Microglia Responses to Pro-inflammatory Stimuli (LPS, IFNγ+TNFα) and Reprogramming by Resolving Cytokines (IL-4, IL-10)

Microglia respond to CNS injuries and diseases with complex reactions, often called “activation.” A pro-inflammatory phenotype (also called classical or M1 activation) lies at one extreme of the reactivity spectrum. There were several motivations for this study. First, bacterial endotoxin (lipopolysaccharide, LPS) is the most commonly used pro-inflammatory stimulus for microglia, both in vitro and in vivo; however, pro-inflammatory cytokines (e.g., IFNγ, TNFα) rather than LPS will be encountered with sterile CNS damage and disease. We lack direct comparisons of responses between LPS and such cytokines. Second, while transcriptional profiling is providing substantial data on microglial responses to LPS, these studies mainly use mouse cells and models, and there is increasing evidence that responses of rat microglia can differ. Third, the cytokine milieu is dynamic after acute CNS damage, and an important question in microglial biology is: How malleable are their responses? There are very few studies of effects of resolving cytokines, particularly for rat microglia, and much of the work has focused on pro-inflammatory outcomes. Here, we first exposed primary rat microglia to LPS or to IFNγ+TNFα (I+T) and compared hallmark functional (nitric oxide production, migration) and molecular responses (almost 100 genes), including surface receptors that can be considered part of the sensome. Protein changes for exemplary molecules were also quantified: ARG1, CD206/MRC1, COX-2, iNOS, and PYK2. Despite some similarities, there were notable differences in responses to LPS and I+T. For instance, LPS often evoked higher pro-inflammatory gene expression and also increased several anti-inflammatory genes. Second, we compared the ability of two anti-inflammatory, resolving cytokines (IL-4, IL-10), to counteract responses to LPS and I+T. IL-4 was more effective after I+T than after LPS, and IL-10 was surprisingly ineffective after either stimulus. These results should prove useful in modeling microglial reactivity in vitro; and comparing transcriptional responses to sterile CNS inflammation in vivo

Szempontok és adatok a szellemi világ néprajzának keresztény értelmezéséhez

A dolgozat állást foglal és érveket sorakoztat fel a keresztény világkép alapján való tudományos elemzés létjogosultsága mellett, s megfogalmaz néhány szempontot az ilyen irányú kutatásokhoz

Sex- and Development-Dependent Responses of Rat Microglia to Pro- and Anti-inflammatory Stimulation

Addressing potential sex differences in pre-clinical studies is crucial for developing therapeutic interventions. Although sex differences have been reported in epidemiological studies and from clinical experience, most pre-clinical studies of neuroinflammation use male rodents; however, sexual dimorphisms in microglia might affect the CNS inflammatory response. Developmental changes are also important and, in rodents, there is a critical period of sexual brain differentiation in the first 3 weeks after birth. We compared rat microglia from sex-segregated neonates (P1) and at about the time of weaning (P21). To study transitions from a basal homeostatic state (untreated), microglia were subjected to a pro-inflammatory (IFNγ + TNFα) or anti-inflammatory (IL-4) stimulus. Responses were compared by quantifying changes in nitric oxide production, migration, and expression of nearly 70 genes, including inflammatory mediators and receptors, inflammasome molecules, immune modulators, and genes that regulate microglial physiological functions. No sex differences were seen in transcriptional responses in either age group but the IL-4-evoked migration increase was larger in male cells at both ages. Protein changes for the hallmark molecules, NOS2, COX-2, PYK2 and CD206 correlated with mRNA changes. P1 and P21 microglia showed substantial differences, including expression of genes related to developmental roles. That is, P21 microglia had a more mature phenotype, with higher basal and stimulated levels of many inflammatory genes, while P1 cells had higher expression of phagocytosis-related molecules. Nevertheless, cells of both ages responded to IL-4 and IFNγ + TNFα. We examined the Kv1.3 potassium channel (a potential target for modulating neuroinflammation) and the Kir2.1 channel, which regulate several microglia functions. Kv1.3 mRNA (Kcna3) was higher at P21 under all conditions and male P21 cells had higher mRNA and Kv currents in response to IFNγ + TNFα. Overall, numerous transcriptional and functional responses of microglia changed during the first 3 weeks after birth but few sex-dependent changes were seen

Circulating microRNAs differentiate fast-progressing from slow-progressing and non-progressing knee osteoarthritis in the Osteoarthritis Initiative cohort

