Tiam1^–/– (35), Rac-FRET (Rac-FRET^ki/ki) (44), Prex1^–/– Vav1^–/– (11) and Dock2^–/– (22) mouse strains were described previously. Rac-FRET mice were crossed to Dock2^–/–, Prex1^–/– Vav1^–/– and Tiam1^–/– mice to generate homozygous Rac-FRET Dock2^–/– , Rac-FRET Prex1^–/– Vav1^–/– and Rac-FRET Tiam1^–/– strains, respectively (25). RubyLifeact (Lifeact-mRFPruby, RLA) mice (45) were kindly provided by Dr Max Nobis (CRUK Cancer Research Institute, Glasgow, UK), and were bred as heterozygous RLA^tg/+ animals by crossing with C57Bl6. The Tiam1-RLA strain was generated by crossing Tiam1^–/– and RLA. All mice were on C57Bl6 genetic background. The Biological Support Unit operates as recently described (42). Mice were bred and group-housed (up to 5) under Specific Opportunistic Pathogen Free conditions, with unrestricted access to chow diet and water. They were used in experiments at young-adult age (8-14 weeks). Male mice were used for in vivo experiments, and mice of both sexes for in vitro experiments, with sex-and age-matching between genotypes. To infect mice with S. pneumoniae, they were group-housed in isocages within a biosafety level 2 containment facility. The breeding of animals and experiments were carried out with approval from the local Animal Welfare Ethical Review Body under the British Home Office Animal Scientific Procedures Act 1986.

Mature primary neutrophils were purified daily from mouse bone marrow using a Percoll^PLUS gradient at 4°C following our previously described method (13). Endotoxin-free reagents were used throughout. The marrow of mouse femurs, tibias, and pelvic bones was flushed out with ice-cold Hank’s Balanced Salt Solution without Ca²⁺ or Mg²⁺ (HBSS^–, Sigma H6648) supplemented with 15 mM HEPES, pH 7.4 (RT) (Sigma, H3784) and 0.25% fatty acid-free (FAF) BSA (Sigma, A8806) (HBSS^–++), triturated, and filtered through a 40 μm cell strainer. A 58% isotonic Percoll^PLUS solution (GE Healthcare, 17544501) in HBSS^–++ was added as an underlayer, and the samples were centrifuged at 1620 × g without brake for 30 min at 4°C. After centrifugation, the lower 3 ml were resuspended in 40 ml HBSS^–++ and centrifuged at 326 × g for 10 min at 4°C. Erythrocytes were lysed using Geye’s solution (130 mM NH₄Cl, 5 mM KCl, 780 µM Na₂HPO₄, 176 µM KH₂PO₄, 5.5 mM glucose, 1 mM MgCl₂, 280 µM MgSO₄, 1.54 mM CaCl₂, 13.4 mM NaHCO₃) for 3 min at RT. 10 volumes of ice-cold HBSS^–++ were added, and the cells were centrifuged again. The neutrophils were resuspended in ice-cold Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca²⁺ and Mg²⁺ (Sigma, D8662), 1g/l glucose (Sigma, G8769) and 4 mM NaHCO₃ (Sigma, S8761) (DPBS⁺⁺⁺⁺) and kept on ice. Aliquots were counted using a haemocytometer and their purity assessed by Kwik-Diff staining (Thermo Scientific Shandon, 9990700) of cytospins. The preparations typically had a purity over 90%. Neutrophils were centrifuged again and resuspended as appropriate for the subsequent assay.

Pulmonary infection with S. pneumoniae (TIGR4 serotype 4) was carried out essentially as described (13). S. pneumoniae of known CFU stored in PBS/20% glycerol at −80°C were thawed, washed twice in DPBS without Ca²⁺ and Mg²⁺ (DPBS^–, Invitrogen, 14190-094), and resuspended at 4 × 10⁷/ml in ice-cold DPBS^–. The suspension was kept on ice and used within 1 h. Mice were lightly anaesthetized with 3% isoflurane inhalation, infected intranasally (i.n.) with 50 µl S. pneumoniae (2 × 10⁶ CFU), or mock-treated with 50 µl DPBS^–, returned to their home cages, and euthanized by CO₂ inhalation 6 h later. The femoral artery was severed for confirmation of death, and in some experiments this blood was collected into EDTA-coated microvettes (Sarstedt, 20.1288) for analysis of peripheral neutrophils. Bronchoalveolar lavage (BAL) was performed by three slow injections and aspirations of 1 ml DPBS^– through a Venflon cannula (Becton Dickinson, 391452) in the trachea. The BAL fluid was stored on ice. Lungs were perfused with 10 ml DPBS^– through the right ventricle, removed using aseptic techniques, and stored on ice. The lungs were minced and homogenised in GentleMACS C tubes (Miltenyi Biotec, 130093237) containing Mouse Lung Dissociation kit enzymes (Miltenyi, 130095927) in DPBS^–, using a GentleMACS homogenizer (setting 2.1, lung) for 40 s, incubated at 37°C for 30 min, and a second round of homogenisation was performed. The perfused lung homogenate was washed with 10 ml ice-cold DPBS^– at 500 x g for 5 min at 4°C. BAL and perfused lung homogenate were serially diluted in ice-cold DPBS^–, plated onto LB agar containing 5% defibrinated sheep blood (Trafalgar Scientific, DSC025), and CFU were counted. Flow cytometry was performed using a BD Biosciences Fortessa flow cytometer with antibodies, standard beads, and gating as previously described (13). Data were analysed using FlowJo (Tree Star). Neutrophils were identified among single, live CD45⁺ leukocytes, by their characteristic CD11b^hi, Ly6G^hi staining. The volume of BAL fluid recovered was taken into account. To analyse neutrophils in the periphery, blood was collected as described here-above. Red blood cells were lysed in Geye’s solution for 5 min. Leukocytes were washed, incubated in DPBS⁺⁺⁺⁺, 1% Fc block (BD Biosciences, 553142) for 15 min on ice, washed, stained with BV421-Ly6G (clone 1A8, BioLeged, 127628, 1:500) and AF647-Cd11b (clone M1/70, BD Pharmingen, 557686, 1:1000) antibodies in DPBS⁺⁺⁺⁺, 1% Fc block for 30 min on ice, washed again, and analysed on a Cytek Aurora flow cytometer.

Histological analysis was done essentially as described (13). Mice were infected with S. pneumoniae, or mock-infected, and euthanized 6 h later, as described above. Lungs were inflated with 0.5 ml 10% neutral-buffered formalin (Sigma, HT501128), excised, and fixed in formalin. Samples were paraffin embedded, 5 μm sections were prepared, and H&E staining was done by Abbey Veterinary Services (Newton Abbot). Three sections per mouse were imaged on a Nikon Eclipse Ti-E Widefield microscope using the 20× objective and 3 × 3 image-stitching function, generating 2 images of 3 × 3 mm per lung. For image analysis, 6 grids of 40,000 μm² were placed within each image, comprising vascular, interstitial, and epithelial tissue where possible. Neutrophils within the grids were identified by their characteristic nuclear morphology and quantified according to their localisation within 10 μm of the vasculature, bronchiolar or alveolar epithelium, or within the interstitium, using Fiji (ImageJ) (46). 36 grids were quantified per mouse.

