Fig 5. The immune response is compromised by heme supplementation in vitro and in vivo.

(A) Schematic figure representing the regulation of immune pathway genes by heme incubation from microarray data. (B) Aag2 cells were incubated with vehicle (control—CTR) or 50 μM heme and then challenged with heat-killed M. luteus or E. cloacae. The bar charts show the defensin and peptidoglycan recognition protein gene expression levels relative to the ribosomal protein Rp49 and normalized by a non-challenged sample as measured by qPCR. (C) Gram-negative (E. cloacae) bacteria were inoculated into the cell culture medium in 24-well plates containing Aag2 cells pre-incubated with vehicle or 50 μM heme. The graph shows CFU numbers as a measure of bacterial growth after 3 h incubation at 28°C. (D) Evaluation of immune gene expression in orally infected (S. marcescens) mosquitoes fed with substitute blood meal (SBM) with or without 50 μM heme. (E) Culture-independent evaluation of midgut natural microbiota in unchallenged mosquitoes fed with SBM with or without 50 μM heme through qPCR for bacterial ribosomal 16S RNA normalized by a control sample (sugar-fed). At least 4 independent experiments are shown. Error bars indicate the standard error of the mean. ** = p<0.01; * = p<0.05 (by Student’s t-test).