

NKG2D is an activating or coactivating receptor expressed on human natural killer (NK) cells, CD8+ T cells, and γ/δ T cells. NKG2D ligands have been detected on many tumor cell types and can be induced on nontransformed cells by environmental signals including DNA damage and inflammation. We investigated the contribution of NKG2D–NKG2D ligand interaction on CNS-directed immune responses. We observed that primary cultures of human adult oligodendrocytes and fetal astrocytes expressed ligands for NKG2D in vitro whereas neurons, microglia, and adult astrocytes did not. Disruption of the NKG2D–NKG2D ligand interaction using blocking antibodies significantly inhibited killing of primary human oligodendrocytes mediated by activated human NK cells, γ/δ T cells, and allo-reactive CD8+ T cells. NKG2D ligands [major histocompatibility complex class I chain-related molecules A and B (MICA/B)] were detected in groups of cells and colocalized with an oligodendrocyte marker (adenomatous polyposis coli) in white matter sections obtained from multiple sclerosis lesions but not in normal control samples. CD8+ T cells could be detected in close proximity to MICA/B+ cells within multiple sclerosis lesions, supporting an in vivo interaction between these immune effectors and stressed MICA/B-expressing oligodendrocytes. These results imply that NKG2D–NKG2D ligand interaction can potentially contribute to cytotoxic responses mediated by activated immune effector cells in the inflamed CNS, as observed in multiple sclerosis.

Effect of tumor necrosis factor α and β on human oligodendrocytes and neurons in culture

Cytokines produced by infiltrating hematogenous cells or by glial cells activated during the course of central nervous system disease or trauma are implicated as mediators of tissue injury. In this study, we have assessed the extent and mechanism of injury of human‐derived CNS oligodendrocytes and neurons in vitro mediated by the cytokines tumor necrosis factor α and β and compared these with the tumor necrosis factor independent effects mediated by activated CD4+ T‐cells. We found that activated CD4+ T‐cells, but not tumor necrosis factor α or β, could induce significant release of lactate dehydrogenase, a measure of cell membrane lysis, from oligodendrocytes within 24 hr. Neither induced DNA fragmentation as measured using a fluorescence nick‐end labelling technique. After a more prolonged time period (96 hr), tumor necrosis factor α did induce nuclear fragmentation changes in a significant proportion of oligodendrocytes without increased lactate dehydrogenase release. The extent of DNA fragmentation was comparable to that induced by serum deprivation. Tumor necrosis factor β effects were even more pronounced. In contrast to oligodendrocytes, the extent of DNA fragmentation, assessed by propidium iodide staining, induced in neurons by tumor necrosis factor α was less than that induced by serum deprivation. In‐situ hybridization studies of human adult glial cells in culture indicated that astrocytes, as well as microglia, can express tumor necrosis factor α mRNA.

Expression of a homologue of rat NG2 on human microglia

The expression of NG2 chondroitin sulfate has been widely associated with oligodendrocyte precursors in rodents. We used a monoclonal antibody (9.2.27) against the human homologue of the rat NG2 to determine whether expression of this molecule was associated with a specific glial cell population present in dissociated cell preparations derived from adult and fetal human brain tissue. Our data, derived using FACS and immunocytochemical analyses of immediately ex vivo or cultured glial cells, indicate that the large majority of NG2 expressing cells belonged to the microglial lineage (CD68, CD11c) rather than to the oligodendrocyte lineage (O4, A2B5, GalC). In situ immunohistochemistry performed on non‐fixed normal spinal cord tissue confirmed the observation that NG2 is expressed by mononuclear phagocytes of the CNS. In contrast, peripheral blood‐derived monocytes were NG2−. Cells from fetal brain tissue showed only small numbers of NG2+ cells, which was consistent with the number of microglial cells in this preparation. In absence of additional markers, we cannot exclude that this anti‐NG2 mAb might also recognize human oligodendrocyte progenitor cells. GLIA 27:259–268, 1999.

Distinctive Properties of Human Adult Brain-Derived Myelin Progenitor Cells

We used expression of the ganglioside A2B5 to isolate putative myelin progenitor cells from adult human central nervous system parenchyma and compared their phenotypic (expression of myelin lineage molecules) and functional (survival, proliferation) properties with mature oligodendrocytes (OLGs) derived from the same adult material and with A2B5(+) cells isolated from human fetal brain. A2B5(+) cells represented 3 to 5% of the total cell suspension derived from adult specimens. Results of protein (immunostaining) and RNA (polymerase chain reaction) analyses indicated that the adult A2B5(+) cells were more committed to the OLG lineage than their fetal counterparts while continuing to retain properties of progenitor cells compared to the postmitotic mature OLGs. Although the adult A2B5(+) cells retained the capacity to divide, albeit at a reduced rate compared to fetal A2B5(+) cells, they showed reduced survival and process outgrowth compared not only to fetal cells but also to mature OLGs. Our results confirm the presence of progenitor cells committed to the OLG lineage in the adult human central nervous system but raise the issues regarding the intrinsic capacity of these cells to contribute to the process of remyelination that may be necessary during demyelinating diseases.

