Colony forming units (CFU) where counted for several
dilutions for each condition. The Aa23T cells at 3h post-E coli addition had cleared 99% of bacteria from the culture medium in comparison with only 14% of bacteria cleared in 4a3A cell culture when compared to the same amount of bacteria incubated in cell-free (CF) medium. Figure 3 4a3A and Aa23T immune response to bacterial challenges. (A) qRT-PCR analysis at 3, 6, 9 and 24h after cell challenge with a mixture of heat-killed E. coli and E. faecalis show both early and late phase induction of DEF and TEP in both mosquito species. The time of early phase induction varies between species. Upregulation levels for each gene are similar between the two cell lines. Relative expressions were calculated to PBS-challenged cells and represent the average of 3 biological repeats +/- SE (B) 99% of E. coli is rapidly cleared by Aa23T cell line Nepicastat solubility dmso at 3h post-infection while for 4a3A only about 14% have been killed when compared
to the same amount of bacteria incubated in cell-free (CF) medium. The starting amount used in each case was 25µl per well of culture with an OD600 reading of 0.05, which represents approximately 15-18M CFU/ml. I -Set I; II -Set II. Discussion Obtaining a better understanding of Wolbachia-host immune interactions in insects is particularly JPH203 manufacturer important at the current time given the recently described effects of Wolbachia in inhibiting the development or dissemination of several very important mosquito-borne human pathogens. This study shows
that, as previously observed using VRT752271 clinical trial mammalian cells, the Wolbachia WSP protein is a potent innate immune elicitor in insects. The responses between the two mosquito cell lines to WSP challenge are mechanistically similar: 1) they are dosage dependent, increasing with increasing amounts of WSP up to 5μg/ml; 2) peak induction is seen at 5μg/ml, while higher concentrations sometimes reduce the mRNA levels; and 3) the immune gene transcription was at a maximum at 3h post challenge (early phase induction) and do not show late phase induction. The major difference is the level of upregulation between the two species: Methamphetamine detected peak induction of 3 to 5-fold in the naturally Wolbachia-uninfected cell line compared to just 2-fold induction in the naturally infected one. Tolerance effects due to previous natural Wolbachia exposure have been described  and seem likely to be contributing to the differences observed between these cell lines in their response to WSP. The control experiments also show that Aa23T can show strong induction of immune gene transcription and can effectively clear a bacterial infection. Thus the differences seen between WSP-associated immune induction between these cell lines are not due to impaired immune responses in Aa23T.