The effect of different strains of LPS on the surface expression of CD14 and TLR4 in MonoMac6 cells
Cardiff Metropolitan University
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Toll-like Receptors (TLRs) recognise microbial pathogen-associated molecular patterns (PAMPs) and are a vital component of the innate immune system. TLR4, along with mCDl4 and MD2, recognises lipopolysaccharide (LPS) expressed by Gram-negative bacteria, whilst TLR2, with TLR1 or TLR6, recognises bacterial lipoproteins and lipopeptides. TLR4 and TLR2 are associated with a range of inflammatory disorders including sepsis. The effect of different strains of LPS and Pam3Cys4 on surface expression of mCD14, TLR4 and TLR2 in MM6 cells was analysed by Flow cytometry. Inflammatory cytokine production was assessed by ELISA. Flow cytometry analysis demonstrated LPS (1000ng/ml) significantly upregulated mCD14 at 24 hours (>-460%). LPS stimulation caused a transient upregulation of surface TLR4 at 1.5 hours, followed by pronounced downregulation at 24 hours (-40%). Significant differences were observed between Rough- (R-) and Smooth-form (S-form) LPS strains. Pam3Cys4 (500ng/ml) treatment downregulated surface TLR2 at 24 hours. LPS also altered TLR2 causing downregulation at 2-6 hours. Pam3Cys4 upregulated mCDl4 at 24 hours (288%). ELISA analysis showed both LPS Pam3Cys4 induced significant upregulation in TNF-a and Il-8 expression, with significant differences observed between R- and S-form LPS. This study therefore provides evidence for regulation of mCD14, TLR4 and TLR2 surface by LPS and Pam3Cys4in MM6 cells, and demonstrates differences in this regulation between R- and S-form LPS. Improved knowledge in the regulation of TLRs and their role in the pathogenesis of inflammatory disorders may provide novel therapies.
MSc Biomedical Science (Immunohaematology)
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