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18-Wheelerin Innate Immunity
By
Joe Peraza
California State University, Long Beach
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
• 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
• 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
• 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
Humoral antimicrobial defenseRecognition of pathogenic
organism
Serine protease cascades
Extracellular signaling events
Humoral immune response reception
Signal transduction
Gene expression
Antimicrobial peptides
• 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
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
Materials and MethodsImmunohistochemistry
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
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
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
Drosophila Fat Bodies
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
Drosophila Innate Immune Response
Marker kDa
1 2 3 4 5 6 18W kDa
200
175
120
83
62
43
0h 2h 4h 6h
220
76
4333
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
Acknowledgments
• Dr. Eldon for her patience and help
• Howard Hughes Medical Institute
• Dr. Merryfield for organizing this program
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