Bovine PMN isolation
Blood was collected from cows that were venipunctured for diagnostic purposes at the Ruminant Clinic of the Vetsuisse Faculty, University of Bern (Switzerland). Donor cows were hospitalized for treatment of non-infectious conditions or localized inflammations/infections. Available clinical data on donor cows are given in Additional file 8: Table S1. Ethical approval for this study (number: BE33/17) was obtained from the Ethics Commission of the Canton of Bern.
PMN were isolated as described previously  with slight modifications. Briefly, blood was collected aseptically by jugular venipuncture into an equal volume of Alsever’s solution, distributed into conical 50 ml tubes and centrifuged at 1000×g for 20 min at 4 °C. The plasma, buffy coat and the upper third of the red cell pack were discarded and the red cell pack was resuspended in up to 35 ml ice-cold phosphate-buffered saline (PBS). The red cell pack suspension was gently overlaid on top of 10 ml of Ficoll–Paque PREMIUM 1.084 (GE Healthcare, Chicago, IL) and centrifuged at 400×g for 40 min at RT, after which the supernatant containing residual mononuclear cells was discarded. The red cell pack was resuspended in hypotonic lysis buffer (8.29 g/l NH4Cl, 1 gl NaHCO3, pH 7.4) to lyse residual erythrocytes and centrifuged at 800×g for 5 min at 4 °C. Then, the supernatant was discarded, and the pellet resuspended a second time in 10 ml of lysis buffer followed by centrifugation at 600×g for 5 min at 4 °C. The purified PMN pellet was washed twice in ice-cold PBS and centrifuged at 400×g for 5 min at 4 °C, after which PMN were resuspended in PBS for further use.
Staining and preparation of bovine PMN
Before use, PMN were stained with CellTracker Green CMFDA (CTG, 0.5 μM; Invitrogen) and LIVE/DEAD fixable Near-IR Dead Cell Stain (LD, 1:300; Invitrogen) in PBS for 30 min at 37 °C, 5% CO2. PMN were washed twice and resuspended in chemotaxis assay medium (CAM) containing Dulbecco's modified Eagle's medium (DMEM; low glucose, pyruvate, no glutamine, no phenol red; Thermo Fisher Scientific) adjusted to pH 6.8 and supplemented with 10% fetal calf serum (FCS; Bioswisstec, Schaffhausen, Switzerland) and 1% l-Glutamine 0.2 M (Merck Millipore, Darmstadt, Germany). PMN number and viability were assessed using a CASY Cell Counter (OLS OMNI Life Science, Bremen, Germany) before PMN concentration was adjusted for each assay.
Culture of primary cells and cell lines, and immunophenotypic characterization of primary cells
Primary bovine astrocytes were isolated from fresh cattle brains collected at local slaughterhouses adopting a previously described method  with slight modifications. Briefly, cerebral cortex was mechanically dissociated and enzymatically digested with collagenase Type I (final concentration 500 µg/ml; Sigma, C0130), after which cells were isolated through repeated bovine serum albumin (BSA, 25%; Sigma #A2153) density gradient separations and sequentially passed through 100, 70 and 30 µM nylon screens. Cells were seeded in flasks and grown to ~ 80% confluency, after which they were trypsinized, passaged 1:1 and allowed to adhere for 30 min. This selective attachment, in which slow-adhering cells were collected and discarded, allowed for enrichment of fast-adhering astrocytes, as previously reported . Astrocytes were seeded in poly-l-lysine-coated 24-well plates (Corning, Vitaris, Baar, Switzerland) in complete medium (DMEM-FCS) composed of DMEM supplemented with 10% FCS, 1% l-Glutamine 0.2 M and 0.5% gentamicin and grown to > 90% confluence. For immunophenotypic characterization, primary bovine astrocytes grown on poly-l-lysine-coated 12 mm glass coverslips were fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich) for 10 min at RT. Methods, primary and secondary antibodies used are indicated in Additional file 9: Table S2. Cell imaging was carried out using an Olympus Fluoview FV3000 confocal laser scanning microscope (Olympus, Tokyo, Japan), equipped with 405‐nm, 488‐nm and 561-nm laser channels. Obtained images were processed using the open-source software Fiji . The percentage of GFAP, vimentin, S100 and Iba-1 immunolabeled cells, respectively, was evaluated in two–three random fields of view (FOV) from 6 independent experiments (17 FOV in total) at 20× magnification.
