Analysis of IL‐6/gp130 family receptor expression reveals that in contrast to astroglia,microglia lack the oncostatin M receptor and functional responses to oncostatin M

The interleukin (IL)‐6/gp130 family of cytokines (e.g., IL‐6, IL‐11, leukemia inhibitory factor (LIF) and oncostatin M (OSM)) play important roles in the central nervous system (CNS) during neuroinflammation and neurodevelopment. However, little is known regarding the responses by astroglia and microglia to this family of cytokines. Here the expression of the IL‐6/gp130 cytokine receptors and subsequent signal pathway activation was examined in murine astrocytes and microglia in vitro. Astrocytes had high levels of OSMR mRNA while lower levels of IL‐6R, LIFR and IL‐11R mRNAs were also present. In comparison, in microglia there was no detectable OSMR mRNA, higher levels of IL‐6R mRNA and lower levels of the LIFR and IL‐11R mRNAs. The OSMR protein was present in astrocytes but was undetectable in microglia. Conversely, the IL‐6R protein was present in microglia but not detectable in astrocytes. In astrocytes but not microglia, phosphorylation of STAT1 and STAT3 occurred in response to OSM, whereas both microglia and astrocytes responded to hyper‐IL‐6 (IL‐6 linked to the soluble IL‐6 receptor). Finally, in both microglia and astrocytes, OSM failed to activate NFκB or induce iNOS and nitrite production. We conclude: (1) notable differences exist in the expression of receptors utilized by the IL‐6/gp130 family of cytokines in astrocytes and microglia, and (2) the findings provide a molecular basis for the differential response to OSM by astrocytes versus microglia and demonstrate a fundamental means for achieving cellular specificity in the response of these glial cells to this cytokine. GLIA 2015;63:132–141     

The majority of infiltrating CD8 T lymphocytes in multiple sclerosis lesions is insensitive to enhanced PD-L1 levels on CNS cells

Central nervous system (CNS) cells locally modulate immune responses using numerous molecules that are not fully elucidated. Engagement of programmed death-1 (PD-1), expressed on activated T cells, by its ligands (PD-L1 or PD-L2) suppresses T-cell responses. Enhanced CNS PD-1 and PD-L1 expression has been documented in inflammatory murine models; however, human CNS data are still incomplete. We determined that human primary cultures of astrocytes, microglia, oligodendrocytes, or neurons expressed low or undetectable PD-L1 under basal conditions, but inflammatory cytokines significantly induced such expression, especially on astrocytes and microglia. Blocking PD-L1 expression in astrocytes using specific siRNA led to significantly increased CD8 T-cell responses (proliferation, cytokines, lytic enzyme). Thus, our results establish that inflamed human glial cells can express sufficient and functional PD-L1 to inhibit CD8 T cell responses. Extensive immunohistochemical analysis of postmortem brain tissues demonstrated a significantly greater PD-L1 expression in multiple sclerosis (MS) lesions compared with control tissues, which colocalized with astrocyte or microglia/macrophage cell markers. However, more than half of infiltrating CD8 T lymphocytes in MS lesions did not express PD-1, the cognate receptor. Thus, our results demonstrate that inflamed human CNS cells such as in MS lesions express significantly elevated PD-L1, providing a means to reduce CD8 T cell responses, but most of these infiltrating immune cells are devoid of PD-1 and thus insensitive to PD-L1/L2. Strategies aimed at inducing PD-1 on deleterious activated human CD8 T cells that are devoid of this receptor could provide therapeutic benefits since PD-L1 is already increased in the target organ.     