To analyse degranulation and NETs, we performed immunohistochemistry (IHC) analysis. Unstained histology slides were deparaffinised in xylene, rehydrated in ethanol, and washed in H₂O. For antigen retrieval, slides were boiled in 10 mM sodium citrate, pH 6.0, 0.05% Tween-20 for 10 min and allowed to cool to 25°C, and this was repeated with fresh buffer. Samples were washed 3 × 5 min in PBS, permeabilised in PBS, 0.5% Triton-X100 (Sigma, T9284) for 5 min, and washed again. Slides were blocked in 50 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.6 (TBS-T) containing 5% BSA for 1 h at RT. A hydrophobic ring was drawn around the tissue using a PAP pen (Abcam, ab2601), and samples were incubated with myeloperoxidase (MPO, R&D Systems, AF3667, 1:400) and citrullinated histone H3 (CitH3, Abcam, ab5103, 1:1000) antibodies in blocking buffer overnight at 4°C. Samples were washed 3 × 5 min in TBS-T, incubated with anti-goat-IgG-AF647 (ThermoFisher Scientific, A-21447, 1:500) for 30 min at RT, washed again, and incubated with anti-rabbit-IgG-AF488 (ThermoFisher Scientific, A-11034, 1:500) and Hoechst 33342 (ThermoFisher Scientific, 62249, 1:1000) for 30 min at RT. Slides were washed 3 × 5 min in TBS-T, then 2 × 5 min in H₂O, and mounted using ProLong Gold Antifade (ThermoFisher Scientific, P36934). Samples were imaged as here-above, except using the 4 × 4 stitching function. Image analysis was performed with QuPath, using tissue auto-fluorescence to calculate the total tissue area, and expressing the AF647-positive tissue area as %. In addition, MPO puncta were counted and their relative fluorescence intensities were quantified.

Peritonitis experiments were conducted as described (13). Mice were injected i.p. with 0.25 ml sterile 3% thioglycollate (TGC, Sigma, T9032) in H₂O, or mock-treated with H₂O, and returned to their home cages, with food and water ad libitum. 3 h later, the mice were euthanized using CO₂ asphyxiation, peritoneal lavages performed by two i.p. injections, and aspirations of 8 ml DPBS^–, 5 mM EDTA, and lavage samples pooled and stored on ice. Cells were sedimented at 450 × g for 10 min at 4°C and erythrocytes lysed in Geye’s solution for 3 min at RT. DPBS⁺⁺⁺⁺ was added, and leukocytes were pelleted and resuspended in DPBS⁺⁺⁺⁺. Part of each sample was stained with AF647-Cd11b (1:800) and FITC-Gr1 (BD Biosciences, 553126, 1:800) antibodies in DPBS⁺⁺⁺⁺, 1% Fc block for 20 min on ice in the dark, washed in DPBS⁺⁺⁺⁺, 5 mM EDTA and resuspended in 500 μl DPBS⁺⁺⁺⁺, 1 μg/ml DAPI, and 1.25 × 10⁵ Spherotech ACBP-50-10 beads/ml (5.0-5.9 μm). Flow cytometry was done on a BD Biosciences LSRII flow cytometer, and data were analysed in FlowJo. Neutrophils were identified by their Cd11b^hi, Gr1^hi staining. Leukocytes in the remaining sample were counted by haemocytometer and analysed by Kwik-Diff staining of cytospins as an alternative method. The lavage volume recovered was taken into account. Both methods gave similar results.

Where bone marrow transplantation was performed prior to inducing TGC peritonitis, 6-8 week old male C57Bl6 recipient mice were treated with 2 doses of 5 Gray by ¹³⁷Cs γ-irradiation, and their drinking water was supplemented with 2 mg/ml neomycin for two weeks. One day after irradiation, bone marrow cells were purified from 12-week-old male C57Bl6 or Tiam1^–/–donor mice using aseptic technique. Cells were flushed from bones with DPBS^–, containing 10% heat-inactivated foetal bovine serum (FBS), filtered through a 40 μm cell strainer, and adjusted to 5 × 10⁷ cells/ml. 100 μl were i.v. injected into an irradiated recipient mouse. After 10 weeks, to allow reconstitution of the haemopoietic system, TGC peritonitis experiments were performed as described above.

To measure ROS production between adherent neutrophils, a 96-well luminometer plate was coated with 0.75 µg/ml recombinant mouse ICAM1 (ICAM1/hFc, R&D Systems, 796-IC) in DPBS^– overnight at RT, washed 3 times in DPBS^–, blocked with 2% FAF-BSA in DPBS^– for 1 h at RT, and washed 3 × in DPBS^– prior to use. Isolated neutrophils at 5 × 10⁶/ml in DPBS⁺⁺⁺⁺ were primed with 100 ng/ml GM-CSF, 5 ng/ml murine TNFα for 45 min at 37°C. 75 μl cells were added per ICAM1-coated well for 40 min at 37°C, and non-adherent cells were removed by gentle washing with DPBS⁺⁺⁺⁺. Other primed neutrophils were kept in suspension for 40 min at 37°C, for direct comparison, and 75 μl were dispensed into uncoated wells of the luminometer plate just before the assay. 75 μl prewarmed detection buffer (DPBS⁺⁺⁺⁺, 16 units/ml horseradish peroxidase (HRP, Sigma, P8375), 120 µM luminol (Sigma, 123072)) was added, and samples were mixed gently and incubated for 3 min at 37°C. 100 μl of prewarmed 7.5 μM fMLP (2.5×, Sigma, F3506) in DPBS⁺⁺⁺⁺, or DPBS⁺⁺⁺⁺ control, were added by automatic injection port, and real-time ROS production was recorded at 37°C in a Berthold MicroLumat Plus luminometer (Berthold Technologies). ROS were quantified as the area under the curve (AUC) of the response over 2 min.

ROS production by neutrophils in suspension was measured as described (13). Neutrophils were resuspended and primed with GM-CSF and TNFα as here-above, or were mock-primed in DPBS⁺⁺⁺⁺. Unprimed neutrophils were maintained on ice and prewarmed to 37°C for 3 min before the assay. fMLP, C5a (Sigma, C5788) and PMA (Sigma, P1585) were prepared as 2.5× stocks in DPBS⁺⁺⁺⁺. Zymosan A (S. cerevisiae, Thermo Fisher, Z2849) were washed twice in DPBS⁺⁺⁺⁺, opsonised in DPBS⁺⁺⁺⁺, 50% mouse serum for 1 h at 37°C, or kept unopsonised, and resuspended at 1.125 × 10⁷/ml. Prewarmed detection buffer was added to the cells for 3 min at 37°C as above, before 150 μl were dispensed into a prewarmed, uncoated luminometer plate. 100 μl of prewarmed 2.5× stimuli, or DPBS⁺⁺⁺⁺, were added by automatic injection port (fMLP, C5a) or manually (PMA, zymosan), and ROS production was recorded in real-time. Final concentrations were 1.5 × 10⁶ neutrophils/ml with 3 μM fMLP, 25 nM C5a, 500 nM PMA, or 4.5 × 10⁶ zymosan A particles/ml. ROS was quantified as the AUC of the response over 2 min for fMLP or C5a, 10 min for PMA, and 90 min for zymosan.

To measure degranulation in adherent neutrophils, a 12-well plate (Nunc) was coated overnight with 3 µg/ml ICAM1, blocked in 2% FAF and endotoxin-free BSA, DPBS^– for 1 h, and washed 3 times in DPBS^– before use. An overnight culture of E. coli DH5α (New England Biolabs, 2527) was diluted 1:50 in LB and grown to log phase. Bacteria were counted by OD₆₀₀, sedimented, opsonised at 1 × 10⁹/ml in DPBS⁺⁺⁺⁺, 10% mouse serum for 15 min at 37°C, washed in ice-cold DPBS⁺⁺⁺⁺, resuspended in DPBS⁺⁺⁺⁺ at 1 × 10⁹/ml, kept on ice, and prewarmed for 3 min at 37°C before the experiment. Neutrophils at 8 × 10⁶/ml in DPBS⁺⁺⁺⁺ were allowed to adhere to the ICAM1-coated plate for 30 min at 37°C. In parallel, total lysate controls were prepared by sedimenting cells, resuspending in boiling 1.3× SDS sample buffer, boiling for 10 min, and snap-freezing in liquid nitrogen. After the 30 min incubation, non-adherent cells were carefully aspirated, and 250 µl DPBS⁺⁺⁺⁺ containing 10 µl (2.5 × 10⁷) opsonised E. coli were added to the 3 h timepoint sample. DPBS⁺⁺⁺⁺ was added to the other samples, and the plate was incubated for 3 h at 37°C. For other timepoints, the DPBS⁺⁺⁺⁺ was replaced with E. coli at 45 and 15 min prior to the end of the incubation, respectively. The supernatant was harvested and cleared by centrifugation for 1 min at 10,000 × g to remove any detached cells. Boiling 4× sample buffer was added to 1.3× final and the sample treated like the total lysates. Samples were western blotted with goat MPO antibody (R&D Systems, AF3667, 1:3000), and the blots were quantified by densitometry using Fiji.

