Tlr and inflammation 2013

829 views

Published on

0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total views
829
On SlideShare
0
From Embeds
0
Number of Embeds
1
Actions
Shares
0
Downloads
35
Comments
0
Likes
0
Embeds 0
No embeds

No notes for slide

Tlr and inflammation 2013

  1. 1. Sample & Assay Technologies Toll-like Receptors in Inflammation Raed Samara, Ph.D. R&D Scientist
  2. 2. Sample & Assay Technologies QIAGEN webinars Access recorded webinars and slides http://sabiosciences.com/home.php Title, Location, Date 2
  3. 3. Sample & Assay Technologies QIAGEN webinars Access recorded webinars and slides Title, Location, Date 3
  4. 4. Sample & Assay Technologies Keep up to date: Follow Pathway focused biology on Facebook Latest information on pathway and disease research, resources and demos.
  5. 5. Sample & Assay Technologies Biology-Focused Solutions for Pathway Analysis SABiosciences is now a Company http://sabiosciences.com/home.php Title, Location, Date 5
  6. 6. Sample & Assay Technologies Introduction: Inflammation Definition: a protective tissue response to tissue damage or microbes, which serves to destroy, dilute, or wall off both the injurious agent and the injured tissues. ̣ Microbes/Infection Tissue Damage TLR Acute Inflammation Infection Clearance Tissue Homeostasis Epigenetic Changes mRNA Changes Cytokines & Chemokines Signaling Pathways Immune system composition Chronic Inflammation TLR Pre-cancer & Cancer Chronic Inflammatory Diseases Title, Location, Date 6
  7. 7. Sample & Assay Technologies Toll-like Receptors (TLR) Recognize molecular patterns derived from: - Microbes - Endogenous molecules Either: - Directly - Indirectly (with the help of accessory molecules) TLR8 TLR10 Both at: - Plasma membrane - Intracellular compartments Signal through: - MYD88 - TRIF TLR activation leads to: - Regulation of innate immune response - Regulation of adaptive immune responses - Inflammation - Tissue repair Rakoff-Nahoum & Medzhitov. 2009. Nat Rev Cancer
  8. 8. Sample & Assay Technologies Ligands of TLR Chen et al. 2007. Int’l Immunopharm Title, Location, Date 8
  9. 9. Sample & Assay Technologies Expression of TLR TLR are expressed in a wide variety of cells Ospelt & Gay. 2010. Int’l J Biochem Cell Biol Title, Location, Date 9
  10. 10. Sample & Assay Technologies TLR signaling TAK1 Foster & Medzhitov. 2009. Clin Imm Title, Location, Date 10
  11. 11. Sample & Assay Technologies TLR signaling Uematsu & Akira. 2007. JBC Title, Location, Date 11
  12. 12. Sample & Assay Technologies TLR signaling Induction of 3 classes of transcription factors Post-translational activation of transcription factors after TLR stimulation Transcription factors are induced during the primary response Transcription factors are cellspecific Medzhitov & Horng. 2009. Nat Rev Imm Title, Location, Date 12
  13. 13. Sample & Assay Technologies TLR in inflammation TLR activation leads to a complex module-specific inflammatory transcriptional response Medzhitov & Horng. 2009. Nat Rev Imm Title, Location, Date 13
  14. 14. Sample & Assay Technologies TLR in inflammation TLRs form a bridge between innate and adaptive immune responses Parker, Prince, Sabroe. 2007. Clin Exp Imm Title, Location, Date 14
  15. 15. Sample & Assay Technologies TLR in inflammation TLR DC T cells Mills. 2011. Nat Rev Imm Title, Location, Date 15
  16. 16. Sample & Assay Technologies Title, Location, Date 16
  17. 17. Sample & Assay Technologies Expression of TLR Expression of TLR and MYD88 in the CNS of EAE-diseased mice EAE is an animal model of multiple sclerosis Detection of mRNA expression by RTPCR Title, Location, Date 17