TGFβ produced by IL‐10 redirected astrocytes attenuates microglial activation

While there clearly is an intimate relationship between astrocytes and microglia, few studies have examined these potentially dynamic interactions. In this study, cytokine‐mediated communication between microglia and astrocytes under inflammatory conditions was investigated. We have previously shown that activated microglia produce Interleukin (IL)‐10, a regulatory cytokine that plays an important role in resolving neuroinflammation. Nonetheless, the mechanism by which IL‐10 attenuates pro‐inflammatory cytokine expression in the brain is unclear. Here, we show that IL‐10 redirected astrocytes regulate the activation of microglia in a transforming growth factor (TGF)‐β dependent manner. In support of this concept, astrocytes in the brain maintained higher IL‐10 receptor (IL‐10R1) expression and primary astrocytes in culture were markedly more sensitive to the anti‐inflammatory effects of IL‐10 compared with microglia. Moreover, studies using primary cultures and an astrocyte‐microglia coculture system revealed that astrocytes mediated the anti‐inflammatory effects of IL‐10 on microglia through the production of TGFβ. For instance, only when astrocytes were present did IL‐10 stimulation reduce the expression of IL‐1β and increase expression of anti‐inflammatory mediators fractalkine receptor (CX₃CR1) and interleukin 4 receptor‐α (IL‐4Rα) in microglia. Importantly, these IL‐10‐astrocyte dependent effects on microglia were blocked by a TGFβ inhibitor. Furthermore, inhibition of TGFβ signaling in the brain resulted in prolonged sickness behavior and amplified pro‐inflammatory cytokine expression in mice challenged with lipopolysaccharide. Taken together, IL‐10 stimulated the production of TGFβ by astrocytes, which in turn, attenuated microglial activation. Overall, these findings provide novel insight into the mechanisms by which astrocytes modulate microglia under inflammatory conditions. GLIA 2014;62:881–895     

The effects of C5aR1 on leukocyte infiltration following pilocarpine‐induced status epilepticus

This study aimed to determine the role C5aR1 plays in mediating immune responses acutely after pilocarpine‐induced status epilepticus (SE), specifically those of brain‐infiltrating leukocytes. Three days following pilocarpine SE, we determined by flow cytometry the brain immune cell phenotypes and measured key proinflammatory and antiinflammatory cytokine expression by infiltrating leukocytes and microglia in C5aR1‐deficient and wild‐type mice. Absence of C5aR1 reduced by 47% the numbers of Ly6G⁺ neutrophils in the brains of No‐SE mice and decreased neutrophil entry after SE to levels found in wild‐type brains that did not undergo SE (No‐SE). Moreover, C5aR1‐deficient mice showed increased interleukin (IL)‐4 expression in infiltrating leukocytes, but not in microglia. Increases in IL‐4 expression in infiltrating leukocytes coupled with decreased neutrophil invasion in C5aR1‐deficient mice after SE is likely to contribute to the reduced neuronal loss previously found in these mice compared to their wild‐type littermates. Although other SE models need to be investigated to substantiate our findings, this study provides further evidence that C5aR1 is an inflammatory mediator and may play a role in epileptogenesis.     

Distinctive responses of brain tumor cells to TLR2 ligands:

Malignant brain tumor mass contains significant numbers of infiltrating glial cells that may intimately interact with tumor cells and influence cancer treatments. Understanding of characteristic discrepancies between normal GLIA and tumor cells would, therefore, be valuable for improving anticancer therapeutics. Here, we report distinct differences in toll‐like receptors (TLR)?2‐mediated responses between normal glia and primary brain tumor cell lines. We found that tyrosine phosphorylation of STAT1 by TLR2 ligands and its downstream events did not occur in mouse, rat, or human brain tumor cell lines, but were markedly induced in normal primary microglia and astrocytes. Using TLR2‐deficient, interferon (IFN)‐γ‐deficient, and IFNγ‐receptor‐1‐deficient mice, we revealed that the impaired phosphorylation of STAT1 might be linked with defective TLR2 system in tumor cells, and that a TLR2‐dependent pathway, not IFNγ‐receptor machinery, might be critical for tyrosine STAT1 phosphorylation by TLR2 ligands. We also found that TLR2 and its heterodimeric partners, TLR1 and 6, on brain tumor cells failed to properly respond to TLR2 ligands, and representative TLR2‐dependent cellular events, such as inflammatory responses and cell death, were not detected in brain tumor cells. Similar results were obtained in in vitro and in vivo experiments using orthotopic mouse and rat brain tumor models. Collectively, these results suggest that primary brain tumor cells may exhibit a distinctive dysfunction of TLR2‐associated responses, resulting in abnormal signaling and cellular events. Careful targeting of this distinctive property could serve as the basis for effective therapeutic approaches against primary brain tumors. GLIA 2015;63:894–905     

