Macrophages in inflammatory multiple sclerosis lesions have an intermediate activation status

BACKGROUND: Macrophages play a dual role in multiple sclerosis (MS) pathology. They can exert neuroprotective and growth promoting effects but also contribute to tissue damage by production of inflammatory mediators. The effector function of macrophages is determined by the way they are activated. Stimulation of monocyte-derived macrophages in vitro with interferon-γ and lipopolysaccharide results in classically activated (CA/M1) macrophages, and activation with interleukin 4 induces alternatively activated (AA/M2) macrophages. METHODS: For this study, the expression of a panel of typical M1 and M2 markers on human monocyte derived M1 and M2 macrophages was analyzed using flow cytometry. This revealed that CD40 and mannose receptor (MR) were the most distinctive markers for human M1 and M2 macrophages, respectively. Using a panel of M1 and M2 markers we next examined the activation status of macrophages/microglia in MS lesions, normal appearing white matter and healthy control samples. RESULTS: Our data show that M1 markers, including CD40, CD86, CD64 and CD32 were abundantly expressed by microglia in normal appearing white matter and by activated microglia and macrophages throughout active demyelinating MS lesions. M2 markers, such as MR and CD163 were expressed by myelin-laden macrophages in active lesions and perivascular macrophages. Double staining with anti-CD40 and anti-MR revealed that approximately 70% of the CD40-positive macrophages in MS lesions also expressed MR, indicating that the majority of infiltrating macrophages and activated microglial cells display an intermediate activation status. CONCLUSIONS: Our findings show that, although macrophages in active MS lesions predominantly display M1 characteristics, a major subset of macrophages have an intermediate activation status

Inflammogenesis of Secondary Spinal Cord Injury

Spinal cord injury (SCI) and spinal infarction lead to neurological complications and eventually to paraplegia or quadriplegia. These extremely debilitating conditions are major contributors to morbidity. Our understanding of SCI has certainly increased during the last decade, but remains far from clear. SCI consists of two defined phases: the initial impact causes primary injury, which is followed by a prolonged secondary injury consisting of evolving sub-phases that may last for years. The underlying pathophysiological mechanisms driving this condition are complex. Derangement of the vasculature is a notable feature of the pathology of SCI. In particular, an important component of SCI is the ischemia-reperfusion injury (IRI) that leads to endothelial dysfunction and changes in vascular permeability. Indeed, together with endothelial cell damage and failure in homeostasis, ischemia reperfusion injury triggers full-blown inflammatory cascades arising from activation of residential innate immune cells (microglia and astrocytes) and infiltrating leukocytes (neutrophils and macrophages). These inflammatory cells release neurotoxins (proinflammatory cytokines and chemokines, free radicals, excitotoxic amino acids, nitric oxide (NO)), all of which partake in axonal and neuronal deficit. Therefore, our review considers the recent advances in SCI mechanisms, whereby it becomes clear that SCI is a heterogeneous condition. Hence, this leads towards evidence of a restorative approach based on monotherapy with multiple targets or combinatorial treatment. Moreover, from evaluation of the existing literature, it appears that there is an urgent requirement for multi-centered, randomized trials for a large patient population. These clinical studies would offer an opportunity in stratifying SCI patients at high risk and selecting appropriate, optimal therapeutic regimens for personalized medicine.Grant #NPRP 4-571-3-171 from the Qatar National Research Fund(a member of Qatar Foundation)

Fetal microglial phenotype in vitro carries memory of prior in vivo exposure to inflammation

Objective. Neuroinflammation in utero may result in life-long neurological disabilities. The molecular mechanisms whereby microglia contribute to this response remain incompletely understood. Methods. Lipopolysaccharide (LPS) or saline were administered intravenously to non-anesthetized chronically instrumented near-term fetal sheep to model fetal inflammation in vivo. Microglia were then isolated from in vivo LPS and saline (naïve) exposed animals. To mimic the second hit of neuroinflammation, these microglia were then re-exposed to LPS in vitro. Cytokine responses were measured in vivo and subsequently in vitro in the primary microglia cultures derived from these animals. We sequenced the whole transcriptome of naïve and second hit microglia and profiled their genetic expression to define molecular pathways disrupted during neuroinflammation.Results. In vivo LPS exposure resulted in IL-6 increase in fetal plasma 3 h post LPS exposure. Even though not histologically apparent, microglia acquired a pro-inflammatory phenotype in vivo that was sustained and amplified in vitro upon second hit LPS exposure as measured by IL-1β response in vitro and RNAseq analyses. While NFKB and Jak-Stat inflammatory pathways were up regulated in naïve microglia, heme oxygenase 1 (HMOX1) and Fructose-1,6-bisphosphatase (FBP) genes were uniquely differentially expressed in the second hit microglia. Microglial calreticulin/LRP genes implicated in microglia-neuronal communication relevant for the neuronal development were up regulated in second hit microglia.Discussion. We identified a unique HMOX1down and FBPup phenotype of microglia exposed to the double-hit suggesting interplay of inflammatory and metabolic pathways as a memory of prior inflammatory insult. These findings suggest new therapeutic targets for early postnatal intervention to prevent brain injury

