Chemokines and Inflammatory Mediators Interact to Regulate Adult Murine Neural Precursor Cell Proliferation, Survival and Differentiation

Adult neural precursor cells (NPCs) respond to injury or disease of the CNS by migrating to the site of damage or differentiating locally to replace lost cells. Factors that mediate this injury induced NPC response include chemokines and pro-inflammatory cytokines, such as tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ), which we have shown previously promotes neuronal differentiation. RT-PCR was used to compare expression of chemokines and their receptors in normal adult mouse brain and in cultured NPCs in response to IFNγ and TNFα. Basal expression of many chemokines and their receptors was found in adult brain, predominantly in neurogenic regions, with OB≫SVZ>hippocampus and little or no expression in non-neurogenic regions, such as cortex. Treatment of SVZ-derived NPCs with IFNγ and TNFα (alone and in combination) resulted in significant upregulation of expression of specific chemokines, with CXCL1, CXCL9 and CCL2 most highly upregulated and CCL19 downregulated. Unlike IFNγ, chemokine treatment of NPCs in vitro had little or no effect on survival, proliferation or migration. Neuronal differentiation was promoted by CXCL9, CCL2 and CCL21, while astrocyte and total oligodendrocyte differentiation was not affected. However, IFNγ, CXCL1, CXCL9 and CCL2 promoted oligodendrocyte maturation. Therefore, not only do NPCs express chemokine receptors, they also produce several chemokines, particularly in response to inflammatory mediators. This suggests that autocrine or paracrine production of specific chemokines by NPCs in response to inflammatory mediators may regulate differentiation into mature neural cell types and may alter NPC responsiveness to CNS injury or disease

P2Y12 expression and function in alternatively activated human microglia

Objective: To investigate and measure the functional significance of altered P2Y12 expression in the context of human microglia activation. Methods: We performed in vitro and in situ experiments to measure how P2Y12 expression can influence disease-relevant functional properties of classically activated (M1) and alternatively activated (M2) human microglia in the inflamed brain. Results: We demonstrated that compared to resting and classically activated (M1) human microglia, P2Y12 expression is increased under alternatively activated (M2) conditions. In response to ADP, the endogenous ligand of P2Y12, M2 microglia have increased ligand-mediated calcium responses, which are blocked by selective P2Y12 antagonism. P2Y12 antagonism was also shown to decrease migratory and inflammatory responses in human microglia upon exposure to nucleotides that are released during CNS injury; no effects were observed in human monocytes or macrophages. In situ experiments confirm that P2Y12 is selectively expressed on human microglia and elevated under neuropathologic conditions that promote Th2 responses, such as parasitic CNS infection. Conclusion: These findings provide insight into the roles of M2 microglia in the context of neuroinflammation and suggest a mechanism to selectively target a functionally unique population of myeloid cells in the CNS

Cudraflavone C induces tumor-specific apoptosis in colorectal cancer cells through inhibition of the phosphoinositide 3-kinase (PI3K)-AKT pathway

Cudraflavone C (Cud C) is a naturally-occurring flavonol with reported anti-proliferative activities. However, the mechanisms by which Cud C induced cytotoxicity have yet to be fully elucidated. Here, we investigated the effects of Cud C on cell proliferation, caspase activation andapoptosis induction in colorectal cancer cells (CRC). We show that Cud C inhibits cell proliferation in KM12, Caco-2, HT29, HCC2998, HCT116 and SW48 CRC but not in the non-transformed colorectal epithelial cells, CCD CoN 841. Cud C induces tumorselective apoptosis via mitochondrial depolarization and activation of the intrinsic caspase pathway. Gene expression profiling by microarray analyses revealed that tumor suppressor genes EGR1, HUWE1 and SMG1 were significantly up-regulated while oncogenes such as MYB1, CCNB1 and GPX2 were down-regulated following treatment with Cud C. Further analyses using Connectivity Map revealed that Cud C induced a gene signature highly similar to that of protein synthesis inhibitors and phosphoinositide 3-kinase (PI3K)-AKT inhibitors, suggesting that Cud C might inhibit PI3K-AKT signaling. A luminescent cell free PI3K lipid kinase assay revealed that Cud C significantly inhibited p110?/p85? PI3K activity, followed by p120?, p110?/p85?, and p110?/p85? PI3K activities. The inhibition by Cud C on p110?/p85? PI3K activity was comparable to LY-294002, a known PI3K inhibitor. Cud C also inhibited phosphorylation of AKT independent of NF?B activity in CRC cells, while ectopic expression of myristoylated AKT completely abrogated the anti-proliferative effects, and apoptosis induced by Cud C in CRC. These findings demonstrate that Cud C induces tumor-selective cytotoxicity by targeting the PI3K-AKT pathway. These findings provide novel insights into the mechanism of action of Cud C, and indicate that Cud C further development of Cud C derivatives as potential therapeutic agents is warranted

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

Summary of chemokine ligand and receptor expression in adult brain.