Role of interleukin‐6 and soluble IL‐6 receptor in region‐specific induction of astrocytic differentiation and neurotrophin expression

Increasing evidence supports an essential role for interleukin‐6 (IL‐6) in the development, differentiation, as well as de‐ and re‐generation of neurons in the central nervous system (CNS). Both IL‐6 and its specific receptor (IL‐6R) are expressed on neurons and glial cells including astrocytes. In this study, we have analyzed the responses of primary rat astrocytes of various brain regions to IL‐6 with respect to morphological changes and neurotrophin expression. Since IL‐6 alone failed to initiate effects on astrocytes, we have examined whether the soluble IL‐6R (sIL‐6R) can modulate the responsiveness of to IL‐6 in these cells. For this purpose, we used a highly active fusion protein of IL‐6 and sIL‐6R, which is designated Hyper‐IL‐6 (H‐IL‐6). We show that treatment of cultured astrocytes with Hyper‐IL‐6 promotes region‐specific morphological changes of GFAP‐positive astrocytes from typical stellate‐ to fibrous‐like cells. In addition, we find that Hyper‐IL‐6 induces expression of neurotrophins (NTs) of the nerve growth factor (NGF)‐family in a dose‐dependent manner. Interestingly, astrocytes of various brain regions show differing patterns of cytokine‐induced NT expression: NGF is maximally induced in cortex and hippocampus, NT‐3 in hippocampus, and NT‐4/5 in cortex and cerebellum. In summary, our results indicate that IL‐6 in conjunction with sIL‐6R regulates specific neurotrophin expression in astrocytes in a brain region dependent manner. Thus, the IL‐6 system provides a local supply of neurotrophins that participate in diverse CNS functions such as protection of neurons from insults, neuronal survival, and neuro‐immune responses. GLIA 26:191–200, 1999. © 1999 Wiley‐Liss, Inc.     

Sequential activation of microglia and astrocyte cytokine expression precedes increased iba‐1 or GFAP immunoreactivity following systemic immune challenge

Activation of the peripheral immune system elicits a coordinated response from the central nervous system. Key to this immune to brain communication is that glia, microglia, and astrocytes, interpret and propagate inflammatory signals in the brain that influence physiological and behavioral responses. One issue in glial biology is that morphological analysis alone is used to report on glial activation state. Therefore, our objective was to compare behavioral responses after in vivo immune (lipopolysaccharide, LPS) challenge to glial specific mRNA and morphological profiles. Here, LPS challenge induced an immediate but transient sickness response with decreased locomotion and social interaction. Corresponding with active sickness behavior (2–12 h), inflammatory cytokine mRNA expression was elevated in enriched microglia and astrocytes. Although proinflammatory cytokine expression in microglia peaked 2‐4 h after LPS, astrocyte cytokine, and chemokine induction was delayed and peaked at 12 h. Morphological alterations in microglia (Iba‐1⁺) and astrocytes (GFAP⁺), however, were undetected during this 2–12 h timeframe. Increased Iba‐1 immunoreactivity and de‐ramified microglia were evident 24 and 48 h after LPS but corresponded to the resolution phase of activation. Morphological alterations in astrocytes were undetected after LPS. Additionally, glial cytokine expression did not correlate with morphology after four repeated LPS injections. In fact, repeated LPS challenge was associated with immune and behavioral tolerance and a less inflammatory microglial profile compared with acute LPS challenge. Overall, induction of glial cytokine expression was sequential, aligned with active sickness behavior, and preceded increased Iba‐1 or GFAP immunoreactivity after LPS challenge. GLIA 2016;64:300–316     

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     

Signaling through JAK2-STAT5 pathway is essential for IL-3-induced activation of microglia

