Effects of low dose GM-CSF on microglial inflammatory profiles to diverse pathogen-associated molecular patterns (PAMPs)

BACKGROUND: It is well appreciated that obtaining sufficient numbers of primary microglia for in vitro experiments has always been a challenge for scientists studying the biological properties of these cells. Supplementing culture medium with granulocyte-macrophage colony-stimulating factor (GM-CSF) partially alleviates this problem by increasing microglial yield. However, GM-CSF has also been reported to transition microglia into a dendritic cell (DC)-like phenotype and consequently, affect their immune properties. METHODS: Although the concentration of GM-CSF used in our protocol for mouse microglial expansion (0.5 ng/ml) is at least 10-fold less compared to doses reported to affect microglial maturation and function (≥ 5 ng/ml), in this study we compared the responses of microglia derived from mixed glial cultures propagated in the presence/absence of low dose GM-CSF to establish whether this growth factor significantly altered the immune properties of microglia to diverse bacterial stimuli. These stimuli included the gram-positive pathogen Staphylococcus aureus (S. aureus) and its cell wall product peptidoglycan (PGN), a Toll-like receptor 2 (TLR2) agonist; the TLR3 ligand polyinosine-polycytidylic acid (polyI:C), a synthetic mimic of viral double-stranded RNA; lipopolysaccharide (LPS) a TLR4 agonist; and the TLR9 ligand CpG oligonucleotide (CpG-ODN), a synthetic form of bacteria/viral DNA. RESULTS: Interestingly, the relative numbers of microglia recovered from mixed glial cultures following the initial harvest were not influenced by GM-CSF. However, following the second and third collections of the same mixed cultures, the yield of microglia from GM-CSF-supplemented flasks was increased two-fold. Despite the ability of GM-CSF to expand microglial numbers, cells propagated in the presence/absence of GM-CSF demonstrated roughly equivalent responses following S. aureus and PGN stimulation. Specifically, the induction of tumor necrosis factor-α (TNF-α), macrophage inflammatory protein-2 (MIP-2/CXCL2), and major histocompatibility complex (MHC) class II, CD80, CD86 expression by microglia in response to S. aureus were similar regardless of whether cells had been exposed to GM-CSF during the mixed culture period. In addition, microglial phagocytosis of intact bacteria was unaffected by GM-CSF. In contrast, upon S. aureus stimulation, CD40 expression was induced more prominently in microglia expanded in GM-CSF. Analysis of microglial responses to additional pathogen-associate molecular patterns (PAMPs) revealed that low dose GM-CSF did not significantly alter TNF-α or MIP-2 production in response to the TLR3 and TLR4 agonists polyI:C or LPS, respectively; however, cells expanded in the presence of GM-CSF produced lower levels of both mediators following CpG-ODN stimulation. CONCLUSION: We demonstrate that low levels of GM-CSF are sufficient to expand microglial numbers without significantly affecting their immunological responses following activation of TLR2, TLR4 or TLR3 signaling. Therefore, low dose GM-CSF can be considered as a reliable method to achieve higher microglial yields without introducing dramatic activation artifacts

Patient Specific Seizure Prediction System Using Hilbert Spectrum and Bayesian Networks Classifiers

The aim of this paper is to develop an automated system for epileptic seizure prediction from intracranial EEG signals based on Hilbert-Huang transform (HHT) and Bayesian classifiers. Proposed system includes decomposition of the signals into intrinsic mode functions for obtaining features and use of Bayesian networks with correlation based feature selection for binary classification of preictal and interictal recordings. The system was trained and tested on Freiburg EEG database. 58 hours of preictal data, 40-minute data blocks prior to each of 87 seizures collected from 21 patients, and 503.1 hours of interictal data were examined resulting in 96.55% sensitivity with 0.21 false alarms per hour, 13.896% average proportion of time spent in warning, and 33.21 minutes of average detection latency using 30-second EEG segments with 50% overlap and a simple postprocessing technique resulting in a decision (a seizure is expected/not expected) every 5 minutes. High sensitivity and low false positive rate with reasonable detection latency show that HHT based features are acceptable for patient specific seizure prediction from intracranial EEG data. Time spent for testing an EEG segment was 4.1451 seconds on average, which makes the system viable for use in real-time seizure control systems

Microglia and Astrocyte Activation by Toll-Like Receptor Ligands: Modulation by PPAR-γ Agonists

Microglia and astrocytes express numerous members of the Toll-like receptor (TLR) family that are pivotal for recognizing conserved microbial motifs expressed by a wide array of pathogens. Despite the critical role for TLRs in pathogen recognition, when dysregulated these pathways can also exacerbate CNS tissue destruction. Therefore, a critical balance must be achieved to elicit sufficient immunity to combat CNS infectious insults and downregulate these responses to avoid pathological tissue damage. We performed a comprehensive survey on the efficacy of various PPAR-γ agonists to modulate proinflammatory mediator release from primary microglia and astrocytes in response to numerous TLR ligands relevant to CNS infectious diseases. The results demonstrated differential abilities of select PPAR-γ agonists to modulate glial activation. For example, 15d-PGJ2 and pioglitazone were both effective at reducing IL-12 p40 release by TLR ligand-activated glia, whereas CXCL2 expression was either augmented or inhibited by 15d-PGJ2, effects that were dependent on the TLR ligand examined. Pioglitazone and troglitazone demonstrated opposing actions on microglial CCL2 production that were TLR ligand-dependent. Collectively, this information may be exploited to modulate the host immune response during CNS infections to maximize host immunity while minimizing inappropriate bystander tissue damage that is often characteristic of such diseases

Microglia and Astrocyte Activation by Toll-Like Receptor Ligands: Modulation by PPAR-gamma Agonists.