Fetal bovine brain cells (FBBC-1)  were grown in flasks in a 1:1 mixture of DMEM and Ham’s F-12 medium (DMEM/F12, Life Technologies) supplemented with 10% FCS, 50 ng/ml epithelial growth factor, 50 ng/ml bFGF, 100 U/ml penicillin, 10 μg/ml streptomycin, and 0.5% N2 supplement (Life Technologies). After reaching confluence, FBBC-1 cells were trypsinized and reseeded into 24-well plates with DMEM supplemented with 10% FCS without penicillin/streptomycin, grown to confluence and then incubated with 100 μM forskolin (Merck‐Millipore, Schaffhausen, Switzerland) to induce neuronal differentiation 18 h prior to infection.
Primary bovine microglial cells were isolated by mechanical dissociation and Percoll (Cat. No. P1644, Sigma) gradient centrifugation, and were cultured as recently described . As assessed through recently described characterization methods , almost pure bovine microglial cultures were obtained.
Bacterial strains and cultures
Lm strain JF5203 (lineage I, clonal complex 1, sequence type 1, https://www.ncbi.nlm.nih.gov/nuccore/NZ_LT985474.1) isolated from a bovine rhombencephalitis case was used as the wild-type (WT-Lm) reference strain in our experiments. An isogenic deletion mutant for the hly gene (Δhly) generated for this study (Additional file 6: Methods S1) and the EGD-e strain (lineage II, clonal complex 9) were used to compare the effect of bacterial listeriolysin-O (LLO) deficiency and lineage-related differences on PMN chemotaxis, respectively.
Single bacterial colonies were inoculated in brain–heart infusion (BHI) broth and grown overnight at 37 °C on a shaking platform. The day of the experiment, bacteria were pelleted by centrifugation (2100×g, for 5 min) and washed once in PBS, after which they were diluted in PBS to an OD600 of 0.6 (equivalent to ~ 109 CFU/ml) then further diluted in CAM to a final concentration of 6 × 105 CFU/well. Bacterial concentration was retrospectively assessed for each experiment by CFU enumeration following overnight incubation on BHI-agar plates.
Where indicated, pelleted bacteria were opsonized in 10% pooled bovine serum in PBS for 30 min at 37 °C, then washed twice in PBS prior to being diluted in CAM. In selected experiments, where alive bacteria were used, the medium from the upper compartment was collected and plated on BHI-agar for CFU counting, to assess bacterial migration into the upper chamber. As a high number of resuspended Lm were observed to migrate into the upper compartment, thus possibly diluting the chemotactic gradient, we also resuspended bacteria in an agarose solution (final concentration 6.5 mg/ml, Sigma-Aldrich) to a dilution of 6 × 107 CFU/ml, of which 10 μl was plated on the bottom of a 24-well plate and, once solidified, further sealed with 10 μl of agarose solution. CAM was added on top of the agarose drops at the final volume required for the assay, bringing the final concentration of bacteria per well to 6 × 105 CFU/well. To produce heat-killed Lm (HK-Lm), bacteria were incubated at 60 °C for 30 min and loss of viability was checked by lack of overnight growth after plating. Bacterial culture supernatants were obtained by inoculating Lm in CAM, after which bacteria were grown overnight at 37 °C on a rocking platform. The following day, the supernatant was separated from bacteria after centrifugation, filter-sterilized through a 0.22 μm pore size syringe filter (Millipore, Switzerland) and assessed for sterility by absence of CFU after plating. Culture supernatants were stored at − 20 °C and finally used for the experiments after being diluted 1:10 in fresh CAM.
Infection of endogenous brain cells and preparation of culture supernatants
To obtain culture supernatant from infected astrocytes, FBBC-1 and microglia, the day prior to the assay wells were washed 3× with sterile PBS and fresh CAM without antibiotics was added. The following day, cells were inoculated with WT-Lm at the equivalent of a multiplicity of infection (MOI) of 10 in FCS-free CAM, and plates were centrifuged at 300×g for 5 min to synchronize infection. Cells were incubated with bacteria for 2 h, the medium was discarded, and cells were washed 3× in PBS to remove extracellular bacteria. Fresh CAM with 0.01 mg/ml gentamicin was added to the wells to kill residual extracellular adhering bacteria and prevent Lm rapid replication inside the medium. Cell culture supernatant was collected following 4 h of infection, filter-sterilized and stored at -20 °C until the day of the experiments. As negative control, supernatants of mock-infected cells undergoing the same experimental procedures were used. In selected experiments, supernatants from infected microglia were pre-incubated for 1 h at 4 °C with a mouse monoclonal anti-sheep IL-8 antibody (final concentration 50 μg/ml, MCA1660, clone 8M6, Bio-Rad) before testing in the chemotaxis assay. To confirm a specific blocking effect of the anti-IL-8 antibody, the same procedures were also repeated on medium containing bovine recombinant IL-8 (Bovine IL-8, 50 ng/ml; Kingfisher Biotech, Inc. St. Paul, MN, USA), while bovine IL-8-containing medium incubated with Mouse IgG1 (MoIgG1, final concentration 50 µg/ml; MAB002, R&D systems) was used as negative control.
