PFAS-exposed zebrafish embryos show impaired innate immune response after infection with mycobacteria

The Per- and polyfluoroalkyl substances PFNA, PFOA, PFOS and PFHxS have been regulated in the EU based on their interference with immune system functions. For many more environmental pollutants possible effects on immune function are unknown, so quick screening methods are required to determine which compounds are of highest immunotoxic concern. The aim of the present study was to optimize a test protocol to assess effects of chemicals on the innate immune system using zebrafish (Danio rerio) embryos. In a second step, the protocol was used to determine the immunotoxic effects of 11 PFAS on the innate immune response to pathogen infection. In the final protocol, zebrafish embryos were manually dechorionated 24 h after fertilization and subsequently infected with a transgenic strain of Mycobacterium marinum expressing the fluorescent protein mCherry. To avoid interference by systemic toxic effects that are not specific to the immune system, embryos were exposed to test compound concentrations that caused no developmental effects. Exposure was started immediately after infection. At 120 h after fertilization, the bacterial load, i.e. the integrated fluorescence intensity of bacteria in embryos exposed to the PFAS was compared to the integrated fluorescence intensity in infected vehicle-treated (control) embryos. In our optimized immunotoxicity assay, exposure to PFHxS and PFOA resulted in increased bacterial loads compared to vehicle treated embryos, indicating an increase in infection severity. Thus, the present study demonstrates that this zebrafish embryo immunotoxicity assay is useful to detect suppression of the innate immune system after chemical exposure.