Degranulation of gelatinase (Mmp9) by neutrophils in suspension was measured using in-gel zymography as previously described (13). Neutrophils at 5 × 10⁶/ml in DPBS⁺⁺⁺⁺ were left unprimed on ice or primed with 50 ng/ml GM-CSF, 20 ng/ml TNFα for 45 min at 37°C. A 96-well plate (Nunc) was blocked with 10% heat-inactivated FBS. 80 μl neutrophils were added to wells containing 20 μl 5× fMLP in DPBS⁺⁺⁺⁺, or DPBS⁺⁺⁺⁺alone, and samples were incubated for 30 min at 37°C, 5% CO₂, followed by centrifugation at 300 × g for 10 min at 4°C. Controls containing cytochalasin B were processed in parallel. The conditioned supernatant was recovered, and 40 μl were mixed with 20 μl 3× non-reducing SDS-PAGE sample buffer (160 mM Tris, pH 6.8, 8% SDS, 50% glycerol, bromophenol blue) at RT. Samples were run on an SDS-PAGE gel containing 0.067% gelatine B. Gels were equilibrated in 2.5% Triton X-100 for 30 min and incubated in 50 mM Tris, pH 7.5, 200 mM NaCl, 5 mM CaCl₂, 0.02% Triton X-100 overnight at RT, allowing the gelatinase to digest the gelatine. Gels were stained with coomassie, and gelatinase activity was analysed by densitometry using Fiji.

NETs formation in response to S. aureus (Wood 46) was measured as described (42), except that bacteria were opsonized with 50% mouse serum in Dulbecco’s Modified Eagle Medium with Ca²⁺, Mg²⁺ and 4.5 g/l glucose (Thermo Fisher Scientific, 31053), supplemented with 10 mM Hepes, pH 7.4 (DMEM⁺⁺⁺⁺), for 30 min at 37°C and resuspended at 2.5 × 10⁷ bacteria/ml in DMEM⁺⁺⁺⁺. 250 μl neutrophils at 4 × 10⁵ cells/ml in DMEM⁺⁺⁺⁺, 10% heat-inactivated FBS, were seeded per well of an 8-well μ-slide (ibidi, 80826) and allowed to adhere for 30 min at 37°C, 5% CO₂. Opsonised S. aureus was added at a ratio of 10 bacteria per neutrophil, or cells were mocked-treated with DMEM⁺⁺⁺⁺, for the indicated periods of time. For each timepoint, the non-cell permeable DNA dye Sytox Green (Thermo Fisher Scientific, S7020, 0.1 mM) and cell permeable Hoechst 33342 (1:500) were added 15 min before the end of the incubation, and samples were live-imaged using a Nikon Eclipse Ti-E widefield system. Images were analysed by Fiji, using phase contrast and Hoechst signals to determine total cell numbers and Sytox Green to enumerate NETs.

The ability of neutrophils in suspension to kill S. aureus was tested essentially as described (13), except that 1 × 10⁸ bacteria were opsonised in 200 μl DPBS⁺⁺⁺⁺, 50% mouse serum for 30 min at 37˚C, before 800 μl DPBS was added to give 1 × 10⁸ bacteria/ml. Neutrophils at 2.5 × 10⁷/ml in DPBS⁺⁺⁺⁺ were primed with 50 ng/ml GM-CSF, 20 ng/ml TNFα for 45 min at 37°C. Alternatively, neutrophils were heat-killed for 45 min at 55˚C, then kept on ice, and prewarmed to 37°C for 5 min prior to the assay. 50 μl opsonised bacteria were added to 200 μl primed neutrophils (ratio 1:1), or to heat-killed neutrophils as a control, and incubated at 37°C. 10 μl aliquots were taken after 30 min and 90 min and added to 190 μl ice cold LB, 0.05% saponin, vortexed hard, incubated on ice for 5 min, and vortexed again. Samples containing bacteria but no neutrophils were processed in parallel as an additional control. Serial dilutions in LB were plated onto LB-agar and incubated overnight at 37°C. CFUs of samples with live neutrophils were expressed as % of CFU with heat-killed neutrophils. To measure the ability of adherent neutrophils to kill S. aureus, 13 mm coverslips were coated ON with 1 μg/ml ICAM1 in DPBS⁺⁺⁺⁺. 200 μl neutrophils at 5 × 10⁶/ml in DPBS⁺⁺⁺⁺ were plated on the coverslips during priming with 50 ng/ml GM-CSF, 20 ng/ml TNFα for 45 min. 50 μl opsonised bacteria at 2 × 10⁷/ml in DPBS⁺⁺⁺⁺were added (ratio 1:1) and samples incubated at 37°C. Samples were recovered by scraping and processed further as here-above.

Transwell chemotaxis assays were done essentially as described (11), with 3 μm-pore filters (Millipore, Millicell-PC, PITP01250) in 24-well plates (Costar, 3473). Bone marrow was flushed into HBSS containing Ca²⁺ and Mg²⁺ (HBSS⁺⁺, Sigma, H8264), 0.25% BSA, and 15 mM Hepes, pH 7.5 at 37°C, all endotoxin-free (HBSS⁺⁺⁺⁺), triturated, strained through 40 μm filters, counted by haemocytometer and resuspended 5 × 10⁶/ml. Cells were primed with 50 ng/ml GM-CSF, 20 ng/ml murine TNFα for 45 min at 37°C, or mock-primed in DPBS⁺⁺⁺⁺. 400 μl were pipetted onto the transwell filters in wells containing 600 μl prewarmed 1 μM fMLP, 30 ng/ml CXCL1 (KC, Bio-Techne, R&D Systems, 453-KC-010), or 100 ng/ml CXCL1 in HBSS⁺⁺⁺⁺, or HBSS⁺⁺⁺⁺ alone, and incubated for 40 min or 90 min at 37°C. Cells remaining in the upper chamber were replaced with 400 μl ice-cold HBSS^–++, 3 mM EDTA. 60 µl HBSS^–++, 30 mM EDTA were added to the bottom well, and samples were incubated on iced metal trays for 15 min to detach cells. Cells were collected, centrifuged at 10,000 x g for 30 s, resuspended in ice-cold HBSS^–++, and stained with FITC-Gr1 (1:800) and AF647-CD11b (1:800) antibodies in HBSS^–++, 1% Fc block, in parallel to control cells which had not undergone the procedure. Cells were analysed with Spherotech ACBP-50-10 standard beads in an LSR2 flow cytometer, identifying neutrophils by Gr1^hi/CD11b^hi staining.

ibidi chamber migration assays were performed essentially as described (25). 6-channel ibidi slides (µ-slide VI 0.4, ibidi 80601) were coated with 3 µg/ml ICAM1 and blocked in BSA as described above, or with 20 μg/ml poly-Arg-Gly-Asp (pRGD, Sigma, F5022), or 1 mg/ml fibrinogen (Sigma, F8630) in DPBS^– for 1 h at RT and washed 3 times in HBSS⁺⁺⁺⁺. The central chamber and wells of the slide were filled with HBSS⁺⁺⁺⁺. Neutrophils at 2 × 10⁷/ml in HBSS⁺⁺⁺⁺, 1 g/l glucose were primed with 50 ng/ml GM-CSF, 20 ng/ml TNFα for 45 min at 37°C, or mock-primed. Buffer was removed from the wells, 45 µl cells added into one well, 45 µl liquid removed from the other, and cells were allowed to adhere for 20 min at 37°C. The fMLP gradient was generated by adding 5 μl HBSS^–++ containing 10 µM fMLP and 5 × 10⁶ carboxyl polystyrene beads (Bangs Laboratories, PC06N, 6.9 μm) into one well, and removing 5 µl buffer from the other. HBSS^–++ was used for mock-stimulated samples. The location of the beads showed the steepest part of the gradient, where imaging was done. Neutrophils were live-imaged for 20 min at 37°C in an Olympus CellR microscope using the 20× objective, taking frames every 10 s. Cells were tracked manually using the ‘chemotaxis and migration’ plugin of Fiji to quantify speed and directionality.

