Commercial Hy-Line Brown and CSF1R-eGFP or CSF1R-mApple transgenic birds  were hatched and reared in floor pens at the National Avian Research Facility, The Roslin Institute, Edinburgh (UK). Animals were reared under conventional conditions but were not vaccinated. The chickens were housed in groups and received food and water ad libitum. Animals were housed in premises licensed under a UK Home Office Establishment License (PEL 60/4604) in full compliance with the requirements of the Animals (Scientific Procedures) Act 1986. Breeding of transgenic chickens was carried out under the authority of Project License PPL 70/8940 and application of substances was conducted under PPL 70/7860 with the consent of The Roslin Institute Animal Welfare and Ethical Review Board. White Leghorn chickens from the PA12 outbred line were raised in closed breeding since 1968 (Aycardi and Schellenberg, 1970). PA12 birds were hatched and reared under SPF conditions at INRAE (Plate-Forme d’Infectiologie Expérimentale, PFIE, Nouzilly, France) in full compliance with the requirements of the French regional ethics committee number 19 (Comité d’Ethique en Expérimentation Animale Val de Loire). Food and water were provided ad libitum.
Eight-week-old Hy-Line Brown chickens and CSF1R-eGFP or CSF1R-mApple transgenic chickens, and 4-week-old SPF PA12 were culled by cervical dislocation and death confirmed by decapitation. The chicken carcasses were inverted, held in position with a clamp stand and the trachea located with minimal dissection of the area. The trachea was intubated, tied off to create a seal and the lungs inflated with molten, pre-warmed (41 °C) 30–40 mL of 2% ultra-low gelling point agarose (Sigma Aldrich, Irvine, UK). The whole carcass was chilled at 4 °C for 1–2 h to allow the agarose to set prior to isolating the lungs. The lungs were dissected and biopsies generated using an 8 mm disposable biopsy punch (Integra Life science services, Saint-Priest, France). The 8 mm biopsy of lung tissue was attached to the piston of a Compresstome™ VF-300 OZ (Precisionary, Natick, MA, USA) vibrating microtome with superglue and embedded in 6% ultra-low gelling point agarose, following which 500 µm slices were cut. Alternatively, the 8 mm biopsies of the inflated SPF PA12 lungs were cut into 200–300 µm slices using a Krumdieck MD6000 Tissue Slicer (Alabama Specialty Products, Inc., Munford, AL, USA). All PCLS were collected in chilled Hanks’ Balanced Salt solution (HBSS) (Life Technologies, Paisley, UK) before being transferred into a 24 well plate, washed 3 times in appropriate media and cultured in 500 µL. Three different media were tested to optimise culture conditions: (1) high glucose Dulbecco’s Modified Eagles Media (DMEM) supplemented with 100 U/mL penicillin 100 µg/mL streptomycin, 4 mM l-glutamine, 20 µg/mL gentamycin, and 1.25 µg/mL Amphotericin B (Thermo Fisher, Paisley, UK), (2) DMEM F-12 Nutrient mixture (Ham; DMEM/F12) supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 4 mM l-glutamine and (3) DMEM/F12 supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, 4 mM l-glutamine and 5% heat inactivated FCS (DMEM/F12/FCS). Media and supplements were all purchased from Thermo Fisher Scientific (Paisley, UK) unless otherwise stated. The PCLS were incubated at 41 °C and 5% CO2 for 1 h per wash. The PCLS were then cultured at 41 °C and 5% CO2 in the different media, hereafter referred to as DMEM, DMEM/F12 and DMEM/F12/FCS, replacing the media every 24 h, with the day of slice preparation defined as day 0 post slice.
AlamarBlue cell viability assays (Thermo Fisher Scientific, Paisley, UK) were carried out as per manufacturer’s instructions. Briefly the supernatant was removed from the PCLS and replaced with 450 μL of either DMEM, DMEM/F12 or DMEM/F12/FCS and 50 μL of AlamarBlue added per well of the 24 well plate, before incubating for 1 h at 41 °C. The supernatant was then harvested and the fluorescence assessed by excitation at 530 nm and the emission read at 590 nm using a Synergy HT plate reader (Biotek, Winooski, VT, USA). The supernatant from the PCLS was replaced with fresh media and the PCLS maintained in culture at 41 °C and 5% CO2. The viability assessment was repeated up to 44 days of culture.