Distinct immune regulatory properties of human adult microglia and blood-derived macrophages: relevance for multiple sclerosis disease processes

In the human central nervous system (CNS), the two major populations of myeloid cells are the blood-derived macrophages and the CNS-resident microglia. Under physiologic conditions, macrophages occupy the perivascular spaces and microglia populate the CNS parenchyma. Both cell types may be recruited to participate in neuroinflammatory responses such as in multiple sclerosis (MS) lesions, as macrophages access the parenchyma and the cell types become difficult to distinguish. Differential responses of macrophages and microglia to environmental conditions and to therapeutic agents could be important determinants of outcome in neuroinflammatory diseases of the CNS of which multiple sclerosis is the prototype. Experiments described in this thesis were based on use of microglia derived from the adult human CNS and myeloid cells generated from peripheral blood samples. Comparisons were made with regard to responses to the novel MS therapeutic FTY720, experimental polarizing conditions, and exposure to the CNS breakdown product myelin. As an indication of their propensity to respond to FTY720 treatment, the myeloid cell subsets were examined for their expression of sphingosine-1-phosphate (S1P) receptor mRNA. 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. Despite these similar receptor expression patterns, the S1P agonist FTY720 decreased extracellular-signal-regulated kinases (ERK) phosphorylation and induced myosin light chain (MLC) II phosphorylation in macrophages and DCs but not in microglia. FTY720 inhibited interleukin (IL)-12p70 production (CD40L induced) by DCs and macrophages but not microglia (poly I:C induced). IL-10 production was increased in DCs following FTY720 treatment and unaffected in the other myeloid cells. In terms of responses to polarizing conditions, macrophages can be polarized into a continuum of activation phenotypes, the extremes of which are the “pro-inflammatory” M1 and “anti-inflammatory” M2 phenotypes. These phenotypes have been linked to functional properties including production of inflammation association molecules and phagocytic activity. The phenotypic and functional properties of microglia were compared with macrophages derived from peripheral blood monocytes in response to M1 and M2 inducing conditions. Under M1 conditions, microglia and macrophages up-regulated expression of the M1 markers CCR7 and CD80. M2 treatment of microglia induced expression of CD209 but not the additional M2 markers CD23, CD163 and CD206 expressed by M2 macrophages. M1-polarizing conditions induced production of IL-12p40 by both microglia and macrophages; microglia produced higher levels of IL-10 under M1 conditions than did macrophages. Under M2 conditions, microglia produced comparable levels of IL-10 as under M1 conditions, whereas IL-10 was induced in M2 macrophages subsequently activated with lipopolysaccharide (LPS). Myelin phagocytosis was significantly greater by microglia than macrophages under all conditions; activity was higher for M2 treated cells for both cell types. Our findings delineate distinctive properties of microglia compared to exogenous myeloid cells both under physiological conditions and in response to signals derived from an inflammatory environment in the CNS. The results that myeloid cells exhibit differential responses to an approved MS therapeutic agent suggest that targeting the distinct myeloid populations and promoting M2 polarization, especially in the infiltrating macrophages, may be a potential therapeutic target to control inflammation in the CNS.Les principales populations de cellules myeloïdes du système nerveux central (SNC) sont les macrophages, provenant de monocytes sanguins, et les microglies, résidant au sein du SNC. Les macrophages occupent normalement l'espace périvasculaire alors que les microglies peuplent le parenchyme du SNC. Le rôle de ces deux types de cellules diffère probablement dans le contexte neuro-inflammatoire caractérisant la sclérose en plaques (SP). Les macrophages accèdent alors au parenchyme et il devient difficile de les distinguer des microglies. Les expériences décrites dans la présente thèse ont été effectuées sur des microglies prélevées du SNC d'humains adultes et de cellules myéloïdes provenant du sang périphérique. Leurs réponses à différents stimuli ont été comparées. Les microglies et macrophages ont été exposés à la molécule FTY720, à des conditions expérimentales de polarisation ou à la myéline, une composante détruite dans la SP. La présence de l'acide ribonucléique codant pour les récepteurs de sphingosine-1-phosphate 1 à 5 (S1P1 à 5) a été utilisée comme indicateur du potentiel de réponse des cellules à l'agoniste FTY720. Il fut observé que les microglies, cellules dendritiques et macrophages dérivés de monocytes exprimaient le récepteur S1P1 à un niveau relativement plus élevé que les autres récepteurs (S1P2-5). En dépit de ce profil d'expression similaire, les voies de signalisation engagées par FTY720 ont différé entre les types de cellules. Les macrophages et cellules dendritiques traités avec FTY720 ont vu leur niveau de phospho-ERK baisser et leur phospho-MLC II augmenter tandis que les niveaux sont restés inchangés chez les microglies. FTY720 a mitigé la production d'interleukine (IL)-12p70 chez les cellules dendritiques et les macrophages (stimulées avec CD40L), mais pas chez les microglies (stimulées avec poly I:C). FTY720 a augmenté la production d'IL-10 chez les cellules dendritiques, mais est demeurée la même chez les autres cellules myéloïdes. Concernant les réponses à des conditions de polarisation, les macrophages peuvent être polarisés dans un spectre continu de phénotypes dont les extrêmes sont les phénotypes pro-inflammatoires « M1 » et anti-inflammatoires « M2 ». Ces phénotypes sont liés à la production de molécules inflammatoires et à l'activité phagocytaire des cellules. La fonction et le phénotype des microglies soumises aux conditions de polarisation pro-M1 et pro-M2 ont été comparés à celles des macrophages dérivés de monocytes sanguins périphériques dans des conditions semblables. Dans des conditions pro-M1, les microglies et les macrophages augmentaient leur expression des marqueurs M1 CCR7 et CD80. Les macrophages soumis à des conditions pro-M2 exprimaient les marqueurs C209, CD23, CD163 et CD206. Par contre, les microglies soumises à de telles conditions n'exprimaient que CD209. Les conditions pro-M1 ont amené les microglies et les macrophages à produire IL-12p40. Cependant, les microglies produisaient davantage d'IL-10 que les macrophages dans ces mêmes conditions. Les microglies stimulées par les signaux pro-M1 et pro-M2 produisaient des niveaux comparables de IL-10 tandis que les macrophages produisaient IL-10 avec des signaux M2 plus LPS. L'activité phagocytaire était plus élevée chez les microglies que chez les macrophages, peu importe la condition polarisante. Elle était cependant augmentée chez les deux types de cellules par des conditions pro-M2. Nos résultats mettent en évidence certaines propriétés distinctes des microglies et des cellules myéloïdes sanguines dans le contexte physiologique et face aux signaux provenant d'un SNC inflammé. Les donnés démontrant un comportement unique chez chacun des types de cellules myéloïdes en réponse à un agent thérapeutique approuvé pour la SP suggèrent que de cibler les populations myéloïdes peut avoir un impact. Favoriser une polarisation M2 chez les macrophages pourrait s'avérer une stratégie visant à contrôler l'inflammation du SNC