Microglia and astrocytes express numerous members of the Toll-like receptor (TLR) family that are pivotal for recognizing conserved microbial motifs expressed by a wide array of pathogens. Despite the critical role for TLRs in pathogen recognition, when dysregulated these pathways can also exacerbate CNS tissue destruction. Therefore, a critical balance must be achieved to elicit sufficient immunity to combat CNS infectious insults and downregulate these responses to avoid pathological tissue damage. We performed a comprehensive survey on the efficacy of various PPAR-gamma agonists to modulate proinflammatory mediator release from primary microglia and astrocytes in response to numerous TLR ligands relevant to CNS infectious diseases. The results demonstrated differential abilities of select PPAR-gamma agonists to modulate glial activation. For example, 15d-PGJ(2) and pioglitazone were both effective at reducing IL-12 p40 release by TLR ligand-activated glia, whereas CXCL2 expression was either augmented or inhibited by 15d-PGJ(2), effects that were dependent on the TLR ligand examined. Pioglitazone and troglitazone demonstrated opposing actions on microglial CCL2 production that were TLR ligand-dependent. Collectively, this information may be exploited to modulate the host immune response during CNS infections to maximize host immunity while minimizing inappropriate bystander tissue damage that is often characteristic of such diseases

Type‐I interferons suppress microglial production of the lymphoid chemokine, CXCL13

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/108063/1/glia22692.pd

Complexity of the microglial activation pathways that drive innate host responses during lethal alphavirus encephalitis in mice

Microglia express multiple TLRs (Toll-like receptors) and provide important host defence against viruses that invade the CNS (central nervous system). Although prior studies show these cells become activated during experimental alphavirus encephalitis in mice to generate cytokines and chemokines that influence virus replication, tissue inflammation and neuronal survival, the specific PRRs (pattern recognition receptors) and signalling intermediates controlling microglial activation in this setting remain unknown. To investigate these questions directly in vivo, mice ablated of specific TLR signalling molecules were challenged with NSV (neuroadapted Sindbis virus) and CNS viral titres, inflammatory responses and clinical outcomes followed over time. To approach this problem specifically in microglia, the effects of NSV on primary cells derived from the brains of wild-type and mutant animals were characterized in vitro. From the standpoint of the virus, microglial activation required viral uncoating and an intact viral genome; inactivated virus particles did not elicit measurable microglial responses. At the level of the target cell, NSV triggered multiple PRRs in microglia to produce a broad range of inflammatory mediators via non-overlapping signalling pathways. In vivo, disease survival was surprisingly independent of TLR-driven responses, but still required production of type-I IFN (interferon) to control CNS virus replication. Interestingly, the ER (endoplasmic reticulum) protein UNC93b1 facilitated host survival independent of its known effects on endosomal TLR signalling. Taken together, these data show that alphaviruses activate microglia via multiple PRRs, highlighting the complexity of the signalling networks by which CNS host responses are elicited by these infections

MyD88 expression by CNS-resident cells is pivotal for eliciting protective immunity in brain abscesses

MyD88 KO (knockout) mice are exquisitely sensitive to CNS (central nervous system) infection with Staphylococcus aureus, a common aetiological agent of brain abscess, exhibiting global defects in innate immunity and exacerbated tissue damage. However, since brain abscesses are typified by the involvement of both activated CNS-resident and infiltrating immune cells, in our previous studies it has been impossible to determine the relative contribution of MyD88-dependent signalling in the CNS compared with the peripheral immune cell compartments. In the present study we addressed this by examining the course of S. aureus infection in MyD88 bone marrow chimaera mice. Interestingly, chimaeras where MyD88 was present in the CNS, but not bone marrow-derived cells, mounted pro-inflammatory mediator expression profiles and neutrophil recruitment equivalent to or exceeding that detected in WT (wild-type) mice. These results implicate CNS MyD88 as essential in eliciting the initial wave of inflammation during the acute response to parenchymal infection. Microarray analysis of infected MyD88 KO compared with WT mice revealed a preponderance of differentially regulated genes involved in apoptotic pathways, suggesting that the extensive tissue damage characteristic of brain abscesses from MyD88 KO mice could result from dysregulated apoptosis. Collectively, the findings of the present study highlight a novel mechanism for CNS-resident cells in initiating a protective innate immune response in the infected brain and, in the absence of MyD88 in this compartment, immunity is compromised