  18. 18. Sample & Assay Technologies Effects of modulating TLR activity on EAE TLR signaling is critical for the induction of EAE and Th1 responses Induction of EAE by active immunization Knockout of MYD88 protected mice from EAE Title, Location, Date Knockout of MYD88 decreased Th1 responses after induction of EAE 18
  19. 19. Sample & Assay Technologies Which cell type is it? TLR DC T cells Mills. 2011. Nat Rev Imm Title, Location, Date 19
  20. 20. Sample & Assay Technologies Effects of modulating TLR activity on EAE Transfer reactive T cells (responsible for inducing EAE) in to mice of different strains Knockout of TLR9 or MYD88 in non-T cells in the host decreased severity of EAE Title, Location, Date 20
  21. 21. Sample & Assay Technologies TLR in inflammation TLR DC T cells Mills. 2011. Nat Rev Imm Title, Location, Date 21
  22. 22. Sample & Assay Technologies Title, Location, Date 22
  23. 23. Sample & Assay Technologies Effects of TLR activation on Treg cell activity TLR activation in Tregs suppresses their suppressive functions Title, Location, Date 23
  24. 24. Sample & Assay Technologies Effects of TLR activation on Treg cell activity The TLR8-Myd88-IRAK4 pathway is required to reverse the suppressive functions of Tregs Knockout indicated components in Tregs, sort Tregs, co-culture sorted Tregs with naïve CD4+ cells Title, Location, Date 24
  25. 25. Sample & Assay Technologies Regulation of TLR signaling Foster & Medzhitov. 2009. Clin Imm Title, Location, Date 25
  26. 26. Sample & Assay Technologies QIAGEN tools for studying TLR in inflammation RT2 Profiler PCR Arrays & Assays Profile 84 different genes on one array Appropriate controls for data normalization, sample quality, and reaction performance Focus on your questions and papers http://sabiosciences.com/PCRArrayPlate.php Title, Location, Date 26
  27. 27. Sample & Assay Technologies QIAGEN tools for studying TLR in inflammation RT2 Profiler PCR Arrays & Assays Array Species TLR Signaling Pathway Human/Mouse/Rat/Rhesus NFkB Signaling Pathway Human/Mouse/Rat NFkB Signaling Targets Human/Mouse/Rat MAPK Signaling Pathway Human/Mouse/Rat PI3K-Akt Signaling Pathway Human/Mouse/Rat TH1 & Th2 responses Human/Mouse/Rat/Rhesus Interferons & Receptors Human/Mouse/Rat Others (>140) Varies Array Species Antibacterial responses Human/Mouse/Rat Antiviral responses Human/Mouse/Rat Antifungal responses Human/Mouse/Rat Title, Location, Date 27
  28. 28. Sample & Assay Technologies QIAGEN tools for studying TLR in inflammation RT2 Profiler PCR Arrays & Assays Sample Isolation & Preparation Assay Set-up & Analysis Customer support group: 1-888-503-3187 support@SABiosciences.com Title, Location, Date 28
  29. 29. Sample & Assay Technologies Regulation of TLR signaling Quinn & O’Neil. 2011. Int’l Imm Title, Location, Date 29
  30. 30. Sample & Assay Technologies miRNA in TLR & inflammation Title, Location, Date 30
  31. 31. Sample & Assay Technologies miRNA webinars http://sabiosciences.com/home.php Title, Location, Date 31
  32. 32. Sample & Assay Technologies miRNA webinars Title, Location, Date 32
  33. 33. Sample & Assay Technologies Title, Location, Date 33
  34. 34. Sample & Assay Technologies Expression of miR-181 in hematopoietic lineages Northern analyses Chen, …, Bartel et al. 2004. Science Title, Location, Date 34
  35. 35. Sample & Assay Technologies Expression of miR-181 in hematopoietic lineages Ectopic expression Transduce bone marrow cells with Vector or miR-181; reconstitute mice with transduced BM cells; detect cell lineages by flow Monocytes Neutrophils nonmyeloid Chen, …, Bartel et al. 2004. Science Title, Location, Date 35