Interferon regulatory factor 7 participates in the M1-like microglial polarization switch

Microglia are generally considered the immune cells of the central nervous system. Recent studies have demonstrated that under specific polarization conditions, microglia develop into two different phenotypes, termed M1-like and M2-like microglia. However, the phenotypic characteristics of M1-like- and M2-like-polarized microglia and the mechanisms that regulate polarization are largely unknown. In this study, we characterized lipopolysaccharide-treated M1-like and IL-4-treated M2-like microglia and investigated the mechanisms that regulate phenotypic switching. The addition of M2-like microglial conditioned medium (CM) to primary neurons resulted in an increase in neurite length when compared with neurons treated with M1-like microglial CM, possibly because of the enhanced secretion of neurotrophic factors by M2-like microglia. M1-like microglia were morphologically characterized by larger soma, whereas M2-like microglia were characterized by long processes. M2-like microglia exhibited greater phagocytic capacity than M1-like microglia. These features switched in response to polarization cues. We found that expression of interferon regulatory factor 7 (IRF7) increased during the M2-like to M1-like switch in microglia in vitro and in vivo. Knockdown of IRF7 using siRNA suppressed the expression of M1 marker mRNA and reduced phosphorylation of STAT1. Our findings suggest that IRF7 signaling may play an important role in microglial polarization switching