LPS-induced TLR4 neuroinflammatory signaling in CHME-5 microglial cells

Aim: In the field of neuroinflammation, identifying specific effects of pharmacological agents and other factors is problematic given the relative difficulty and expense in obtaining and culturing primary microglia. Immortalized microglial cell lines are very useful, but only a limited number have been characterized for inflammatory signaling. Therefore, characterization of lipopolysaccharide (LPS)-induced toll-like receptor 4 (TLR4) signaling in CHME-5, a microglial cell line, is expected to be of value as an experimental model of inflammatory signaling in the central nervous system (CNS). Methods: It was recently suggested that CHME-5 cells are of rat origin, not human, hence, verification of this claim using short tandem repeat genotype sequencing, along with immunoblotting, reverse transcription-polymerase chain reaction, and immunocytochemistry techniques to validate that CHME-5 retain morphological, phenotypical, and functional characteristics of primary microglia were undertaken. Results: LPS induced inhibitor kappa B-alpha and nuclear factor-kappa B (NF-κB) p65 activation, NF-κB p65 binding activity, and tumor necrosis factor alpha gene expression.Additionally, results also confirmed the maintenance of microglial phenotype as seen with increased cluster of differentiation 68 gene and protein expression, immunofluorescence, and the absence of glial fibrillary acidic protein-immunoreactivity.TLR4 gene expression and immunofluorescence were significantly increased after LPS treatment. Conclusion: These data demonstrate that CHME-5 cells are not human, but are indeed a beneficial tool for studying microglial inflammatory signaling.

Interleukin-1β-induced inflammatory signaling in C20 human microglial cells

Aim: Increased inflammatory signaling in microglia is implicated in the pathogenesis of neurodegenerative diseases, trauma, psychiatric disorders, and anxiety/depression. Understanding inflammatory signaling in microglia is critical for advancing treatment options. Studying rodent-derived microglia has yielded substantial information, yet, much remains to better understand inflammatory signaling in human microglia. Hence, there is great interest in developing immortalized human microglial cell lines. The C20 human microglial cell line was recently developed and our primary objective was to advance our knowledge of inflammatory signaling in these cells. Methods: Expression of the microglia specific marker transmembrane protein 119 (TMEM119) was assessed by western blot analysis. Lipopolysaccharide (LPS)- and interleukin-1β (IL-1β)-induced cytokine/chemokine expression was determined by ELISA. Phosphorylation of inhibitory kappa B alpha (IκBα), nuclear factor (NF)-κB p65, and p38 mitogen-activated protein kinase (p38 MAPK) was measured by western blot analysis. Results: TMEM119 was expressed in unstimulated C20 cells, and to a greater extent in IL-1β-stimulated cells. IL-1βsignificantly induced IL-6, monocyte chemoattractant protein-1/CCL2, and interferon-γ inducible protein 10/CXCL10 expression. LPS induced CCL2 expression, but not IL-6 or CXCL10 expression. IL-1β induced inflammatory signaling as indicated by increased phosphorylation of IκBα, NF-κB p65 and p38 MAPK. Conclusion: We provide the first evidence that C20 microglia express TMEM119. This is the initial report of IL-1β-induced activation of IκBα, NF-κB p65, and p38 MAPK and subsequent CXCL10, CCL2 and IL-6 secretion in C20cells. These findings advance our understanding of inflammatory signaling in C20 cells and support the value of this cell line as a research tool.

Direct choline administration in semi-intensive pisciculture system: A positive contaminant

Present work evaluated the possible impact of choline on metabolic enzymes of cultured fish species into a semi-intensive pond culture system,reared with Catla catla(catla),Labeo rohita(rahu),Clarias batracus(magur)and Anabas testudineus(climbing perch)for 90 days in two seasons.Choline was added directly into the pond water periodically throughout the experimental tenure.The water quality parameters of each experimental pond were monitored and were analyzed in 15 days interval throughout the experimental tenure in both the seasons.Results were compared with treatment as choline-fed and control as non-choline-fed conditions during breeding(June-Aug.)and dry(Nov.-Jan.)seasons.The metabolic enzymes,viz.,LDH(Lactate dehydrogenase),MDH(Malate dehydrogenase),and G6PDH(Glucose-6-phosphate dehydrogenase)activities in the muscle,liver,and AchE(Acetylcholine esterase)activity in the brain and muscle revealed maximum significantly(p<0.01),but ALT(Alanine aminotransferase)and AST(Aspartate aminotransferase)activity showed significant(p<0.01)depletion in the muscle and liver in treatment-breeding(TB)condition.The fishes under treatment-dry(TD)condition presented significant(p<0.01)higher elevation in HK(Hexokinase)activity in the muscle and liver.Additionally,the result of principal component analysis(PCA)depicted the positive as well as in some cases a negative correlation among the enzymatic activities both in dry and breeding seasons.So,it can be inferred that choline enrichment in fishes can substantiate the well nutritionally balanced food-flesh for consumption under this farm culture and the choline behaved as a positive contaminant into the pond eco-system under the semi-intensive culture condition.