The Enhanced activation of innate immunity in Drosophila S2 cells by Micrococcus luteus VKM Ac-2230 is mediated by Relish

The canonical model of immune response activation in Drosophila suggests that the IMD pathway is activated by Gram-negative (Gram (−)) bacteria, while the Toll pathway is activated by both Gram-positive bacteria (Gram (+)) and fungi. However, the mechanisms by which these pathogens promote cross-activation of these pathways remain controversial. In addition, the mechanisms of cross-activation in S2 cell culture remain unstudied. In this study, we investigated the role of two Gram (+) bacteria (Micrococcus luteus and Bacillus subtilis) and fungal spores (Metarhizium anisopliae) in activating the IMD pathway in S2 cell cultures. Cells were treated with Escherichia coli as a control to ensure the specificity of IMD pathway activation. Our results demonstrated a significant involvement of M. luteus in the activation of the IMD pathway in S2 cell cultures. This is evidenced by the marked activation of IMD pathway-dependent genes, as well as the proteolytic cleavage of the Relish protein, which serves as a key transcription factor for this pathway. We also observed a strong recruitment of Relish to the promoters of antimicrobial peptide (AMP) genes, along with a partial recruitment to the genes encoding peptidoglycan recognition proteins (PGRPs). Furthermore, RNA interference targeting Relish resulted in a significant reduction in the transcription levels of all AMP genes and most PGRPs. Similarly, we analyzed the contributions of B. subtilis and M. anisopliae to the cross-activation of the IMD pathway. Our data indicate that both B. subtilis and M. anisopliae also activate the IMD pathway, albeit to a lesser extent compared to M. luteus. At the same time, fungal spores exhibited minimal influence on the activation of the IMD pathway when compared to Gram (+) bacteria. Thus, we have investigated in detail the mechanisms of cross-activation of the immune response in S2 cell culture, suggesting that Relish may play a critical role in inducing a humoral immune response in Drosophila S2 cells, primarily against M. luteus and to a lesser extent against B. subtilis and M. anisopliae.