Microglia, the resident macrophage of the brain, mediates immune and inflammatory responses in the central nervous system (CNS). Activation of microglia and secretion of inflammatory cytokines associate with the pathogenesis of CNS diseases, including multiple sclerosis (MS), Alzheimer's disease (AD), Parkinson's disease, prion disease, and AIDS dementia. Microbial pathogens, cytokines, chemokines, and costimulatory molecules are potent inducers of microglial activation in the CNS. Signaling through its receptor, IL-3 induces the activation of JAK-STAT and MAP kinase pathways in microglial cells. In this study, we found that in vitro treatment of EOC-20 microglial cells with tyrphostin AG490 blocked IL-3-induced tyrosine phosphorylation of JAK2, STAT5A, and STAT5B signaling proteins. Stable transfection of EOC-20 cells with a dominant negative JAK2 mutant also blocked IL-3-induced tyrosine phosphorylation of JAK2, STAT5A, and STAT5B in microglia. The blockade of JAK2-STAT5 pathway resulted in a decrease in IL-3-induced proliferation and expression of CD40 and major histocompatibility complex class II molecules in microglia. These findings highlight the fact that JAK2-STAT5 signaling pathway plays a critical role in mediating IL-3-induced activation of microglia.     

Sleep and immunomodulatory responses to systemic lipopolysaccharide in mice selectively expressing interleukin‐1 receptor 1 on neurons or astrocytes

Sleep‐wake behavior is altered in response to immune challenge. Although the precise mechanisms that govern sickness‐induced changes in sleep are not fully understood, interleukin‐1β (IL‐1) is one mediator of these responses. To better understand mechanisms underlying sleep and inflammatory responses to immune challenge, we used two transgenic mouse strains that express IL‐1 receptor 1 (IL1R1) only in the central nervous system and selectively on neurons or astrocytes. Electroencephalographic recordings from transgenic and wild‐type mice reveal that systemic challenge with lipopolysaccharide (LPS) fragments sleep, suppresses rapid eye movement sleep (REMS), increases non‐REMS (NREMS), diminishes NREM delta power, and induces fever in all genotypes. However, the magnitude of REMS suppression is greater in mice expressing IL1R1 on astrocytes compared with mice in which IL1R1 is selectively expressed on neurons. Furthermore, there is a delayed increase in NREM delta power when IL1R1 is expressed on astrocytes. LPS‐induced sleep fragmentation is reduced in mice expressing IL1R1 on neurons. Although LPS increases IL‐1 and IL‐6 in brain of all genotypes, this response is attenuated when IL1R1 is expressed selectively on neurons or on astrocytes. Collectively, these data suggest that in these transgenic mice under the conditions of this study it is neuronal IL1R1 that plays a greater role in LPS‐induced suppression of REMS and NREM delta power, whereas astroglial IL1R1 is more important for sleep fragmentation after this immune challenge. Thus, aspects of central responses to LPS are modulated by IL1R1 in a cell type‐specific manner. GLIA 2016;64:780–791     

Interactions between TLR7 and TLR9 agonists and receptors regulate innate immune responses by astrocytes and microglia

Toll‐like receptors 7 (TLR7) and 9 (TLR9) are important mediators of innate immune responses. Both receptors are located in endosomal compartments, recognize nucleic acids, and signal via Myeloid differentiation factor 88 (MyD88). In the current study, we analyzed TLR7 and TLR9 induced activation of astrocytes and microglia, two cell types that contribute to innate immune responses in the CNS. TLR7 and TLR9 agonists induced similar cytokine profiles within each cell type. However, there were notable differences in the cytokine profile between astrocytes and microglia, including the production of the anti‐inflammatory cytokine IL‐10 and antiapoptotic cytokines G‐CSF and IL‐9 by microglia but not astrocytes. Costimulation studies demonstrated that the TLR7 agonist, imiquimod, could inhibit TLR9 agonist‐induced innate immune responses, in both cell types, in a concentration‐dependent manner. Surprisingly, this inhibition was not mediated by TLR7, as deficiency in TLR7 did not alter suppression of the TLR9 agonist‐induced responses. The suppression of innate immune responses was also not due to an inhibition of TLR9 agonist uptake. This suggested that imiquimod suppression may be a direct effect, possibly by blocking CpG‐ODN binding and/or signaling with TLR9, thus limiting cell activation. An antagonistic relationship was also observed between the two receptors in microglia, with TLR7 deficiency resulting in enhanced cytokine responses to CpG‐ODN stimulation. Thus, both TLR7 and its agonist can have inhibitory effects on TLR9‐induced cytokine responses in glial cells. © 2009 Wiley‐Liss, Inc.     