Transwell chemotaxis assay
PMN chemotaxis was assessed in 24-well Transwell plates with 5.0 µm size pore polycarbonate membrane inserts (# 3421, Corning). Membranes were coated with rat-tail collagen prepared according to the method of Bornstein , following a previously described procedure . The lower compartment of the Transwell chamber was filled with 600 μl of unstimulated CAM to assess random migration, or with CAM containing bacteria, chemotactic factors, or cell supernatants (Additional file 10: Table S3). Following preliminary tests and titrations, bovine IL-8 at 50 ng/ml was used as a positive control for bovine PMN chemotaxis. Following isolation, PMN were incubated for 30 min at 37 °C, 5% CO2 to allow them to recover. Then, 5 × 105 cells suspended in 100 µl CAM were added to the upper compartment and the assay was started by assembling the Transwell plate. Migration was allowed to occur for 1 h at 37 °C, 5% CO2, and migrated PMN were detached from the lower-side of the filter by incubating the plate 15 min at 4 °C following addition of 60 μl 0.5 M EDTA to the bottom chamber, as previously described . The plate was then gently shaken, the PMN-containing medium was collected from the lower compartment and migrated cells were quantified with an Attune NxT Flow Cytometer (Thermo Fisher Scientific, Switzerland). Data were analyzed using FlowJoTM software (Tree Star, Ashland, OR, USA). PMN purity was on average 90% according to FSC and SSC gating. PMN gating strategy was based on control stainings as illustrated in Additional file 1: Fig. S1. Briefly, PMN were gated according to the FSC and SSC localization of cells positive for the anti-bovine granulocyte monoclonal antibody CH138A (IgG1, Cat. No WS0608B-100, Kingfisher Biotech, Inc.), as illustrated in Additional file 2: Fig. S2. PMN staining procedures with CH138A are indicated in Additional file 7: Methods S2. After excluding debris and contaminants with the FSC vs SSC gating, doublets and aggregates were excluded on an FSC-A vs FSC-H plot. The number of migrated PMN per well was enumerated by gating on viable and CTG-positive cells.
As the number of intact PMN decreased during incubation from the initial 5 × 105 PMN in 100 µl (average: 3.7 × 105 PMN in 100 µl), we inoculated 100 μl of 5 × 105 PMN in 500 μl of CAM and incubated it for 1 h as control for the reduction of PMN number over the course of the incubation. The number of cells recovered from this sample corresponds to the pool of PMN inoculated into the upper well that would have been able to migrate into the lower well (termed “input”). To allow comparison between different experiments, the absolute number of PMN per well was normalized to an input of 5 × 105 PMN in each experiment and the number of migrated PMN was expressed as absolute cell number normalized to the input.
In preliminary experiments assessing the dose effect of bovine IL-8 on PMN chemotaxis (Additional file 4: Fig. S4), PMN collected from the lower well were stained with Trypan blue and viable PMN were enumerated with a hemocytometer. The number of viable PMN migrated in the lower well in experiments assessing the blocking effect of the anti-IL-8 antibody against bovine recombinant IL-8 (Fig. 4C) was assessed with a CASY counter.
ELISA for quantification of bovine IL-8 in microglia supernatant
Secretion of bovine IL-8 in the supernatant of both infected and non-infected microglia was assessed through a sandwich enzyme-linked immunosorbent assays (ELISA) similarly as previously reported  with slight modifications. Briefly, 96-well plates (Corning, Vitaris, Baar, Switzerland) were coated with 5 µg/ml of mouse anti-sheep IL-8 antibody (MCA1660, clone 8M6, Bio-Rad) and incubated overnight at 4 °C. Wells were then washed and incubated with supernatants from infected microglia, and non-infected microglia or bovine IL-8 (Kingfisher Biotech, Inc. St. Paul, MN, USA), which was applied at different concentrations to prepare the standard curve, for 3 h at RT. After washing, wells were incubated with a rabbit anti-sheep IL-8 antibody (AHP425, 1:500; Bio-Rad) for 4 h at RT, then plates were washed, and detection was carried out using the Thermo Scientific TMB QUICK Liquid Substrate for ELISA (10748352, Fisher Scientific). Reactions were stopped by addition of H2SO4 and plates were read at 450 nm using a Cytation 5 imaging multimode reader (BioTek). IL-8 concentration in microglial supernatant samples was calculated in the Gen5 v3.0 software (BioTek) based on the OD values and concentrations of the standard curve.
