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Travis J. Kochan, Jacob Blair, Ashok Kumar; Modulation Of Toll-like Receptor Signaling In Microglia By Tlr2, And Tlr4 Ligands And Their Relevance To Bacterial Endophthalmitis. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2958.
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© ARVO (1962-2015); The Authors (2016-present)
Recently we reported microglial activation in response to TLR2 ligand challenge in B6 mouse retina. To investigate their specific role in endophthalmitis, here we tested how their innate response is modulated by TLR2 and TLR4 ligand pretreatment in vitro.
Immortalized mouse microglia cell line, BV-2 was challenged with S. aureus (SA), TLR2 (Pam3Cys) and TLR4 (LPS) ligands and both concentration and time course studies were performed. Expression of inflammatory cytokines, chemokines and antimicrobial peptides was determined by RT-PCR and ELISA. TLR-mediated downstream signaling pathway activation was assessed by Western blot. To test the modulation of innate response, microglia were pretreated with low doses of Pam3Cys or LPS and then challenged, with higher dose of TLR ligands or SA.
BV-2 cells constitutively expressed TLR2 and TLR4 and their expression is increased upon stimulation with their respective ligands. Both TLR ligands and S. aureus induced the activation of NF-ΚB in a concentration (0.01-10 µg/ml) and time (15-90 min) dependent manner. LPS challenge profoundly induced cytokine (TNF-α, IL-1β and KC) secretion even at low (0.001 µg /ml) concentration followed by SA and Pam3Cys. Pretreatment of BV-2 cells with low dose of Pam3Cys (0.1 or 1 µg/ml) followed by subsequent challenge with either higher dose of Pam3Cys (10 µg/ml) or SA, but not LPS (1 µg/ml), impaired NF-ΚB activation and dramatically reduced the production of cytokines. Similarly, LPS pretreatment also reduced cytokine production in response to LPS re-challenge but not towards Pam3Cys or SA stimulation.
Our data indicate that in microglia TLR2 and TLR4 stimulation results in homo- but not cross-tolerance; further studies are warranted to define the underlying mechanism involved in this phenomenon.
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