Establishment of baseline cytology metrics in nestling American kestrels (Falco sparverius): Immunomodulatory effects of the flame retardant isopropylated triarylphosphate isomers

Abstract

Avian populations must mount effective immune responses upon exposure to environmental stressors such as avian influenza and xenobiotics. Although multiple immune assays have been tested and applied to various avian species, antibody-mediated immune responses in non-model avian species are not commonly reported due to the lack of commercially available species-specific antibodies. The objectives of the present study were to advance methods for studying wild bird immune responses and to apply these to the evaluation of cytological responses after exposure of American kestrels, Falco sparverius, to a commercial flame retardant mixture containing isopropylated triarylphosphate isomers (ITP). Hatchlings were gavaged daily with safflower oil or 1.5 ug/g bw/day of ITP suspended in safflower oil, then bled on days 9, 17, and 21. The ITP treatment group ( n = 18) and a subset of controls (Poly I:C treatment group; n = 10) were injected on days 9 and 15 with a synthetic analog of viral double-stranded RNA, polyinosinic:polycytidylic acid (Poly I:C), a toll-like receptor ligand and synthetic viral mimic, and responses compared to a sham injected control group (n = 8). The hypotheses tested whether kestrels showed immunological differences among treatment groups, genetic sex, and/or white blood cell (WBC) subpopulation type over time. A flow cytometry (FCM) gating strategy categorized heterophils (H), lymphocytes (L), and monocytes (M) and their proportions, and measured relative fluorescence in response to anti-chicken CD4 binding. Fluorescent cell surfaces and some granular/vacuolar inclusions were visualized by epifluorescence microscopy. A fourth subpopulation with higher levels of granularity than M but less than H became increasingly apparent with time and was gated along with the H subpopulation; its frequency of occurrence was lowest in the ITP group ( P = 0.0023). The percentages of cells differed among treatment groups, days, and sexes ( P = 0.0001). For both sexes, percentages of H and L were higher than M in control and Poly I:C. In the ITP group, L percentages were higher than H and M ( P = 0.0457), and H and L were higher than M on days 9 and 21 ( P = 0.0001). The ratios of H:L and H:WBC, indicators of robust immunity, were also higher on days 9 and 21 than on 17 ( P = 0.0079). For each sex, the highest levels of activity measured by FCM geometric means (GEO) of fluorescence (indicative of antibody binding) were observed on day 9 ( P = 0.0001 female, and P = 0.0011 male) in H over both L and M ( P < 0.0001 for each). In males, GEO of the Poly I:C group was higher than that of the ITP group ( P = 0.0374), with no difference observed among females over all days. By using a FCM algorithm for population comparisons of fluorescence to investigate binding within H, the T(x) scores indicated higher fluorescence in control and Poly I:C groups over ITP ( P = 0.0001). Unlike chickens, Gallus gallus , which express CD4 primarily on L, kestrels bound the commercial antibody primarily within the gated H subpopulation, suggesting an immunophenotypic difference between taxa, despite a ~60% identity of Falco CD4 amino acid sequences with chicken CD4. The emergent cell subset within the gated H presented dendritic-like cell (DLC) morphological and functional properties, apparently serving as an effector cell. This study adds interpretive context to ecological investigations of infection and of potential immunomodulation by emerging compounds, whereby the early innate responses are mediated by the various cell subsets serving as useful quantitative markers of immunological condition. Data showed that dietary exposure to ITP was immunosuppressive for male and female kestrels over the course of the experiment, reducing DLC frequency compared to the Poly I:C controls. Heterophils and DLC were important in facilitating innate immunological responses.