title: Validation of Aedes aegypti Aag-2 cells as a model for insect immune studies.
abstract: BACKGROUND
  The understanding of mosquito immune responses can provide valuable tools for development of novel mosquito control strategies. Aiming the study at insect innate immunity, continuous insect cell lines have been established and used as research tools due to the fact that they constitute more homogeneous, sensitive, and reproducible systems than the insects from which they originated. More recently, Aag-2, an Aedes aegypti cell lineage, began to be frequently used as a model for studies of mosquito immunity. Nevertheless, to our knowledge, no study has systematically characterized the responses of Aag-2 cell line against different kinds of pathogens and compared its response to those exhibited by whole mosquitoes. For this reason, in this study we characterized gene expression profiles of the Aag-2 cell line in response to different kinds of immune challenges, such as Gram negative and positive bacteria, fungi and viruses, comparing the obtained results with the ones already described in the literature for whole mosquitoes.
  
  
  METHODS
  Aedes aegypti Aag-2 cells were exposed to different immune stimuli (gram-positive and gram negative heat inactivated bacteria, zymosan or Sindbis virus) for 24 hours and the expression of selected marker genes from toll, IMD and Jak/STAT pathways was analyzed by qPCR. Also, cells were incubated with fluorescent latex beads for evaluation of its phagocytosis capacity.
  
  
  RESULTS
  Aag-2 cells were stimulated with two concentrations of heat-killed Gram negative (Enterobacter cloacae) or Gram positive (Micrococcus luteus) bacteria, Zymosan or infected with Sindbis virus and the expression of key genes from the main immune related pathways, Toll, IMD and Jak/STAT, were investigated. Our results suggest that Toll and IMD pathways are activated in response to both Gram positive and negative bacteria and Zymosan in Aag-2 cells, displaying an immune profile similar to those described in the literature for whole mosquitoes. The same stimuli were also capable of activating Jak/STAT pathway in Aag-2 cells. Infection with Sindbis virus led to an up-regulation of the transcription factor STAT but was not able to induce the expression of any other gene from any of the pathways assayed. We also showed that this cell line is able to phagocytose latex beads in culture.
  
  
  CONCLUSIONS
  Our results characterize the expression profile of Aag-2 cells in response to different immune stimuli and demonstrate that this cell lineage is immune-competent and closely resembles the response described for whole Ae. aegypti mosquitoes. Hence, our findings support the use of Aag-2 as a tool to comprehend Ae. aegypti immune response both at cellular and humoral levels.
authors: Laboratório de Bioquímica de Artrópodes Hematófagos, Instituto de Bioquímica Médica, Programa de Biologia Molecular e Biotecnologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors: Ana Beatriz Ferreira
authors: AB
authors: Barletta
authors: Maria Clara L Nascimento
authors: MC
authors: Silva
authors: Marcos H Ferreira
authors: MH
authors: Sorgine
mesh_terms: D000330:Aedes / Q000166:cytology* / Q000276:immunology*; D000818:Animals; D002460:Cell Line; D016972:Enterobacter cloacae; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000276:immunology; D016982:Micrococcus luteus; D010587:Phagocytosis; D015203:Reproducibility of Results; D012845:Sindbis Virus; D015054:Zymosan
chemical_list: D015054:Zymosan