In situ immunohistochemistry and immunofluorescence of bovine listeriosis cases
To assess microglial IL-8 expression in acute lesions of naturally infected cattle, formalin-fixed paraffin-embedded (FFPE) sections of brains from listeriosis cases were processed for immunohistochemistry (IHC) and immunofluorescence (IF).
Following screening of archive neurolisteriosis cases for the presence of acute microabscesses, classified as such according to previously published histologic criteria , 9 neurolisteriosis cases were selected for immunohistochemical processing. Of such cases, 5 were further selected for IF analysis following the assessment of marked IL-8 positivity centered on microabscesses on IHC.
For both IHC and IF, dewaxing, antigen retrieval (citrate buffer, pH 6) and blocking with 5% normal goat serum (NGS) were performed as previously described .
For IHC, sections were incubated overnight at 4 °C in PBS-Tween (PBS-T) containing 10% NGS and mouse anti-sheep IL-8 antibody (1:1000, MCA1660, clone 8M6, Bio-Rad). Signal detection was carried out using the Mouse and Rabbit Specific HRP/DAB (ABC) Detection IHC kit (ab64264; Abcam plc, Cambridge, UK) according to manufacturer’s instructions. Slides were then counterstained with Mayer's hemalum solution (Merck KGaA, Darmstadt, Germany), mounted with Aquatex (Merck KGaA) and finally imaged with a Zeiss microscope. As negative control, neurolisteriosis tissue was subjected to the same protocol using unspecific mouse IgG instead of the specific IL-8 antibody. As positive control, neurolisteriosis tissue containing numerous PMN was used.
For IF, incubation with rabbit anti-P2RY12 (1:200, 55043A, AnaSpec), rabbit anti-Iba1 (1:500, 013-27593, Wako) and/or mouse Factor XIII A (F13A1, 1:100, MA5-11751, Thermo Fisher) were performed as previously described . In addition, sections were incubated with mouse anti-sheep IL-8 antibody (1:400, MCA1660, clone 8M6, Bio-Rad) overnight at 4 °C, following antigen retrieval in Target Retrieval solution, pH 9 (S2367, Agilent Technologies) for 15 min at 95 °C in a H2850 Microwave Processor (EBSciences, East Granby, Hartford County, Connecticut). Labeling was visualized by 1 h incubation at RT with secondary antibodies coupled to Alexa Fluor 555 or Alexa-Fluor 647 (Invitrogen), diluted at either 1:400 (P2RY12, Iba1 and F13A1) or 1:100 (IL-8). Moreover, staining for Lm was performed using a rabbit anti-Listeria polyclonal antibody (1:200, Difco Laboratories, Detroit MI, USA) pre-labeled with the Zenon Alexa Fluor 488 Rabbit IgG Labeling Kit (Z23502, Invitrogen) according to the manufacturer’s instructions. As negative control, neurolisteriosis tissue was subjected to the same protocol using unspecific mouse IgG instead of the specific IL-8 antibody. As positive control, neurolisteriosis tissue containing numerous PMN was used.
All tissue slides were additionally stained with DAPI (1:1000, Invitrogen). Finally, slides were mounted with coverslips using glycergel (Agilent) and Z-stack images were captured using an Olympus Fluoview FV3000 confocal laser scanning microscope (Olympus, Tokyo, Japan). Images were processed with the software Fiji .
PMN chemotaxis was evaluated in a minimum of three independent experiments performed in triplicates and conducted with PMN from a minimum of three different individual donor cows for each condition tested (with the exception of IL-8 blocking and IL-8 dose effect assays which were conducted in duplicates or triplicates with PMN from one to three donor cows). PMN chemotaxis rates displayed variability depending on the donor animal. Therefore, data from chemotaxis experiments are represented as superimposed scatter dot plots highlighting donor cows, from which PMN were extracted. Statistical analysis was carried out using GraphPad Prism v.9 (GraphPad Software, CA, USA). Nonparametric Mann–Whitney U tests were performed in each assay by comparing PMN chemotaxis in tested conditions with random migration towards medium alone. Mann–Whitney U tests were also conducted to compare CFU collected from the upper well. Differences were considered statistically significant at P < 0.05 and P values are indicated in the figures by asterisks: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Non-significant differences are indicated in the figures as “ns”.