Differential responses of human microglia and blood-derived myeloid cells to FTY720

Human microglia, monocyte-derived dendritic cells (DCs) and macrophages ex vivo express relatively higher levels of sphingosine-1-phosphate (S1P) receptor 1 (S1P1) mRNA as compared to other receptor subtypes. The S1P agonist FTY720 decreased ERK phosphorylation and induced myosin light chain (MLC) II phosphorylation only in macrophages and DCs. FTY720 inhibited IL-12p70 production (CD40L induced) by DCs and macrophages but not microglia (poly I:C induced). IL-10 production was increased in DCs and unaffected in other myeloid cells. Despite similar receptor expression patterns, the distinct myeloid cell populations present in the human CNS, under steady-state or inflammatory conditions, exhibit differential responses to FTY720.

Reduction of the peripheral blood CD56(bright) NK lymphocyte subset in FTY720-treated multiple sclerosis patients.

FTY720 (fingolimod) treatment of multiple sclerosis (MS) results in lymphopenia due to increased recruitment into and decreased egress from secondary lymphoid organs of CCR7+ lymphocytes. Although absolute numbers of NK lymphocytes were reported as being unaltered in FTY720-treated MS patients (MS-FTY), such analyses did not detect a change in a minor subset. Because expression of CCR7 has been described on CD56bright NK cells, a minority population of NK cells, we investigated the effect of FTY720 treatment on the phenotype and function of human NK cells in the peripheral circulation of MS patients. MS-FTY patients displayed a decreased proportion of peripheral CD56brightCD62L+CCR7+ NK cells compared with untreated MS and healthy donors. In vitro treatment with FTY720-P increased migration of untreated donor NK cells to CXCL12 while reducing the response to CX3CL1 with similar migration responses seen in NK cells from MS-FTY patients. FTY720-P inhibited sphingosine 1-phosphate–directed migration of CD56bright and CD56dim NK cells subsets from untreated healthy donors. IL-12– and IL-15–stimulated NK cells from MS-FTY patients displayed similar capacity to produce IFN-γ, TNF, IL-10, and MIP-1α cytokines/chemokines compared with NK cells from untreated healthy donors and displayed comparable levels of degranulation in response to K562 tumor cells compared with untreated donors. Subset alterations and function of NK cell populations will need to be considered as part of assessing overall immunosurveillance capacity of patients with MS who will receive sustained FTY720 therapy.

Differential effects of Th1 and Th2 lymphocyte supernatants on human microglia

We assessed the effects of soluble molecules (supernatants) produced by pro‐ (Th1) and anti‐ (Th2) inflammatory T‐cell lines on the capacity of adult human CNS‐derived microglia to express or produce selected cell surface and soluble molecules that regulate immune reactivity or impact on tissue protection/repair within the CNS. Treatment of microglia with supernatants from allo‐antigen and myelin basic protein‐specific Th1 cell lines augmented expression of cell surface molecules MHC class II, CD80, CD86, CD40, and CD54, enhanced the functional antigen‐presenting cell capacity of microglia in a mixed lymphocyte reaction, and increased cytokine/chemokine secretion (TNFα, IL‐6, and CXCL10/IP‐10). These Th1‐induced effects were not reproduced by interferon‐γ (IFNγ) alone and were only incompletely blocked by anti‐IFNγ antibody. Th2 cell supernatant treatments did not alter costimulatory/adhesion molecule expression or induce cytokine/chemokine production by microglia. Th2 treatment, furthermore, failed to reduce the induction observed in response to Th1 supernatants. Neither Th1 nor Th2 supernatants induced production of the neurotrophin molecules, nerve growth factor, or brain‐derived neurotrophic factor. Our results suggest that soluble molecules released by Th1 and not Th2 cells that infiltrate the CNS can stimulate resident microglia to acquire enhanced effector and accessory cell functions; the Th1‐induced effects were not downregulated by Th2 supernatant‐mediated bystander suppression. GLIA 42:36–45, 2003.