<p>SVZ, Subventricular zone; OB, olfactory bulb; Hipp, hippocampus; Ctx, cortex.</p><p>Expression levels: − none; +/− very low; + low; ++ moderate, +++ high; ++++ very high.</p

Effect of selected chemokines on NPC differentiation.

<p>(A) The effect of chemokines (10 ng/ml) on neuronal differentiation as assessed by quantifying the relative percentage of cells that expressed the neuronal marker βIII-tubulin at 3 days under treatment conditions compared to the basal control. (B) The effect on astrocyte differentiation was assessed by counting the percentage of cells immunostained for the astrocyte marker GFAP at 7 days, expressed as a relative percentage compared to the basal control. (C) The effect on oligodendrocyte differentiation was assessed by counting the number of cells immunostained for the oligodendrocyte marker O4 at 7 days and expressing this as a percentage relative to the basal control. (D) Representative micrographs of βIII-tubulin, GFAP and DAPI labeling of cultures differentiated for 3 days in the presence of 10 ng/ml CXCL9, CCL2 or CCL21, compared to basal conditions. Results in A–C are expressed as mean+/−sem from at <i>n</i>≥3 independent experiments, *<i>p</i><0.05, **<i>p</i><0.01. Scale bar in D, 50 µm.</p

Expression of CXCR chemokine receptors in neurogenic versus non-neurogenic regions of adult brain.

<p>qPCR analysis of chemokine receptor CXCR1–7 expression in neurogenic subventricular (SVZ), olfactory bulb, and hippocampus and non-neurogenic cortex. Due to large differences in the level of expression of the different receptors, results are presented using a log₁₀ scale and show mean+/−SEM of 3 independent experiments in which all samples were run together and normalized against GAPDH. All samples were compared to expression of CXCR5 in the olfactory bulb as the reference sample (denoted by #), which was expressed at just detectable levels (C_T<32) and given a value of 1. Receptors that were not expressed (C_T≥32) are denoted by 0 in their respective columns.</p

Regulation of chemokine and chemokine receptor expression in adult NPCs by IFNγ and TNFα.

<p>(A) Selected representative gels of RT-PCR for chemokine (CXCL and CCL) and chemokine receptor (CXCR and CCR) expression in adult neurospheres under control (C) conditions or treated with 100 U/ml IFNγ (I), 100 U/ml TNFα (T) or IFNγ plus TNFα (I+T). (B–D) The relative expression level of each chemokine or receptor in response to addition of (B) IFNγ, (C) TNFα or (D) IFNγ plus TNFα was normalized by densitometric analysis to control expression levels for each chemokine or receptor and expressed as fold difference compared to control. Results show mean+/−SEM of at least n = 3 individual experiments; *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

Confirmation of chemokine induction by qPCR.

<p>qPCR was used to confirm the upregulation or induction of chemokine expression by IFNγ and/or TNFα. Due to large differences in the level of expression of the different chemokines, results are presented using a log₁₀ scale and show mean+/−SEM of 3 independent experiments in which all samples were run together and normalized against GAPDH. CXCL1 and CCL2 were detectable in the neurospheres under basal conditions, therefore their expression following incubation with IFNγ and TNFα was compared to the level of expression under basal conditions (which was set to 1). CXCL9 and CXCL13 were not expressed at detectable levels under basal conditions (C_T≥32). CXCL9 was detectable following addition of TNFα, which was used as the reference condition in this case (and set to 1; denoted by #) for comparison with expression induced by IFNγ and IFNγ+TNFα. CXCL13 was not detectable with TNFα treatment (denoted by 0) but was induced by IFNγ which was used as the reference condition (and set to 1; denoted by #) for comparison with levels of expression induced by TNFα plus IFNγ.</p

Regulation of NPC proliferation survival and migration by chemokines and IFNγ.

<p>(A,B) Effect on neurosphere growth. Neurospheres were grown for 7 days in the presence of chemokines (100 ng/ml) and/or IFNγ (100 U/ml) and neurosphere growth determined by assessment of viable cell number using the MTS assay. Results of a representative experiment are shown in A and the combined results of three independent experiments, which are expressed as a percentage of neurosphere growth under basal conditions are shown in B. Effect on NPC proliferation (C,D), survival (E,F) and migration (G,H): neurospheres were pre-treated for 24 hrs with IFNγ then plated onto fibronectin in proliferation medium alone (IFNγ pre), with IFNγ alone (IFNγ+IFNγ), with chemokines alone or with chemokines plus IFNγ for 24 hrs. Basal controls were not pre-treated with IFNγ nor treated with any factor at the time of plating. Representative results from individual experiments are shown in C, E and G and the combined results of three independent experiments, expressed as a percentage of basal, are shown in D, F and H. (C,D) Effect on proliferation was assessed by counting the percentage of cells immunostained for the proliferation marker Ki67. (E,F) Effect on cell survival was assessed by counting the percentage of cells that were labeled by TUNEL staining. (G,H) Effect on cell migration was assessed by measuring the area of spread of cells from individual neurospheres and dividing by cell number per neurosphere. Results are expressed as mean+/−sem, *<i>p</i><0.05, **<i>p</i><0.01.</p