Live/Dead cell imaging
Fresh, unfixed, PCLS from PA12 chickens were prepared as described above, kept in culture for 1, 2, 3, 6 or 7 days in DMEM or DMEM/F12/FCS and stained with the LIVE/DEAD® Cell Imaging Kit (488/570; Thermo Fisher Scientific, Paisley, UK) as per manufacturer’s instructions. Briefly, PCLS cultured in 24 wells plates were washed and the LIVE/Dead reagent added to an equal volume of cell culture media for 2 h. Next, PCLS were directly imaged in the plates under an Axiovert 200 M inverted epifluorescence microscope (Zeiss, Munich, Germany) at 200× magnification. Images were captured with an Axiocam MRm camera (Zeiss). For the quantification of dead cells within the PCLS at each time-point, images were acquired using the AxioVision SE64 software (Zeiss). Next, red fluorescent cells (dead cells) were counted in representative images acquired from 3 to 4 randomly selected PCLS. The numbers of dead cells were assessed using the Fiji-ImageJ image processing software, which permits practicable cell counting .
Luciferase-based cytokine reporter assays
Type I interferons (IFNs) and IL-1β production by PCLS were measured in the supernatants using luciferase-based Mx- or NFκB-reporter bioassays, respectively [23, 24]. The CEC32-Mx-Luc and the CEC32-NFκB-Luc reporter cell lines are quail fibroblast cell lines carrying the luciferase gene under the control of the chicken Mx promoter  or carrying an NFκB-activated luciferase reporter gene  respectively. CEC32-Mx-Luc and the CEC32-NFκB-Luc were kindly provided by Prof. P. Stäheli (University of Freiburg, Germany). CEC32 luciferase reporter cells were cultured in DMEM GlutaMAX™-I supplemented with 8% heat-inactivated FCS, 2% heat-inactivated chicken serum, 4.5 mg/mL d-glucose, 100 U/mL penicillin, 100 μg/mL streptomycin and 50 μg/mL geneticin (all purchased from Thermo Fisher Scientific, Paisley, UK) and grown in 25-cm2 flasks (Corning Life Sciences, Bedford, MA, USA) at 41 °C and 5% CO2. To perform reporter assays, CEC32-Mx or CEC32-NFκB cells were seeded at 2.5 × 105 cells/well in 24 well plates and incubated overnight at 41 °C and 5% CO2. The next day, cells were incubated for 6 h with the diluted supernatants (1/5 of total volume in fresh media) from chicken PCLS supernatants. Medium was removed and cells were washed twice with Phosphate Buffered Saline (PBS). Cells were lysed using the cell culture lysis reagent according to the manufacturer’s instructions, and luciferase activity was measured using the Luciferase assay reagent and a GloMax-Multi Detection System (all purchased from Promega, Charbonnières-les-Bains, France). Data were expressed as IFN-I or IL-1β activity (fold increase as compared to control group, which received fresh cell culture media only).
Production of nitrite, a primary breakdown product of nitric oxide (NO), was assessed in the supernatant of the PCLS culture using the Griess Reagent Kit (Promega, Southampton, UK) as per manufacturer’s instructions. A nitrite standard curve was prepared by serially diluting a concentrated nitrite solution in DMEM. Supernatant from the PCLS was harvested after 24 h of incubation over the duration of the culture. As a positive control, supernatant was harvested from PCLS incubated for 24 h with 1 µg/mL LPS (E. coli 055:B5 Sigma Aldrich) on day 8 post slice. The supernatant was stored at −20 °C until use in the assay. Absorbance was then read at 550 nm using a Multiskan Ascent plate reader (Thermo Fisher Scientific, Paisley, UK). The limit of detection was 2.5 μM (125 pmol) nitrite.
Chicken IL-10 was detected by capture ELISA as described by Wu et al.  using anti-chicken IL-10 capture antibody, clone ROS-AV164, and biotinylated detection antibody, clone ROS-AV163. Plates were incubated with twofold serially diluted standards (recombinant chicken IL-10) or supernatant. The absorbance was read at 450 nm (650 nm as a reference). The standard was fitted by linear regression and final concentration measures determined using Graph Pad Prism 8. The limit of detection was 70 pg/mL IL-10.
