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PAMPs and PRRs drive mucosa immune response : tolerance activation or pathology

PAMPs and PRRs drive mucosa immune response : tolerance activation or pathology. Dr.ihsan edan alsaimary Dr.awatif h.issa Dr.naema y.alyasiry Dr.andrew foey. Overview . The role of macrophages and epithelial cells in directing the mucosal immune response. Types of macrophages.

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PAMPs and PRRs drive mucosa immune response : tolerance activation or pathology

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  1. PAMPs and PRRs drive mucosa immune response : tolerance activation or pathology Dr.ihsanedanalsaimary Dr.awatifh.issa Dr.naemay.alyasiry Dr.andrewfoey

  2. Overview • The role of macrophages and epithelial cells in directing the mucosal immune response. • Types of macrophages. • Types of macrophages stimulates. • Cytokines produce by macrophages and epithelial cells in respones to simulus. • Role of probiotic in modulation of immune respone.

  3. Aim and objectives • Identify types of macrophages • Determine the role of stimulus in cytokine production by macrophage and epithelial cellds • Determine the role of probiotic in modulation of immune response by macrophage and epithelial cells.

  4. Methodolgy • THP-1 (monocytic cell line ) diffrentiated in to : • M1-like macrophage cells in presence of (PhorbolMyristateAcetate) PMA for 3 days. • M2-like macrophage cells in the presence of 25-hydroxyvitamine D3(ViD3) for 7 days. • Caco-2 epithelial cells differentiated for 7 days.

  5. Methodolgy • THP-1 (monocytic cell line ) differentiated in to cultured in to RPMI : • M1-like macrophage cells in presence of (PhorbolMyristateAcetate) PMA for 3 days. • M2-like macrophage cells in the presence of 25-hydroxyvitamine D3(ViD3) for 7 days. • Caco-2 epithelial cells differentiated for 7 days cultured in DMED medium.

  6. Methodolgy • Macrophages and Caco-2 cells pre-treated with HK Lactobacillus casei strain Shirota ( LcS) at 3x10 8 CFU/ml for 18 hr. • Followed by stimulation with different PAMPs (LPS,PGN,LTA, ect) for 18 hr. • The supernatant were collected for ELISA to detect cytokine production • mRNA from macrophage and Caco-2 cells were extracted by Sigma gene elute kit modified by naemayassirHabil (Ph.D. student)et al, 2011 (unpublished data).

  7. Methodolgy • Briefly : Cells were harvested then lysed by probultrasonicate ( 3 sec, 13,000rpm) with lysis solution added with mercaptoethanol. • DNAase column were added to exclude DNA. • The amount of mRNA were eluted in 20 µl. • mRNA quality and quantity were cheeked by nanovue. • qPCR were done using Syber green. • Applied bio system soft ware was used to analyze mRNA expression by all the treatments.

  8. Methodology • To detect TLRs , flow cytometry were used. Abs for TLR2,and TLR4 were used to stain immune cells. To apply the TLRs ,Bio Rad (BR) system was used.

  9. Results and conclusion

  10. Ladder 1a 2a 3a 4a 5a 6a 1b 2b 3b 4b 5b 6b 1c 2c 3c 4c 5b 6b 100 bp 1: Resting cells,2:Cells+ LPS,3: Cells+ LPS+LcS ,4: Cells+PGN,5:PGN+LcS,6: LcS: a, IL-10 expresion; b: TNF-a expression;c: TLR4 expression IL-10,TNF-a,and TLR4 expression by M1-lilke macrophage

  11. To conclude the results • THP-1 monocytic cell line performed good model to assimilate the two types of macrophage cells. • LcS as heat killed bacteremia , which is one of the main bacteria used as probiotic showed nice modulation of mRNA expression by macrophage and epithelial cells.

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