For imaging under shear-stress, 6-channel ibidi slides (µ-slide VI 0.4, ibidi 80601) were coated with 3 µg/ml ICAM1 and blocked with BSA as described above. Neutrophils at 2 × 10⁶/ml in DPBS⁺⁺⁺⁺ were stained using CellTracker Deep Red Dye (Invitrogen C34565, 1:2000) for 15 min in the dark, primed with 50 ng/ml GM-CSF, 20 ng/ml TNFα for 45 min at 37°C, and plated into the coated ibidi slide. Cells were allowed to adhere for 15 min at 37°C in a prewarmed environment chamber before non-adherent cells were gently aspirated and the slide was connected to an ibidi flow system. A shear flow of 6 dyn was applied for 10 min during which samples were live-imaged on a Nikon widefield microscope using the 20× objective, acquiring frames every 10 s. Images were analysed using Fiji, with StarDist for cell detection and TrackMate for automated generation of cell tracks. Adhesion was quantified as neutrophils/fields of view (fov) in each frame. Migration was quantified for neutrophils that adhered throughout the experiment. The directionality of migration was expressed as the degree of deviation from a perfect migration path against the shear flow.

Adhesion and spreading were assayed essentially as described (25). Sterile 13 mm glass coverslips (VWR, 631) in a Nunc 24-well plate (Thermo Fisher, 142475) were coated overnight at RT with 20 µg/ml pRGD, 3 µg/ml ICAM1, or 1 mg/ml fibrinogen in DPBS^–. ICAM1-coated coverslips were blocked with FAF-BSA as described above and all coverslips washed 3 times in DPBS^– before use. Neutrophils at 2 × 10⁶/ml in DPBS⁺⁺⁺⁺ were primed with 50 ng/ml GM-CSF and 20 ng/ml TNFα for 45 min at 37°C, or mock-primed in DPBS⁺⁺⁺⁺ for 45 min at 37°C, or kept unprimed on ice. Unprimed cells were pre-warmed for 5 min at 37°C before use. 250 µl neutrophils/well were added to 250 µl prewarmed 3 µM fMLP in DPBS⁺⁺⁺⁺ (2×, for 1.5 µM final), or DPBS⁺⁺⁺⁺, and incubated for 10 min (pRGD, fibrinogen) or 15 min (ICAM1) at 37°C, 5% CO₂. Non-adherent cells were aspirated and adherent cells were fixed in 4% paraformaldehyde (PFA), DPBS⁺⁺⁺⁺ for 15 min at RT. Samples were washed twice in DPBS^– and stained with FITC-Gr1 (1:400) antibody, Hoechst 33342 (1:400), and 1% Fc block in DPBS^– for 30 min at RT. Coverslips were washed 3 times in DPBS^–, rinsed in H₂O, and mounted using Aqua-Poly/Mount (Polysciences, 18806). Samples were imaged on a Nikon Ti-E wide-field fluorescence microscope, using the 3 × 3 image-stitching function at 60× magnification, imaging duplicate coverslips. Images were blinded prior to analysis, a mask was generated for each cell, and all particles larger than 40 μm² were analysed for number, surface area, and circularity using the ‘Set Measurements’ analysis tools of Fiji.

Sterile 13 mm glass coverslips were coated overnight at RT with 20 µg/ml pRGD or 3 µg/ml ICAM1, and ICAM1-coated coverslips were blocked in FAF-BSA as described above. Neutrophils at 1 × 10⁶/ml were prewarmed for 3 min and allowed to adhere to the coverslips in DPBS⁺⁺⁺⁺, 0.75 µM fMLP, or in DPBS⁺⁺⁺⁺ alone, for 15 min at 37°C, 5% CO₂. Non-adherent cells were aspirated, adherent cells fixed with 4% PFA, DPBS⁺⁺⁺⁺ for 15 min at RT, and samples washed 3 times in DPBS^–. Cells were permeabilised for 10 min with 0.1% Triton X-100 in DPBS^–, washed twice in DPBS^–, and stained with vinculin-FITC antibody (clone hVIN-1, Sigma, F7053, 1:100) or phalloidin-Atto 655 (Sigma, 18846, 1:100) in DPBS^– for 60 min at RT in the dark. Samples were washed twice in DPBS^–, stained with Hoechst 33342 (1:400) in DPBS^– for 5-10 min, washed twice in DPBS^– and once in H₂O, and mounted using Aqua-Poly/Mount. Imaging was done using a Nikon Ti-E wide-field fluorescence microscope with the 100× oil immersion objective. Image analysis was done in a blinded manner to assess neutrophil polarity and F-actin localisation. For TIRF microscopy of focal complexes, neutrophils at 4.5 × 10⁶/ml were primed with 50 ng/ml GM-CSF, 20 ng/ml TNFα in DPBS⁺⁺⁺⁺ for 45 min at 37°C and seeded onto ICAM1-coated ibidi μ-slide 8-well glass bottom chambers (ibidi, 80827). Neutrophils were stimulated, fixed, and focal complexes stained as here-above, except that samples were kept in DPBS⁺⁺⁺⁺ during imaging. Neutrophils were imaged on a Nikon Ti2 TIRF microscope using a 60× objective. Images were analysed using Fiji.

To image adhesion, morphology, and F-actin dynamics in live neutrophils, ibidi μ-slide 8-well glass bottom chambers were coated with ICAM1 or pRGD, and ICAM1-coated chambers were blocked in FAF-BSA as described above, and prewarmed in the chamber (37°C, 5% CO₂) of a Nikon Eclipse Ti-E widefield fluorescence microscope. Neutrophils from RLA^tg/+ and Tiam1^–/– RLA^tg/+ mice at 4 × 10⁶ cells/ml in DPBS ⁺⁺⁺⁺ were prewarmed for 3 min at 37°C (pRGD) or primed with 50 ng/ml GM-CSF, 20 ng/ml TNFα for 45 min at 37°C (ICAM1). Neutrophils from one genotype (alternating) were stained with CellTracker as described above and both genotypes were combined in equal quantities, to allow simultaneous live-imaging within one sample. 150 µl mixed RLA^tg/+ and Tiam1^–/– RLA^tg/+ neutrophils were added to 150 µl prewarmed 1.5 µM fMLP (2×, for final 0.75 µM) in DPBS⁺⁺⁺⁺ per well. 4 areas of each slide were live-imaged over 20 min, using the 100× oil immersion objective of a Nikon Eclipse Ti-E widefield system with 400 ms exposure times, acquiring frames every 10 s. Adhesion, spreading, polarisation, and F-actin dynamics were quantified by Fiji, as well as manually measuring actin features using blinded analysis. After analysis, the genotype of each neutrophil was revealed by the CellTracker.

Neutrophils from RLA^tg/+ and Tiam1^–/– RLA^tg/+ mice at 1 × 10⁷/ml in DPBS⁺⁺⁺⁺ were primed with 50 ng/ml GM-CSF, 20 ng/ml TNFα for 45 min at 37°C, or kept on ice and prewarmed to 37°C for 3 min prior to the assay. 300 μl cells were incubated with 1.5 μM fMLP, or mock-stimulated, for 15 min at 37°C, before being added to 4 ml of ice-cold 4% PFA, DPBS⁺⁺⁺⁺ and fixed for 15 min at RT. 40 ml PBS was added, and samples were centrifuged at 500 × g for 10 min at 4°C. Samples were washed in PBS and analysed on a Cytek Amnis Imagestream MkII flow cytometer, using a 200 mW 561 nm laser to excite RLA⁺ cells. 10,000 events were acquired per sample. Gating was done on single, RLA⁺ cells in focus on the brightfield. Cells were analysed using IDEAS software for area, circularity (arbitrary scale, high values mean high circularity), aspect ratio (cell width/length), and perimeter.