INTRODUCTION: The objective of this study is to identify circulating microRNAs that distinguish fast-progressing radiographic knee osteoarthritis (OA) in the Osteoarthritis Initiative cohort by applying microRNA-sequencing. METHODS: Participants with Kellgren-Lawrence (KL) grade 0/1 at baseline were included (N = 106). Fast-progressors were defined by an increase to KL 3/4 by 4-year follow-up (N = 20), whereas slow-progressors showed an increase to KL 2/3/4 only at 8-year follow-up (N = 35). Non-progressors remained at KL 0/1 by 8-year follow-up (N = 51). MicroRNA-sequencing was performed on plasma collected at baseline and 4-year follow-up from the same participants. Negative binomial models were fitted to identify differentially expressed (DE) microRNAs. Penalized logistic regression (PLR) analyses were performed to select combinations of DE microRNAs that distinguished fast-progressors. Area under the receiver operating characteristic curves (AUC) were constructed to evaluate predictive ability. RESULTS: DE analyses revealed 48 microRNAs at baseline and 2 microRNAs at 4-year follow-up [false discovery rate (FDR) \u3c 0.05] comparing fast-progressors with both slow-progressors and non-progressors. Among these were hsa-miR-320b, hsa-miR-320c, hsa-miR-320d, and hsa-miR-320e, which were predicted to target gene families, including members of the 14-3-3 gene family, involved in signal transduction. PLR models included miR-320 members as top predictors of fast-progressors and yielded AUC ranging from 82.6 to 91.9, representing good accuracy. CONCLUSION: The miR-320 family is associated with fast-progressing radiographic knee OA and merits further investigation as potential biomarkers and mechanistic drivers of knee OA

Age-Related Comparisons of Evolution of the Inflammatory Response After Intracerebral Hemorrhage in Rats

In the hours to days after intracerebral hemorrhage (ICH), there is an inflammatory response within the brain characterized by the infiltration of peripheral neutrophils and macrophages and the activation of brain-resident microglia and astrocytes. Despite the strong correlation of aging and ICH incidence, and increasing information about cellular responses, little is known about the temporal- and age-related molecular responses of the brain after ICH. Here, we monitored a panel of 27 genes at 6 h and 1, 3, and 7 days after ICH was induced by injecting collagenase into the striatum of young adult and aged rats. Several molecules (CR3, TLR2, TLR4, IL-1β, TNFα, iNOS, IL-6) were selected to reflect the classical activation of innate immune cells (macrophages, microglia) and the potential to exacerbate inflammation and damage brain cells. Most of the others are associated with the resolution of innate inflammation, alternative pathways of macrophage/microglial activation, and the repair phase after acute injury (TGFβ, IL-1ra, IL-1r2, IL-4, IL-13, IL-4Rα, IL-13Rα1, IL-13Rα2, MRC1, ARG1, CD163, CCL22). In young animals, the up-regulation of 26 in 27 genes (not IL-4) was detected within the first week. Differences in timing or levels between young and aged animals were detected for 18 of 27 genes examined (TLR2, GFAP, IL-1β, IL-1ra, IL-1r2, iNOS, IL-6, TGFβ, MMP9, MMP12, IL-13, IL-4Rα, IL-13Rα1, IL-13Rα2, MRC1, ARG1, CD163, CCL22), with a generally less pronounced or delayed inflammatory response in the aged animals. Importantly, within this complex response to experimental ICH, the induction of pro-inflammatory, potentially harmful mediators often coincided with resolving and beneficial molecules

Data from: Microglia responses to pro-inflammatory stimuli (LPS, IFNγ+TNFα) and reprogramming by resolving cytokines (IL-4, IL-10)

Microglia respond to CNS injuries and diseases with complex reactions, often called “activation.” A pro-inflammatory phenotype (also called classical or M1 activation) lies at one extreme of the reactivity spectrum. There were several motivations for this study. First, bacterial endotoxin (lipopolysaccharide, LPS) is the most commonly used pro-inflammatory stimulus for microglia, both in vitro and in vivo; however, pro-inflammatory cytokines (e.g., IFNγ, TNFα) rather than LPS will be encountered with sterile CNS damage and disease. We lack direct comparisons of responses between LPS and such cytokines. Second, while transcriptional profiling is providing substantial data on microglial responses to LPS, these studies mainly use mouse cells and models, and there is increasing evidence that responses of rat microglia can differ. Third, the cytokine milieu is dynamic after acute CNS damage, and an important question in microglial biology is: How malleable are their responses? There are very few studies of effects of resolving cytokines, particularly for rat microglia, and much of the work has focused on pro-inflammatory outcomes. Here, we first exposed primary rat microglia to LPS or to IFNγ+TNFα (I+T) and compared hallmark functional (nitric oxide production, migration) and molecular responses (almost 100 genes), including surface receptors that can be considered part of the sensome. Protein changes for exemplary molecules were also quantified: ARG1, CD206/MRC1, COX-2, iNOS, and PYK2. Despite some similarities, there were notable differences in responses to LPS and I+T. For instance, LPS often evoked higher pro-inflammatory gene expression and also increased several anti-inflammatory genes. Second, we compared the ability of two anti-inflammatory, resolving cytokines (IL-4, IL-10), to counteract responses to LPS and I+T. IL-4 was more effective after I+T than after LPS, and IL-10 was surprisingly ineffective after either stimulus. These results should prove useful in modeling microglial reactivity in vitro; and comparing transcriptional responses to sterile CNS inflammation in vivo