RNA extraction and cDNA synthesis
RNA was extracted by homogenising the fresh 8 mm biopsy of lung tissue or cultured PCLS using a pestle and mortar in a small volume of liquid nitrogen, then adding 350 µL of RLT lysis buffer (Qiagen, Manchester, UK) supplemented with β2-mercaptoethanol (Sigma, UK). The homogenate in lysis buffer was transferred to a Qiashredder column and RNA extracted using the RNEasy kit (Qiagen) as per manufacturer’s instructions. The RNA quality and quantity was assessed using a Nanodrop spectrophotometer ND-1000 (Thermo Fisher Scientific, Paisley, UK) and cDNA was synthesized from 156 ng of RNA using Superscript III reverse transcriptase (Life Technologies, Paisley, UK) with a random oligonucleotide primer as previously described . cDNA was stored at −20 °C.
Quantitative PCR was performed using TaqMan Universal PCR Master Mix (Applied Biosystems, Thermo Fisher Scientific, Paisley, UK), EvaGreen dye (Biotium, Freemont, CA, USA) and the following custom oligonucleotide primers; iNOS forward 5′-CAGCGGAAGGAGACAAACAGAG, iNOS reverse 5′-AACTCTTCCAGGACCTCCAGG, IL-1β forward 5′-CAGCAGCCTCAGCGAAGAG, IL-1β reverse 5′-CTGTGGTGTGCTCAGAATCCA, 28S forward 5′-GGCGAAGCCAGAGGAAACT and 28S reverse 5′-GACGACCGATTTGCACGT C (Sigma Aldrich). Each reaction contained 2 μL of cDNA diluted 1:5 in RNase/DNase free water. Quantitative PCR was carried out using an Applied Biosystems 7500 Fast Real-Time PCR System with the following cycle profile: 2 min at 50 °C, 10 min at 95 °C, followed by 40 cycles with denaturing for 15 s at 95 °C, and annealing/elongation for 1 min at 60 °C. Melting curves were generated to confirm a single-PCR product for each reaction as previously described . All reactions were performed in duplicate.
To determine the structural integrity and phenotype of the cells present, PCLS were examined by immunofluorescent staining. PCLS generated from the PA12 chickens were fixed for 4 h in a 4% paraformaldehyde (PFA) solution in PBS at day 1 post slice. Fixed PCLS were washed in PBS and then stored in PBS containing 0.01% sodium azide at 4 °C. Next, the slices were washed in PBS followed by 30 min incubation at room temperature in a permeabilisation solution (0.25% Triton X-100 in PBS). PCLS were then washed in PBS and incubated for 30 min at room temperature with a blocking solution (PBS with 10% Bovine Serum Albumin, BSA, Sigma-Aldrich, Irvine, UK). For specific staining, PCLS were incubated for 3 h at room temperature with rhodamine phalloidin (F-actin probe conjugated to the red–orange fluorescent dye, tetramethylrhodamine—TRITC, Abcam, Cambridge, UK), rabbit anti-von Willebrand Factor (vWF) polyclonal antibody (A0082, Dako, Santa Clara, CA, USA,), mouse anti-chicken actin monoclonal antibody (JLA20, DSHB, Iowa, IA, USA,), mouse anti-β-tubulin monoclonal antibody (MA5-16308, Thermo Fisher Scientific, Paisley, UK), mouse anti-chicken monocytes/macrophages monoclonal antibody specific for the mannose receptor MRC1L-B (KUL01, BioRad, California, CA, USA) or mouse anti-chicken CD45 monoclonal antibody (UM16-6, BioRad, California, CA, USA), in order to identify endothelial cells, cytoskeleton, phagocytes and leukocytes, respectively. Corresponding isotype controls were used as recommended by the manufacturer. All antibodies were titrated and used at the optimal dilution. The PCLS were washed in PBS, followed by a 2 h incubation with goat anti-mouse IgG (H + L) Alexa Fluor 488 or goat anti-rabbit IgG (H + L) Alexa Fluor Plus 594 (both from Thermo Fisher Scientific, Paisley, UK) at room temperature. Following further washes in PBS, the PCLS were incubated for 10 min at room temperature in the presence of Hoechst 33342 dye (Thermo Fisher Scientific, Paisley, UK), then washed in PBS and mounted on glass slides using a Lab Vision™ PermaFluor™ Aqueous Mounting Medium (Thermo Fisher Scientific, Paisley, UK) prior to imaging by confocal microscopy.