  36. 36. Sample & Assay Technologies Title, Location, Date 36
  37. 37. Sample & Assay Technologies Expression of miRNAs after TLR activation Microarray analyses Induce THP-1 cells (human acute monocytic leukemia) with LPS Microarray results are validated by RT-PCR Taganov, …, Baltimore et al. 2006. PNAS Title, Location, Date 37
  38. 38. Sample & Assay Technologies Expression of miR-146a is NFkB-dependent Mutations in NFkB binding site in the genomic loci of miR-146a Taganov, …, Baltimore et al. 2006. PNAS Title, Location, Date 38
  39. 39. Sample & Assay Technologies miR-146 targets mediators downstream of TLR Transfect cells with TRAF6 or IRAK1 reporter constructs, transfect cells with miRNAs, read luciferase activity Taganov, …, Baltimore et al. 2006. PNAS Title, Location, Date 39
  40. 40. Sample & Assay Technologies QIAGEN tools for studying TLR in inflammation miScript miRNA PCR Arrays & Assays Profile 84 different miRNAs on one array Appropriate controls for data normalization, sample quality, and reaction performance Focus on your questions and papers http://sabiosciences.com/mirna.php Title, Location, Date 40
  41. 41. Sample & Assay Technologies QIAGEN tools for studying TLR in inflammation miScript miRNA PCR Arrays & Assays http://sabiosciences.com/mirna_pcr_array.php Title, Location, Date 41
  42. 42. Sample & Assay Technologies QIAGEN tools for studying TLR in inflammation miScript miRNA PCR Arrays & Assays Sample Isolation & Preparation Assay Set-up & Analysis Customer support group: 1-888-503-3187 support@SABiosciences.com Title, Location, Date 42
  43. 43. QIAGEN tools for studying TLR in inflammation Sample & Assay Technologies Gene Expression RT-PCR - http://sabiosciences.com/PCRArrayPlate.php Somatic mutations - http://sabiosciences.com/somaticmutation.php BCR and TLR signaling pathways are recurrently targeted by genetic changes in splenic marginal zone lymphomas (MYD88 mutations) (Haematologica. 2012 April; 97(4): 595–598) Copy Number Variations & Alterations http://sabiosciences.com/copynumber.php Activation of toll-like receptors 2, 3, and 4 on human melanoma cells induces inflammatory factors (Mol Cancer Ther 2008;7(11):3642–53) Epigenetics miRNA - http://sabiosciences.com/mirna_pcr_array.php DNA methylation - http://sabiosciences.com/dna_methylation.php Histone modifications - http://sabiosciences.com/chipqpcr.php Transcriptional control of the inflammatory response (Nat Rev Imm, October 2009, Volume 9) Functional Studies Reporter assays - http://sabiosciences.com/cellassay.php siRNA/shRNA - http://sabiosciences.com/RNAi.php Protein Expression Cytokine levels - http://sabiosciences.com/ELISA.php ̣ ̣ ̣ ̣ http://sabiosciences.com/home.php http://sabiosciences.com/seminarlist.php Title, Location, Date 43
  44. 44. Sample & Assay Technologies Keep up to date: Follow Pathway focused biology on Facebook Latest information on pathway and disease research, resources and demos.
  45. 45. Sample & Assay Technologies Thank you for attending Would you like to try a TLR & Inflammation-related PCR Array? PCR Array Starter Pack Promotion PCR Arrays of any Pathway (FREE) • 2 96-well/100-well (2 samples) 2 Profiler (mRNA): FDK-PAFAS22 oRT omiScript (miRNA): FDK-MIFAS22 Required Reagents (w/ Purchase) • RT2 First-Strand cDNA Synthesis Kit, OR • RT2 SYBR Green Mastermix (2-Pack) Call 1-888-503-3187 to order Email: support@SABiosciences.com US and Canada only Webinar-related question? QIAWEBINARS@QIAGEN.COM Title, Location, Date 45

×