Adhesion molecules on the neutrophil surface were quantified by flow cytometry, essentially as described (13). Bone marrow cells were flushed into ice-cold HBSS^–++, strained through a 40 μm filter, and enumerated by haemocytometer. Cells were centrifuged at 326 × g for 10 min at 4°C, resuspended in ice-cold DPBS⁺⁺⁺⁺ at 4 × 10⁷/ml, and either kept on ice to minimise receptor trafficking, or primed with 50 ng/ml GM-CSF, 20 ng/ml TNFα for 45 min at 37°C for maximal receptor upregulation (13, 42, 47). Cells were pelleted and resuspended in an ice-cold cocktail containing FITC-Ly6G (BD Pharmingen, 551460, 1:400), eFluor 450 CD11b (clone M1/70, Invitrogen, 48-0112-82, 1:400) and BV510 CD62L (clone MEL-14, BioLegend, 104441, 1:100) antibodies, 1% Fc block, and one of these PE-conjugated antibodies: PE/Cy7 β1 integrin (CD29, clone HMβ1-1, BioLegend, 102221, 1:100), PE β3 integrin (CD61, clone 2C9.G2, BioLegend, 104307, 1:200), or PE α4 integrin (CD49d, clone R1-2, BioLegend, 103607, 1:200). Another cocktail contained BV510 Ly6G (BioLegend 127633, 1:400) and FITC LFA-1 (clone M17/4, eBioscience, 1:400) antibodies and 1% Fc block. Zombie NIR (BioLegend, 423105, 1:1000) was used as the fixable viability dye. Cells were stained for 20 min on ice, sedimented, resuspended in 1 ml ice-cold HBSS^–++, 1 mM EDTA, and kept on ice. Flow cytometry was performed in a BD Biosciences Fortessa, and data were analysed by FlowJo. Live single cells were gated, and neutrophils identified by their scatter characteristics and Ly6G^hi staining. Adhesion molecule cell surface levels were determined as the mean fluorescence signal of the relevant antibody.

β2-integrin affinity was measured essentially as described (48). Neutrophils at 1 × 10⁷/ml in DPBS⁺⁺⁺⁺ were primed with 50 ng/ml GM-CSF, 20 ng/ml TNFα for 45 min at 37°C, followed by 10 min incubation with 2 μg ICAM1, 0.2 μg APC-Fc antibody (Southern Biotech, 9042-11), and 10 μg/ml CD11b antibody (clone M1/70, BioLegend, 101202) on ice. Cells were stimulated with 1.5 μM fMLP or 100 ng/ml CXCL1 in DPBS⁺⁺⁺⁺, or mock-stimulated in DPBS⁺⁺⁺⁺, for 3 min at 37°C in a thermomixer at 300 rpm. Control cells were treated with 3 mM Mn²⁺ in DPBS⁺⁺⁺⁺. Ice-cold 7.4% PFA was added, and cells were fixed for 10 min on ice in the dark. Ice-cold DPBS^– was added, and cells were centrifuged for 5 min at 400 x g, 4°C, resuspended in rat anti-mouse Ly6B.2-FITC antibody (clone 7/4, Serotec, MCA771FT, 1:100), and incubated for 10 min on ice in the dark. Cells were washed and resuspended in DPBS^–. DAPI (Sigma, 10236276001, 1:400) was added and flow cytometry performed on a ZE5 YETI Cell Analyzer (Bio-Rad), using the DAPI signal to gate for live/dead cell populations, FITC for the neutrophil population, and APC to quantify ICAM1 binding.

β2-integrin avidity was measured essentially as previously described (13, 48). Neutrophils at 3 × 10⁶/ml in DPBS⁺⁺⁺⁺ were primed with 50 ng/ml GM-CSF, 20 ng/ml TNFα for 45 min at 37°C, and incubated with AF594-conjugated activating CD18 antibody (clone M18/2, BioLegend, 101416) (49, 50), during stimulation with 1.5 μM fMLP or 100 ng/ml CXCL1 in DPBS⁺⁺⁺⁺, or mock-stimulation in DPBS⁺⁺⁺⁺, for 3 min at 37°C in a thermomixer at 300 rpm. Treatment with 3 mM Mn²⁺ was used as a control. Ice-cold DPBS^– was added, and cells were centrifuged at 10,000 x g for 30 s at 4°C, resuspended in 4% PFA and fixed for 10 min on ice, centrifuged at 326 x g for 3 min, resuspended in DPBS^–, and allowed to settle onto electrostatically charged slides (Superfrost Plus, VWR, 631-0108) for 30 min at RT before mounting in Aqua-Poly/Mount. Cells were imaged using a Nikon confocal A1-R microscope with a 60× 1.4 NA oil objective. CellProfiler and Fiji were used to create cell masks and plot the AF594 signal intensity along the cell periphery. Areas of ≥1.25x median AF594 pixel intensity were considered integrin clusters.

Rac activity (GTP loading) in total lysates of neutrophils in suspension was measured by Pak-CRIB pull down assay, as described (44). GST-Pak-CRIB was purified from bacteria and stored in GST-FISH buffer (10% glycerol, 50 mM Tris pH 7.4, 100 mM NaCl, 1% NP-40, 2 mM MgCl₂, 2 mM DTT, 100 µM PMSF, and 10 µg/ml each of leupeptin, pepstatin A, aprotinin and antipain) at 4°C for no more than a week. 200 μl neutrophils at 1 × 10⁷/ml in DPBS⁺⁺⁺⁺ were pre-warmed for 3 min at 37°C, and stimulated with various concentrations of fMLP, DPBS⁺⁺⁺⁺ for 10 s, or mock-stimulated in DPBS⁺⁺⁺⁺. 1 ml ice-cold GST-FISH buffer, 1.2% NP-40 (final 1% NP-40) was added, and cells were lysed on ice for 2 min with frequent vortexing. Samples were centrifuged at 12,000 × g for 3 min at 2°C, and the supernatant was transferred into fresh precooled tubes. 2% were kept as total lysates. The remaining sample was incubated with GST-Pak-CRIB beads by rotating end-over-end for 15 min on ice. Samples were washed 5 times in GST-FISH buffer, boiling 1.3× SDS-PAGE sample buffer was added, and samples were boiled for 5 min. For the total lysates, boiling 4× SDS-PAGE sample buffer was added (to final 1.3×). Samples were western blotted with Rac1 and Rac2 antibodies. GTP-bound and total Rac were quantified by densitometry using Fiji.

To measure Rac activity in total lysates of adherent neutrophils, 30 mm glass coverslips were coated overnight with 3 μg/μl ICAM1 and blocked with FAF-BSA as described above. 2 ml neutrophils at 1.5 × 10⁶/ml in HBSS⁺⁺⁺⁺ were plated onto the coverslips in 35 mm tissue culture dishes (Corning) and allowed to adhere for 60 min at 37°C, 5% CO₂, while priming with 50 ng/ml GM-CSF, 20 ng/ml TNFα. 200 μl 15 μM fMLP in HBSS⁺⁺⁺⁺ (10×, for 1.5 μM final) were added for 1 min, or cells were mock-stimulated in HBSS⁺⁺⁺⁺. Dishes were transferred onto an iced metal tray, and the medium was aspirated. 1 ml ice-cold GST-FISH buffer was added, and cells were scraped-off, transferred into pre-cooled Eppendorf tubes, and lysed for 3 min on ice with frequent vortexing. Samples were cleared, incubated with GST-Pak-CRIB, and processed further as described for cells in suspension.

