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18-Wheeler in Innate Immunity
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  1. 18-Wheelerin Innate Immunity By Joe Peraza California State University, Long Beach

  2. Introduction • Immunity • Evolution • Similarities among vertebrates, invertebrates, and plants • Types of Immunity • Acquired • Specifically driven clonal selection of antibodies and T-cell receptors • Memory-ability to respond rapidly on recurring exposure to antigen

  3. Types of immunity • Innate • Evolutionary memory of the species • Essential role in clonal response regulation • Consists of cellular and humoral response • Insect innate immunity • Cellular response • Not thoroughly studied • Shown to have three classes of hemocytes • Plasmatocytes • Lamellocytes • Crystal cells

  4. Plasmatocytes: • Major cell type in larvae hemolymph • Phagocytosis by macrophage like cells • Secretes antimicrobial peptides into hemolymph • Stimulates fat body, primary source of antimicrobial peptide synthesis • Lamellocytes: • Encapsulates pathogenic organisms such as bacteria and fungi • Crystal cells: • Melanizes the encapsulated pathogens and cuticle wounds

  5. Humoral response • Synthesizes antimicrobial peptides secreted by fat bodies in response to infection • Regulated at the level of mRNA transcription • Includes the Rel/NF-ĸB cascade • Rel/NF-ĸB cascade • Responsible for dorsal/ventral patterning in Drosophilia • Analogous to human immunity activation pathway • Contains three Rel domain proteins: Dorsal,Dif, and Relish • Toll predicted to be receptor involved in mediating cascade

  6. Humoral antimicrobial defense Recognition of pathogenic organism Serine protease cascades Extracellular signaling events Humoral immune response reception Signal transduction Gene expression Antimicrobial peptides

  7. Toll receptor • Contains multiple copies of leucine-rich repeats • Flanked by cysteine-rich regions • Cytoplasmic domain shows similarity to mammalian interleukin-1 type I receptor (IL-1RI) • 18-Wheeler • IL-1R-related • Related to Toll • Expressed in the larvae fat body

  8. Purpose of Experiment • Determine location of expression of the 18W protein upon infection • Show that infection stimulates the expression of antimicrobial peptides in response to 18W signaling • Display 18W functions primarily as a receptor

  9. Materials and Methods Immunohistochemistry Formation of 18W antibodies Third star larvae fat body dissection Fixed fat body in formaldehyde Exposed to primary antibodies Exposed to secondary goat antirabbit antibodies Viewed under fluorescence microscope Used to determine the distribution of 18W protein through staining

  10. Materials and Methods Western blot analysis of 18W Homogenization of OregonR wandering third instar larvae Supernatant was separated from pellet Pellet solubilized in sample buffer SDS page protein gel Transferred to PVDF western membranes Exposed to antibodies and then X-ray film

  11. Results and Conclusions • Immunohistochemistry • Expression of 18W proteins was observed to be localized in the fat body after infection • Abundance of stain on plasma membrane consistent with the dynamics of 18W expression • Stain observed in cytoplasm vesicles suggesting receptor role • Expression was noted in lymph gland and garland cells • A role in cell adhesion cannot be excluded due to LRR domains

  12. Drosophila Fat Bodies

  13. Results and Conclusion • Western blot analysis • Presence of bands at 220 kDa over time intervals displays 18W is induced and antibodies are effective • Intensity of bands decrease from pre-infection to 4 hours and and returns to pre-infection intensity at 6 hours, implicating 18W receptor turnover is active

  14. Drosophila Innate Immune Response 220 76 43 33

  15. Future Experiments • Immunoflourescence confocal microscopy to determine location in relation to neighboring intracellular structures • Gain/loss function mutation to determine antimicrobial peptide expression • Coimmunoprecipitation experiments to detect in vivo what proteins interacts within signaling pathway

  16. Acknowledgments • Dr. Eldon for her patience and help • Howard Hughes Medical Institute • Dr. Merryfield for organizing this program

  17. References • Engstrom,Ylva. Induction and Regulation of Antimicrobial Peptides in Drosophila. Developmental and Comparative Immunology1999; 23:345-358 • Dushay, Mitchell., Eldon, Elizabeth. Insights From Model Systems: Drosophilia Immune Responses as Models for Human Immunity 1998; 62:10-14 • Hoffman, Jules., Reichhart, Jean-Marc. Drosophilia innate immunity: an evolutionary perspective 2002; 3:121-125