Abstract
Avian pathogenic Escherichia coli (APEC) cause severe respiratory and systemic disease in chickens, commonly termed colibacillosis. Early immune responses after initial infection are highly important for the outcome of the infection. In this study, the early interactions between GFP-expressing APEC strains of serotypes O1:K1:H7 and O2:K1:H5 and phagocytic cells in the lung of CSF1R-reporter transgenic chickens were investigated. CSF1R-reporter transgenic chickens express fluorescent protein under the control of elements of the CSF1R promoter and enhancer, such that cells of the myeloid lineage can be visualized in situ and sorted. Chickens were separately inoculated with APEC strains expressing GFP and culled 6 h post-infection. Flow cytometric analysis was performed to phenotype and sort the cells that harbored bacteria in the lung, and the response of the sorted cells was defined by transcriptomic analysis. Both APEC strains were mainly detected in CSF1R-transgeneneg (CSF1R-tgneg) and CSF1R-tglow MHC IIneg MRC1L-Bneg cells and low numbers of APEC were detected in CSF1R-tghigh MHC IIpos MRC1L-Bpos cells. Transcriptomic and flow cytometric analysis identified the APECpos CSF1R-tgneg and CSF1R-tglow cells as heterophils and the APECpos CSF1R-tghigh cells as macrophages and dendritic cells. Both APEC strains induced strong inflammatory responses, however in both CSF1R-tgneg/low and CSF1R-tghigh cells, many immune related pathways were repressed to a greater extent or less activated in birds inoculated with APEC O2-GFP compared to APEC O1-GFP inoculated birds. Comparison of the immune pathways revealed the aryl hydrocarbon receptor (AhR) pathway, IL17 and STAT3 signaling, heterophil recruitment pathways and the acute phase response, are modulated particularly post-APEC O2-GFP inoculation. In contrast to in vivo data, APEC O2-GFP was more invasive in CSF1R-tghigh cells in vitro than APEC O1-GFP and had higher survival rates for up to 6 h post-infection. Our data indicate significant differences in the responses induced by APEC strains of prevalent serotypes, with important implications for the design and interpretation of future studies. Moreover, we show that bacterial invasion and survival in phagocyte populations in vitro is not predictive of events in the chicken lung.
Highlights
APEC are the etiologic agent of colibacillosis in chickens, a complex of severe respiratory and systemic infections that constitute an important threat to all sectors of the poultry industry
We aimed to study the first line of cellular defense against APEC in the respiratory tract, a key porte d’entrée for pathogenic E. coli in poultry
The inocula were prepared by collection of bacteria from fresh cultures by centrifugation and resuspension in sterile, apyrogenic phosphate-buffered saline (PBS) and the inoculation dose confirmed by retrospective plating of serial dilutions onto MacConkey agar (MCA) plates
Summary
APEC are the etiologic agent of colibacillosis in chickens, a complex of severe respiratory and systemic infections that constitute an important threat to all sectors of the poultry industry. The mechanisms underlying mucosal colonization and systemic translocation by APEC are ill-defined. Physical barriers, such as mucociliary clearance and host defense peptides may limit APEC from establishing an infection in the respiratory tract [3, 4] and the early innate immune responses, including the response by heterophils and macrophages, are thought to be important in the control of colibacillosis [5,6,7,8,9]. In a recent study we showed that very early responses were instrumental to the fate of the birds, as chickens of a relatively susceptible inbred line succumbed to intra-air sac APEC O1:K1:H7 infection within 14 h post-inoculation whereas birds of a relatively resistant line survived [10]
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