Live-cell imaging of Rac activity was done essentially as described (25). Ibidi μ-slide 8 well glass bottom slides were coated with ICAM1 or pRGD, and ICAM1 slides were blocked with 2% FAF-BSA as described above. Alternatively, slides were coated with activating CD18 antibody, by incubation with 100 µg/ml poly-D-lysine (Sigma, P6407) and 25 μg/ml protein A (Sigma, P6031) for 1 h at RT, washing, addition of 10 μg/ml activating CD18 antibody (clone M18/2, eBioscience, 14-0181-81) for 1 h at 37˚C, washing again, and blocking in BSA. Slides were washed 3 times in DPBS⁺⁺⁺⁺ and pre-warmed in the imaging chamber (37°C, 5% CO₂). For experiments on ICAM1 or pRGD, cells were primed with 50 ng/ml GM-CSF, 20 ng/ml TNFα for 45 min at 37°C, before 150 µl were added to 150 µl prewarmed 1.5 µM fMLP in DPBS⁺⁺⁺⁺ (2x, for 0.75 µM final) in the ibidi chamber and allowed to adhere for 1 h prior to imaging. For experiments with activating CD18 antibody, neutrophils were kept unprimed and plated without chemoattractant.

Live-cell ratiometric TIRF-FRET microscopy was used to assess the spatiotemporal distribution of Rac activity at the basal cell surface during adhesion and migration. Rac-FRET neutrophils of the various genotypes were directly compared within each experiment, in alternating order. A Nikon Ti2 TIRF microscope with 60× 1.49 NA oil TIRF objective was used with laser excitation at 440 nm. An Andor TuCam emission beam splitter comprising 509 nm dichroic mirror, 480/40 nm (CFP) and 542/27 nm (YFP) band-pass filters (Semrock) was used to direct the fluorescence from the donor channel (CFP) and FRET channel (YFP) to Andor iXon 897 EM-CCD cameras. Channel alignment was done at the start of each experiment using the Fiji plugin bUnwarpJ (51), generating a transformation protocol for images of CPN™ 530 Green standard beads (Stream Bio), which fluoresce in the CFP and YFP channels. This protocol was applied to the whole series of CFP and YFP image pairs. To ensure CFP and YFP images were taken simultaneously, an automated external trigger for the ‘slave’ camera was connected to the ‘master’ camera. TIRF-FRET imaging was done for 120 s, taking frames every 5 s. A mean filter of two pixels was applied and the YFP/CFP ratio image generated as described (44). FRET images were depicted using the Fiji 16-colour table, pseudo-colouring high Rac activity (high FRET ratio) in red and low Rac activity (low FRET ratio) in blue. To quantify overall Rac activity at the basal cell surface, the mean FRET ratio (pixel intensity value) for the whole cell was plotted against time. Alternatively, Rac activity was measured by widefield FRET imaging instead of TIRF-FRET, as previously described (44), to evaluate Rac activity throughout the adhering cell.

Imaging of Rac activity during micropipette chemotaxis assays was done essentially as described (44), except using TIRF-FRET imaging. Briefly, Rac-FRET neutrophils of various genotypes were stimulated with a point source of 0.5 μM fMLP delivered from a femtotip microinjection needle (Eppendorf, 5242957008) and imaged for 2 min by live-cell ratiometric TIRF-FRET imaging, as described above, with a frame interval of 1 s. Line scans were performed through the central longitudinal axis and the pixel with the highest Rac-FRET ratio along this axis determined for each frame. The duration of peak Rac activity localising within 0.8 μm of the leading edge or uropod throughout the 2 min observation period, and the number of oscillations of peak Rac activity between leading edge and uropod, were determined as described (44). In addition, the distance of migration was quantified. Cells that translocated at least 7.8 μm (half their mean body length), during the observation period were considered migrating.

GST-Rac1^G15A (52) was used to isolate proteins that interact with nucleotide-free Rac. GST-Rac1^G15A was purified from bacterial culture, coupled to glutathione sepharose 4B (GE Healthcare, 17-0756-01), and stored in 20 mM Hepes, pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 30% glycerol, 1 mM DTT, essentially as described (52). 50 mm glass coverslips were coated with 3 μg/μl ICAM1 in 60 mm tissue culture dishes and blocked with 2% FAF-BSA as described above. 4 ml neutrophils at 2 × 10⁶/ml in HBSS⁺⁺⁺⁺ were allowed to adhere to the coverslips while priming with 50 ng/ml GM-CSF, 20 ng/ml TNFα for 50 min at 37°C, 5% CO₂, inside a chemical fume hood. 7 mM of the irreversible, cell-permeable protease inhibitor diisopropyl-fluorophosphate (DFP) was added for a further 10 min, before neutrophils were stimulated with 1.5 µM fMLP for 1 min. Dishes were transferred carefully onto an iced metal tray inside the fume hood, the medium aspirated, and samples washed with ice-cold HBSS⁺⁺⁺⁺. 1 ml ice-cold lysis buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 1% Triton X-100, 1:100 protease inhibitor cocktail (Sigma, P8340)) was added, cells were scraped-off, and lysates transferred into precooled Eppendorf tubes. The DFP waste was disposed-of safely, and samples were processed further outside the fume hood. Lysates were incubated for 3 min on ice with frequent vortexing and centrifuged at 12,000 x g for 1 min at 2°C. The supernatant was transferred into fresh precooled tubes and samples were divided. Then 5% was used for total lysates, and the rest divided equally for incubation with either GST-Rac1^G15A beads or glutathione sepharose 4B (both prewashed in lysis buffer) by end-over-end rotation for 1 h on ice. Samples were washed 4 times in lysis buffer. For both total lysate and pull down samples, boiling 1.3× SDS-PAGE sample buffer was added, the samples were boiled, and then snap-frozen in liquid nitrogen.

Prior to mass spectrometry, the samples were run ~5 mm into an SDS-PAGE gel, which was stained with Coomassie. The protein-containing region of gel was excised, destained, reduced/alkylated, and trypsin digested as described (53). The resulting peptides were fractionated by high-pH reversed-phase chromatography, using a micro pipette tip packed with Oasis HLB beads. The peptides were loaded in 0.1% ammonium hydroxide and batch eluted with acetonitrile concentrations of 10%, 15%, 20%, and 32%, in 0.1% ammonium hydroxide. The peptide fractions were analysed with an Orbitrap Eclipse mass spectrometer, fitted with a FAIMS Pro interface. Peptides were separated by online reversed-phase nanoLC on an Easy-nLC 1200, with a 90 min gradient of 0-30% acetonitrile (containing 0.1% formic acid), at 300nL/min. The mass spectrometer data acquisition cycle comprised three FAIMS compensation voltages (-40V, -60V, -80V), each of which contained a data-dependent experiment, with one high-resolution MS1 scan followed by 10 MS2 scans, with HCD fragmentation. The mass spectrometric data were searched against the Uniprot mouse proteome using the Mascot search engine within Proteome Discoverer 2.1. Targeted analyses were performed on the same instrument, but without FAIMS and using a 2 h gradient and data-independent analysis. The raw files were processed with DIA-NN software v1.8.1.

Total lysates were western blotted using the following antibodies: 14-3-3 (pan, Cell Signaling Technology, 8312, 1:1000), α-Pix (Cell Signaling Technology, 4573, 1:1000), Git2 (Invitrogen, PA5-78301, 1:1000), Graf1 (Proteintech, 55139-1-AP, 1:1000), Psd4 (Abcam, ab154008, 1:1000), Rap1gds1/SmgGDS (Novus Biologicals, NBP1-87027, 1:1000), Rasa3 (Proteintech, 27835, 1:500), Talin-1 (Cell Signaling Technology, 4021, 1:500), Tiam1 (Bethyl Laboratories, A300-099A, 1:1000) and Tiam2 (54) (1:750).

