Avian Pathogenic Escherichia coli (APEC) Strain-Dependent Immunomodulation of Respiratory Granulocytes and Mononuclear Phagocytes in CSF1R-Reporter Transgenic Chickens.

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 APECposCSF1R-tgneg and CSF1R-tglow cells as heterophils and the APECposCSF1R-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.

Dose-dependent differential resistance of inbred chicken lines to avian pathogenic Escherichia coli challenge.

Avian pathogenic E. coli (APEC) cause severe respiratory and systemic disease. To address the genetic and immunological basis of resistance, inbred chicken lines were used to establish a model of differential resistance to APEC, using strain O1 of serotype O1:K1:H7. Inbred lines 72, 15I and C.B12 and the outbred line Novogen Brown were inoculated via the airsac with a high dose (107 colony-forming units, CFU) or low dose (105 CFU) of APEC O1. Clinical signs, colibacillosis lesion score and bacterial colonization of tissues after high dose challenge were significantly higher in line 15I and C.B12 birds. The majority of the 15I and C.B12 birds succumbed to the infection by 14 h post-infection, whilst none of the line 72 and the Novogen Brown birds developed clinical signs. No difference was observed after low dose challenge. In a repeat study, inbred lines 72 and 15I were inoculated with low, intermediate or high doses of APEC O1 ranging from 105 to 107 CFU. The colonization of lung was highest in line 15I after high dose challenge and birds developed clinical signs; however, colonization of blood and spleen, clinical signs and lesion score were not different between lines. No difference was observed after intermediate or low dose challenge. Ex vivo, the phagocytic and bactericidal activity of lung leukocytes from line 72 and 15I birds did not differ. Our data suggest that although differential resistance of inbred lines 72, 15I and C.B12 to APEC O1 challenge is apparent, it is dependent on the infectious dose. Research Highlights Lines 15I and C.B12 are more susceptible than line 72 to a high dose of APEC O1. Differential resistance is dose-dependent in lines 15I and 72. Phagocytic and bactericidal activity is similar and dose independent.

Visualisation and characterisation of mononuclear phagocytes in the chicken respiratory tract using CSF1R-transgenic chickens.

The respiratory tract is a key organ for many avian pathogens as well as a major route for vaccination in the poultry industry. To improve immune responses after vaccination of chickens through increased uptake of vaccines and targeting to antigen presenting cells, a better understanding of the avian respiratory immune system is required. Transgenic MacReporter birds were used expressing a reporter gene (eGFP or mApple) under the control of the CSF1R promoter and enhancer in cells of the mononuclear phagocyte (MNP) lineage to visualize the ontogeny of the lymphoid tissue, macrophages and dendritic cells, in the trachea, lung and air sac of birds from embryonic day 18-63 weeks of age. Small aggregates of CSF1R-transgene+ cells start to form at the openings of the secondary bronchi at 1 week of age, indicative of the early development of the organised bronchus-associated lymphoid tissue. Immunohistochemical staining revealed subpopulations of MNPs in the lung, based on expression of CSF1R-transgene, CD11, TIM4, LAMP1, and MHC II. Specialised epithelial cells or M cells covering the bronchus-associated lymphoid tissue expressed CSF1R-transgene and type II pneumocytes expressed LAMP1 suggesting that these epithelial cells are phagocytic and transcytose antigen. Highly organised lymphoid tissue was seen in trachea from 4 weeks onwards. Throughout the air sacs at all ages, CSF1R-transgene+ cells were scattered and at later stages, CSF1R-transgene+ cells lined capillaries. These results will serve as a base for further functional characterization of macrophages and dendritic cells and their role in respiratory diseases and vaccine responses.

The role of type I interferons (IFNs) in the regulation of chicken macrophage inflammatory response to bacterial challenge.

Mammalian type I interferons (IFNα/ß) are known to modulate inflammatory processes in addition to their antiviral properties. Indeed, virus-induced type I interferons regulate the mammalian phagocyte immune response to bacteria during superinfections. However, it remains unresolved whether type I IFNs similarly impact the chicken macrophage immune response. We first evidenced that IFNα and IFNß act differently in terms of gene expression stimulation and activation of intracellular signaling pathways in chicken macrophages. Next, we showed that priming of chicken macrophages with IFNα increased bacteria uptake, boosted bacterial-induced ROS/NO production and led to an increased transcriptional expression or production of NOS2/NO, IL1B/IL-1ß and notably IFNB/IFNß. Neutralization of IFNß during bacterial challenge limited IFNα-induced augmentation of the pro-inflammatory response. In conclusion, we demonstrated that type I IFNs differently regulate chicken macrophage functions and drive a pro-inflammatory response to bacterial challenge. These findings shed light on the diverse functions of type I IFNs in chicken macrophages.

The role of macrophages in healing the wounded lung.

Acute tissue injury is often considered in the context of a wound. The host response to wounding is an orchestrated series of events, the fundamentals of which are preserved across all multicellular organisms. In the human lung, there are a myriad of causes of injury, but only a limited number of consequences: complete resolution, persistent and/or overwhelming inflammation, a combination of resolution/remodelling with fibrosis or progressive fibrosis. In all cases where complete resolution does not occur, there is the potential for significant ongoing morbidity and ultimately death through respiratory failure. In this review, we consider the elements of injury, resolution and repair as they occur in the lung. We specifically focus on the role of the macrophage, long considered to have a pivotal role in regulating the host response to injury and tissue repair.