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