Sample size was determined using power calculations to yield 80% power, based on pilot experiments and previously published data. Experiments were conducted at least three times, except where specified. Figure legends contain detailed information on sample size and numbers of independent experiments. Mice were randomly selected for cohorts by the staff of our Biological Support Unit, within specified group size and age parameters. The identity of images was blinded prior to analysis. Excel 2016, GraphPad Prism 9, and R were used for statistical analysis and for making graphs. Data were tested for normal distribution using the Shapiro-Wilk test to determine whether parametric or non-parametric statistical analysis was appropriate. If the variance between groups warranted it, data were log-transformed before statistical analysis, as indicated in the figure legends. Statistical outliers were identified using the ROUT test and excluded from datasets. Other samples were only excluded if there was a known technical problem affecting the analysis. Student’s t-test was used for comparisons between two groups. For in vivo experiments with fewer mock-infected mice than infected animals, one-way ANOVA was used, followed by pairwise comparisons between genotypes with Holm-Sidak multiple comparisons corrections, and the resulting multiplicity-adjusted p-values were reported. For all other data, one-way, two-way ANOVA or three-way ANOVA were used as appropriate to test for the effects of interventions, and p-values were reported from the multiplicity-adjusted comparisons. p<0.05 was considered the threshold for statistical significance. Group sizes (n) are listed in the figure legends. P-values denoting significant differences are indicated in the figures in black, and p-values reporting non-significance in grey.

To study the role of Tiam1 in neutrophils, we compared primary mouse neutrophils from wild type and Tiam1^–/– mice. Western blots showed that Tiam1 is expressed in wild type neutrophils and deleted in Tiam1^–/– ( Figure 1A ). Neutrophil development was normal in Tiam1^–/– mice, judged by the number and density of neutrophils isolated from the bone marrow, and their characteristic doughnut-shaped nucleus ( Figures 1B, C ).

Neutrophils are the first line of defence against bacterial and fungal infections. To assess whether Tiam1 is important for combatting acute bacterial infection, we infected wild type and Tiam1^–/– mice intranasally with 2 x 10⁶ CFU of S. pneumoniae and assessed bacterial titre and neutrophil recruitment into the lung 6 h later. We analysed bronchoalveolar lavages and lung tissue perfused after lavage, to monitor the infection in the airways and the lung tissue. In the Tiam1^–/– mice, twice as many bacteria remained in the perfused lung tissue after 6 h than in wild type, which shows that Tiam1 is important for the clearance of bacteria from the lung interstitium and vasculature ( Figure 1D ). S. pneumoniae-induced neutrophil recruitment was normal, as were leukocyte numbers overall ( Figure 1D ). In contrast to the perfused tissue, the lavage samples showed normal bacterial titre, neutrophil recruitment, and overall leukocyte number ( Supplementary Figure 1A ), suggesting that Tiam1 is dispensable for clearing the infection from the airways. Neutrophil numbers in the peripheral blood were also normal ( Supplementary Figure 1B ). Therefore, Tiam1 plays a role in innate immunity that depends on the tissue environment.

To assess in more detail if Tiam1 affects the recruitment of neutrophils from the blood vessels into the infected lung, we performed histological analysis, using their characteristic nuclear morphology to identify neutrophils in H&E stained sections. This revealed normal numbers of neutrophils being recruited to the blood vessel wall and into the bronchiolar and alveolar epithelium of Tiam1^–/– mice upon S. pneumoniae infection, but reduced numbers in the interstitial space ( Figure 1E ). This suggests that, although overall recruitment was normal, Tiam1 affects the route or speed of neutrophil recruitment during bacterial pneumonia. The reduced neutrophil numbers in the interstitial space likely contribute to the impaired clearance of S. pneumoniae in Tiam1^–/– mice.

We were interested in studying recruitment also in a different inflammation model, because the requirement for Rac-GEFs in this process is highly organ-specific (1). We investigated neutrophil recruitment in thioglycollate (TGC)-induced sterile peritonitis. Tiam1^–/– mice showed reduced TGC-induced peritoneal neutrophil recruitment, which accounted for decreased overall peritoneal leukocyte numbers ( Figure 2A ). Transplantation of Tiam1^–/– bone marrow into irradiated wild type mice prior to induction of sterile peritonitis confirmed that a Tiam1^–/– haematopoietic system, rather than the tissue environment, was sufficient to cause this reduction in neutrophil recruitment ( Figure 2B ).

Together, these data show that Tiam1 is expressed in neutrophils, is required for antibacterial innate immunity in a manner dependent on the tissue environment, and contributes to neutrophil recruitment during acute inflammation in a manner depending on the tissue environment. The finding that neutrophil numbers were normal overall in the infected Tiam1^–/– lung suggested that impaired neutrophil effector responses may contribute to the reduced immunity.

A key neutrophil response for killing bacteria is ROS production. We investigated ROS production in neutrophils in suspension in response to a range of stimuli, including the chemoattractants fMLP and C5a, and zymosan yeast particles either in serum-opsonised or unopsonised form. ROS production in response to all stimuli was normal in Tiam1^–/– neutrophils ( Figure 3A ; Supplementary Figure 2 ). Similarly, fMLP-stimulated ROS production in neutrophils adhering to the β2-integrin ligand ICAM1 was also normal ( Figure 3A ), as was receptor-independent ROS production in response to PMA ( Supplementary Figure 2 ).

Another neutrophil response important for killing bacteria is degranulation. We measured degranulation both in neutrophils in suspension and in adherent cells. In Tiam1^–/– neutrophils in suspension, the fMLP-stimulated secretion of gelatinase (Mmp9) activity was normal ( Figure 3B ). In contrast, in Tiam1^–/– neutrophils adhering to ICAM1, the E. coli-induced degranulation of myeloperoxidase (MPO) was reduced ( Figure 3B ). Hence, Tiam1 is required for degranulation in adherent neutrophils. To test whether this may contribute to the impaired killing of bacteria observed in Tiam1^–/– mice in vivo, we measured MPO by IHC in lung histology sections. However, no significant changes in MPO signal were observed under these conditions, although there was a trend to increased MPO puncta in S. pneumoniae-infected Tiam1^–/– mice ( Supplementary Figure 3 ).

Neutrophils release NETs to kill bacteria. We measured NET release in response to S. aureus, which was impaired in Tiam1^–/– neutrophils ( Figure 4A ). This impairment likely contributes to the reduced clearance of bacteria in vivo, although this could not be verified, as CitH3 signal was too rare to be evaluated in a meaningful way by IHC in lung histology sections of S. pneumoniae-infected mice under the conditions tested (data not shown).

To test directly if Tiam1 is required for the killing of bacteria, we incubated neutrophils in suspension or adhering to ICAM1 with S. aureus for 30 or 90 min. The killing of S. aureus was reduced in Tiam1^–/– neutrophils under all conditions tested ( Figure 4B ). Hence, Tiam1 is required for the killing of bacteria both in neutrophils in suspension and in adherent neutrophils. This defect, together with the altered distribution of neutrophils in the lung, can explain the reduced immunity of Tiam1^–/– mice.

We investigated neutrophil migration, initially using transwell chemotaxis assays where neutrophils migrate through 3 μm pore filters. fMLP-stimulated chemotaxis was impaired in Tiam1^–/– neutrophils at both timepoints investigated, 40 min and 90 min, whereas random neutrophil migration in the absence of chemoattractant was normal ( Figure 5A ). Hence, Tiam1 is required for fMLP-induced neutrophil chemotaxis. To test if a similar defect can be observed in response to another chemoattractant, we did transwell assays with CXCL1 (KC), the mouse equivalent of human IL8. Interestingly, CXCL1-induced chemotaxis was increased in Tiam1^–/– neutrophils, rather than decreased ( Figure 5B ). This shows that Tiam1 plays an interesting role in promoting or limiting neutrophil chemotaxis, depending on the chemoattractant.