The microglial activation state regulates migration and roles of matrix-dissolving enzymes for invasion

Abstract Background Microglial cells are highly mobile under many circumstances and, after central nervous system (CNS) damage, they must contend with the dense extracellular matrix (ECM) in order to reach their target sites. In response to damage or disease, microglia undergo complex activation processes that can be modulated by environmental cues and culminate in either detrimental or beneficial outcomes. Thus, there is considerable interest in comparing their pro-inflammatory (‘classical’ activation) and resolving ‘alternative’ activation states. Almost nothing is known about how these activation states affect the ability of microglia to migrate and degrade ECM, or the enzymes used for substrate degradation. This is the subject of the present study. Methods Primary cultured rat microglial cells were exposed to lipopolysaccharide (LPS) to evoke classical activation or IL4 to evoke alternative activation. High-resolution microscopy was used to monitor changes in cell morphology and aspects of the cytoskeleton. We quantified migration in a scratch-wound assay and through open filter holes, and invasion through Matrigel™. A panel of inhibitors was used to analyze contributions of different matrix-degrading enzymes to migration and invasion, and quantitative real-time reverse transcriptase PCR (qRT-PCR) was used to assess changes in their expression. Results Vinculin- and F-actin-rich lamellae were prominent in untreated and IL4-treated microglia (but not after LPS). IL4 increased the migratory capacity of microglia but eliminated the preferential anterior nuclear-centrosomal axis polarity and location of the microtubule organizing center (MTOC). Microglia degraded fibronectin, regardless of treatment, but LPS-treated cells were relatively immobile and IL4-treated cells invaded much more effectively through Matrigel™. For invasion, untreated microglia primarily used cysteine proteases, but IL4-treated cells used a wider range of enzymes (cysteine proteases, cathepsin S and K, heparanase, and matrix metalloproteases). Untreated microglia expressed MMP2, MMP12, heparanase, and four cathepsins (B, K, L1, and S). Each activation stimulus upregulated a different subset of enzymes. IL4 increased MMP2 and cathepsins S and K; whereas LPS increased MMP9, MMP12, MMP14 (MT1-MMP), heparanase, and cathepsin L1. Conclusions Microglial cells migrate during CNS development and after CNS damage or disease. Thus, there are broad implications of the finding that classically and alternatively activated microglia differ in morphology, cytoskeleton, migratory and invasive capacity, and in the usage of ECM-degrading enzymes

Table_6_Microglia Responses to Pro-inflammatory Stimuli (LPS, IFNγ+TNFα) and Reprogramming by Resolving Cytokines (IL-4, IL-10).pdf

<p>Microglia respond to CNS injuries and diseases with complex reactions, often called “activation.” A pro-inflammatory phenotype (also called classical or M1 activation) lies at one extreme of the reactivity spectrum. There were several motivations for this study. First, bacterial endotoxin (lipopolysaccharide, LPS) is the most commonly used pro-inflammatory stimulus for microglia, both in vitro and in vivo; however, pro-inflammatory cytokines (e.g., IFNγ, TNFα) rather than LPS will be encountered with sterile CNS damage and disease. We lack direct comparisons of responses between LPS and such cytokines. Second, while transcriptional profiling is providing substantial data on microglial responses to LPS, these studies mainly use mouse cells and models, and there is increasing evidence that responses of rat microglia can differ. Third, the cytokine milieu is dynamic after acute CNS damage, and an important question in microglial biology is: How malleable are their responses? There are very few studies of effects of resolving cytokines, particularly for rat microglia, and much of the work has focused on pro-inflammatory outcomes. Here, we first exposed primary rat microglia to LPS or to IFNγ+TNFα (I+T) and compared hallmark functional (nitric oxide production, migration) and molecular responses (almost 100 genes), including surface receptors that can be considered part of the sensome. Protein changes for exemplary molecules were also quantified: ARG1, CD206/MRC1, COX-2, iNOS, and PYK2. Despite some similarities, there were notable differences in responses to LPS and I+T. For instance, LPS often evoked higher pro-inflammatory gene expression and also increased several anti-inflammatory genes. Second, we compared the ability of two anti-inflammatory, resolving cytokines (IL-4, IL-10), to counteract responses to LPS and I+T. IL-4 was more effective after I+T than after LPS, and IL-10 was surprisingly ineffective after either stimulus. These results should prove useful in modeling microglial reactivity in vitro; and comparing transcriptional responses to sterile CNS inflammation in vivo.</p