Endothelial nitric oxide synthase inhibition triggers inflammatory responses in the brain of male rats exposed to ischemia‐reperfusion injury

Nitric oxide (NO) derived from endothelial NO synthase (eNOS) plays a role in preserving and maintaining the brain's microcirculation, inhibiting platelet aggregation, leukocyte adhesion, and migration. Inhibition of eNOS activity results in exacerbation of neuronal injury after ischemia by triggering diverse cellular mechanisms, including inflammatory responses. To examine the relative contribution of eNOS in stroke‐induced neuroinflammation, we analyzed the effects of systemic treatment with l‐N‐(1‐iminoethyl)ornithine (L‐NIO), a relatively selective eNOS inhibitor, on the expression of MiR‐155‐5p, a key mediator of innate immunity regulation and endothelial dysfunction, in the cortex of male rats subjected to transient middle cerebral artery occlusion (tMCAo) followed by 24 hr of reperfusion. Inducible NO synthase (iNOS) and interleukin‐10 (IL‐10) mRNA expression were evaluated by real‐time polymerase chain reaction in cortical homogenates and in resident and infiltrating immune cells isolated from ischemic cortex. These latter cells were also analyzed for their expression of CD40, a marker of M1 polarization of microglia/macrophages.tMCAo produced a significant elevation of miR155‐5p and iNOS expression in the ischemic cortex as compared with sham surgery. eNOS inhibition by L‐NIO treatment further elevated the cortical expression of these inflammatory mediators, while not affecting IL‐10 mRNA levels. Interestingly, modulation of iNOS occurred in resident and infiltrating immune cells of the ischemic hemisphere. Accordingly, L‐NIO induced a significant increase in the percentage of CD40⁺ events in CD68⁺ microglia/macrophages of the ischemic cortex as compared with vehicle‐injected animals. These findings demonstrate that inflammatory responses may underlie the detrimental effects due to pharmacological inhibition of eNOS in cerebral ischemia.     

Microglia and multiple sclerosis

Microglia participate in all phases of the multiple sclerosis (MS) disease process. As members of the innate immune system, these cells have evolved to respond to stranger/danger signals; such a response within the central nervous system (CNS) environment has the potential to induce an acute inflammatory response. Engagement of Toll-like receptors (TLRs), a major family of pattern-recognition receptors (PRRs), provides an important mechanism whereby microglia can interact with both exogenous and endogenous ligands within the CNS. Such interactions modulate the capacity of microglia to present antigens to cells of the adaptive immune system and thus contribute to the initiation and propagation of the more sophisticated antigen-directed responses. This inflammatory response introduces the potential for bidirectional feedback between CNS resident and infiltrating systemic cells. Such interactions acquire particular relevance in the era of therapeutics for MS because the infiltrating cells can be subjected to systemic immunomodulatory therapies known to change their functional properties. Phagocytosis by microglia/macrophages is a hallmark of the MS lesion; however, the extent of tissue damage and the type of cell death will dictate subsequent innate responses. Microglia/macrophages are armed with a battery of effector molecules, such as reactive nitrogen species, that may contribute to CNS tissue injury, specifically to the injury of oligodendrocytes that is associated with MS. A therapeutic challenge is to modulate the dynamic properties of microglia/macrophages so as to limit potentially damaging innate responses, to protect the CNS from injury, and to promote local recovery.     

Self-tolerance in the immune privileged CNS: lessons from the entorhinal cortex lesion model