Influenza A virus infected PCLS were either stained with non-structural protein 1 (NS1)-specific rabbit antiserum  and goat anti-rabbit IgG (H + L) Alexa Fluor 488 (Invitrogen, Paisley, UK) or with mouse anti-Influenza A Virus nucleoprotein (NP) (AA5H, Abcam) and goat anti-mouse Ig-FITC (Southern Biotech, Birmingham, AL, USA). Pre-immune rabbit serum was used as NS1 staining control. The slices were washed in PBS with 1% FCS and then incubated with Hoechst 33342 dye. The slices stained for NP were washed in PBS and imaged in a 35 mm µ-dish (Ibidi, Grafelfing, Germany) by confocal microscopy. The slices stained for NS1 were prepared and imaged as described for the other antibodies.
Imaging of PCLS
The PCLS isolated from CSF1R-eGFP or CSF1R-mApple transgenic birds were imaged floating in DMEM within a 35 mm µ-dish (Ibidi), weighed down with sterile 13 mm coverslips (Scientific Laboratory Supplies Ltd, Coatbridge, UK). Live images were captured using a Zeiss Live Cell Observer with the heated stage set to 37 °C and the imaging chamber set to 37 °C and 5% CO2. PCLS at 3 days post slice were incubated with either red fluorescent 1 μm latex beads (Thermo Fisher Scientific, Paisley, UK) or 5 × 109 CFU/mL APEC O1-eGFP.
Images of PCLS were also captured using a Zeiss Axio Zoom.v16 in the wells of a 24 well plate at 7X or 16X magnification. Confocal images were captured using a Leica TCS P8 confocal microscope at 200X or 630X magnification and the LAS X software (Leica, Wetzler, Germany) or a Zeiss LSM710 inverted confocal microscope and Zen software (Zeiss).
Avian pathogenic Escherichia coli growth
Avian pathogenic E. coli (APEC) strain O1 (serotype O1:K1:H7) expressing enhanced green fluorescent protein (APEC O1-eGFP) was obtained by transformation of APEC strain O1 with plasmid pFVP25.1 and cultured as described previously . The APEC strain O1 strain was kindly provided by Prof. L. Nolan, Iowa State University, USA . APEC O1-eGFP was grown for 20 h in Luria–Bertani broth supplemented with 100 μg/mL ampicillin (Sigma Aldrich) at 37 °C to reach stationary phase. Prior to use, eGFP expression by the bacteria was confirmed by streaking the bacteria on LB-agar plates, incubating overnight at 37 °C and examining the colonies under UV light.
Influenza A virus infection of PCLS
Low pathogenic avian influenza (LPAI) viruses A/chicken/Italy/1067/1999 (H7N1; ) or A/Mallard/Marquenterre/Z237/83 (H1N1) at 106 PFU were added to PCLS from Hy-Line-Brown (H7N1) or PA12 chickens (H1N1) for 1 or 3 h of incubation at 37 °C, respectively. The supernatant was then removed and replaced with DMEM for up to 48 h. The supernatant was harvested and stored at −80 °C and the slices fixed, stored, stained and imaged as described above.
Serial tenfold dilutions of supernatant from virus-infected PCLS were incubated with confluent MDCK cells for 1 h in a standard plaque assay  using an 1.2% Avicel/DMEM overlay supplemented with 0.14% BSA (Sigma Aldrich) and 1 µg/mL TPCK treated trypsin (Worthington Biochemical Corporation, Lakewood, NJ, USA). Briefly, the diluted supernatants were aspirated and the cells incubated for a further 48 h in DMEM at 37 °C and 5% CO2. The cells were fixed in 4% formalin for 20 min at room temperature, stained with 0.1% toluidine blue or crystal violet at room temperature for 20 min, then washed in water. The cells were left to air dry and plaques enumerated.
Where applicable, data are expressed as mean ± SD. Statistical analysis was performed by one way ANOVA with post hoc Tukey test, using Graph Pad Prism 8.0 software (GraphPad, San Diago, CA, USA). The number of slices examined is defined as “n”. Statistical analysis was only performed where 3 or more birds had been used to generate slices and significance was considered at p < 0.05.