Upon peripheral immunization with myelin epitopes, susceptible rats and mice develop T cell-mediated demyelination similar to that observed in the human autoimmune disease multiple sclerosis (MS). In the same animals, brain injury does not induce autoimmune encephalomyelitis despite massive release of myelin antigens and early expansion of myelin specific T cells in local lymph nodes, indicating that the self-specific T cell clones are kept under control. Using entorhinal cortex lesion (ECL) to induce axonal degeneration in the hippocampus, we identified possible mechanisms of immune tolerance after brain trauma. Following ECL, astrocytes upregulate the death ligand CD95L, allowing apoptotic elimination of infiltrating activated T cells. Myelin-phagocytosing microglia express MHC-II and the costimulatory molecule CD86, but lack CD80, which is found only on activated antigen presenting cells (APCs). Restimulation of invading T cells by such immature APCs (e.g. CD80 negative microglia) may lead to T cell anergy and/or differentiation of regulatory/Th3-like cells due to insufficient costimulation and presence of high levels of TGF-beta and IL-10 in the CNS. Thus, T cell -apoptosis, -anergy, and -suppression apparently maintain immune tolerance after initial expansion of myelin-specific T lymphocytes following brain injury. This view is supported by a previous metastatistical analysis which rejected the hypothesis that brain trauma is causative of MS (Goddin et al., 1999). However, concomitant trauma-independent proinflammatory signals, e.g., those evoked by clinically quiescent infections, may trigger maturation of APCs, thus shifting a delicate balance from immune tolerance and protective immune responses to destructive autoimmunity.     

Recent advances in the immunopathology of experimental autoimmune encephalomyelitis: With special reference to cytokine production in the central nervous system

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease that can be induced by immunization with encephalitogenic antigens such as myelin basic protein. Recent in vitro studies have demonstrated that cytokines play an important role in immune reactions in the central nervous system (CNS), suggesting that cytokines released by infiltrating cells and glial cells may contribute to the pathogenesis of EAE. In this review, we focus on the interactions between infiltrating cells and brain cells during the inflammatory process in EAE and discuss the roles of cytokines in the CNS. After immunization with proper myelin antigens, encephalitogenic T cells increase in number and infiltrate the CNS parenchyma via the subarachnoid space or the blood vessels. Once inflammatory cells infiltrate the CNS, microglia and astrocytes are activated, and some of these cells proliferate in response to cytokines released by infiltrating cells. Following this, activated microglia present antigens to induce T cell proliferation and cytokine production. In contrast, astrocytes induce T cell unresponsiveness, probably due to a lack of costimulatory signals. Furthermore, infiltrating T cells are the main producers of Th1 cytokines and are involved in T cell-brain cell interactions. This cascade of events indicates that immune reactions take place in the CNS, although the CNS has previously been considered to be an immunologically privileged site. Based on these findings, we also discuss the feasibility of using various cytokines to stimulate the immunomodulation of brain inflammation as a treatment for autoimmune demyelinating diseases.     

Adoptive transfer of cytokine‐induced immunomodulatory adult microglia attenuates experimental autoimmune encephalomyelitis in DBA/1 mice

Microglia are resident antigen‐presenting cells in the central nervous system (CNS) that either suppress or promote disease depending on their activation phenotype and the microenvironment. Multiple sclerosis (MS) is a chronic inflammatory disease causing demyelination and nerve loss in the CNS, and experimental autoimmune encephalomyelitis (EAE) is an animal model of MS that is widely used to investigate pathogenic mechanisms and therapeutic effects. We isolated and cultured microglia from adult mouse brains and exposed them to specific combinations of stimulatory molecules and cytokines, the combination of IL‐4, IL‐10, and TGF‐β yielding the optimal regime for induction of an immunosuppressive phenotype (M2). M2 microglia were characterized by decreased expression or production of CD86, PD‐L1, nitric oxide, and IL‐6, increased expression of PD‐L2, and having a potent capacity to retain their phenotype on secondary proinflammatory stimulation. M2 microglia induced regulatory T cells, suppressed T‐cell proliferation, and downmodulated M1‐associated receptor expression in M1 macrophages. Myelin oligodendrocyte glycoprotein (MOG)‐induced EAE was induced in DBA/1 mice and at different time points (0, 5, 12, or 15 days postimmunization) 3 × 10⁵ M2 microglia were transferred intranasally. A single transfer of M2 microglia attenuated the severity of established EAE, which was particularly obvious when the cells were injected at 15 days postimmunization. M2 microglia‐treated mice had reduced inflammatory responses and less demyelination in the CNS. Our findings demonstrate that adult M2 microglia therapy represents a novel intervention that alleviated established EAE and that this therapeutic principle may have relevance for treatment of MS patients. GLIA 2014;62:804–817