To study migration in more detail, we tracked the paths of migrating neutrophils in an ibidi chemotaxis chamber. Neutrophils adhere and migrate in an integrin-dependent manner. The main integrin types expressed in neutrophils are β1 and β2 (55), although we also detected β3, β5, and β7 in our recent proteomic analysis (42). Hence, we coated the ibidi chamber with various integrin ligands: ICAM1, the archetypical β2 integrin-ligand; pRGD, a promiscuous ligand for β1 and other integrins; or fibrinogen, which predominantly binds β3 integrins (56–58). In Tiam1^–/– neutrophils plated on ICAM1, fMLP-stimulated chemotaxis was reduced, whereas random migration in the absence of chemoattractant was unchanged ( Figure 5C ), as previously seen in the transwell assay. Chemotaxis was reduced both in terms of accumulated distance and Euclidian distance (as the crow flies). The impairment resulted from decreased velocity of migration, whereas directional sensing and the propensity to migrate were normal ( Figure 5C ; Supplementary Figure 4 ). Migration on pRGD was also impaired, but in a different manner. The fMLP-stimulated chemotaxis of Tiam1^–/– neutrophils on pRGD was normal, whereas random migration in the absence of chemoattractant was increased, due to increased velocity ( Supplementary Figure 5 ). On fibrinogen, both random migration and chemotaxis were normal ( Supplementary Figure 6 ). Hence, Tiam1 plays complex roles in neutrophil migration that vary depending on the chemoattractant and the integrin ligand through which the neutrophils adhere. Judging by cell behaviours on the various ligands, we conclude that Tiam1 is required for β2-integrin dependent neutrophil chemotaxis and may limit β1-dependent random migration, but is likely dispensable for β3-dependent migration.

During recruitment in vivo, neutrophils become adherent to the endothelial wall of venules under shear stress, and then migrate against the direction of blood flow until they find a site suitable for transmigration (59). To mimic this situation, we applied a shear flow of 6 dyn/cm² to GM-CSF/TNFα-primed neutrophils plated on ICAM1 (60). Tiam1^–/– neutrophils adhered normally both before and after the application of flow, and both genotypes migrated against the direction of flow, but the Tiam1^–/– cells migrated much further, due to increased velocity rather than altered directionality ( Figure 5D ). Hence, Tiam1 limits random neutrophil migration on ICAM1 under shear flow, as it does on pRGD under static conditions. Together, the complex, context-dependent roles of Tiam1 in neutrophil migration suggested that Tiam1 may play similarly complex roles in controlling the cytoskeletal dynamics that underlie adhesion and migration.

We investigated the roles of Tiam1 in neutrophil adhesion, polarity, and cytoskeletal properties, both on ICAM1 and pRGD. Adhesion of Tiam1^–/– neutrophils to ICAM1 was increased, whereas adhesion to pRGD was normal, and spreading was normal on both surfaces ( Figure 6A ; Supplementary Figure 7A ). The increased adhesion on ICAM1 was only seen in unprimed Tiam1^–/– neutrophils, whereas GM-CSF/TNFα-primed cells adhered normally ( Figures 5D , 6A , and data not shown).

To investigate neutrophil polarisation and F-actin distribution, we allowed wild type and Tiam1^–/– neutrophils to adhere to ICAM1 or pRGD, fixed them, stained their F-actin, and analysed them by fluorescence microscopy. On pRGD, more Tiam1^–/– neutrophils polarised more than wild type, whereas on ICAM1 it was the inverse ( Figure 6B ; Supplementary Figure 7B ). Consistent with the altered neutrophil polarity, F-actin distribution was also affected. Usually, F-actin is mostly localised at the leading edge of polar neutrophils. However, in Tiam1^–/– neutrophils, there was increased localisation of F-actin also at the uropod ( Figure 6B ; Supplementary Figure 7C ), which may contribute to the impaired migration.

To investigate F-actin dynamics in live cells, we crossed the Tiam1^–/– mouse to a reporter mouse with fluorescently-labelled actin, RubyLifeact (RLA) (45), and compared wild type RLA^tg/+ with Tiam1^–/– RLA^tg/+ neutrophils. We stained cells from one genotype with CellTracker, mixed cells from both genotypes within the same sample, plated them on ICAM1 or pRGD, and live-imaged them in the presence of fMLP. Neutrophils of both genotypes polarised and underwent chemokinesis, but the Tiam1^–/– cells had altered F-actin dynamics and more F-actin at their uropod on both surfaces. On ICAM1, Tiam1^–/– RLA^tg/+ neutrophils maintained their cortical F-actin ring and F-actin accumulation at the uropod even during migration ( Figure 6C ; Supplementary Figure 7C, Movie 1 ). On pRGD, the cortical F-actin ring was more pronounced, maintained for longer, and reformed during chemokinesis ( Supplementary Figures 7C, D, Movie 2 ). Hence, Tiam1 regulates neutrophil polarisation and F-actin dynamics during adhesion and chemokinesis.

To test if Tiam1 also affects the morphology of neutrophils in suspension, we analysed RLA^tg/+ and Tiam1^–/– RLA^tg/+ neutrophils by imagestream flow cytometry. fMLP stimulation and/or priming of neutrophils caused the RLA^tg/+ cells to spread, elongate, and form protrusions. In contrast, Tiam1^–/– RLA^tg/+ neutrophils showed these morphologies constitutively, and fMLP stimulation or priming did not further enhance this ( Supplementary Figure 8 ). Hence, as during adhesion, Tiam1 also controls the morphology of neutrophils in suspension, where it maintains their rounded basal state.

Focal adhesions connect extracellular matrix-bound integrins to the actin cytoskeleton. Neutrophils do not make the long-lived focal adhesions typical of cultured cells but instead form smaller, short-lived adhesive structures, which are sometimes termed focal complexes (43, 61). We investigated focal complexes in wild type and Tiam1^–/– neutrophils adhering to pRGD or ICAM1, by staining with the marker vinculin. Widefield microscopy showed that Tiam1^–/– neutrophils on pRGD had more focal complexes than wild type upon stimulation with fMLP ( Figure 6D ). TIRF microscopy showed that Tiam1^–/– neutrophils on ICAM1 had the same number of focal complexes as wild type, but these focal complexes were smaller under basal conditions ( Supplementary Figure 7E ). Hence, Tiam1 controls focal complex number or size, depending on the integrin ligand through which the cells adhere. As focal complexes reflect the number or activity of integrins, this suggested that Tiam1 may regulate the cell surface levels of integrins, or the affinity and avidity of integrins.

To test for cell surface levels of adhesion molecules, we performed flow cytometry experiments in basal and primed neutrophils. Neutrophils reacted to priming by strongly upregulating the β2 integrin Mac-1 (p=0.0008 in wild type) onto their surface, and shedding L-selectin (p=0.0008), whereas β2 integrin LFA-1 increased only slightly (p=0.0290) ( Figure 7A ), as expected (13, 42, 47). The β-chain common to β1 integrins was also upregulated slightly upon priming (p=0.0106), whereas the α-chain of the β1 integrin VLA-4 (α4β1) and the β-chain common to β3 integrins remained unchanged. Importantly, the cell surface levels of all adhesion molecules tested were the same between wild type and Tiam1^–/– neutrophils ( Figure 7A ). Therefore, Tiam1 does not affect the cell surface levels of major neutrophil adhesion molecules.

β2 integrins must be activated from their bent, closed conformation to their extended, open conformation in order to bind ICAM1, and this activation occurs in response to chemoattractant signalling through GPCRs, or can be artificially induced by exposure to Mn²⁺ (62). We tested the ability of neutrophils to bind ICAM1 by flow cytometry. ICAM1 binding increased upon Mn²⁺ treatment, as expected, and to the same extent in both wild type and Tiam1^–/– neutrophils, suggesting that the overall ICAM1 binding capacity of Tiam1^–/– neutrophils is normal, in line with the normal β2 integrin cell surface levels. Stimulation with fMLP had no apparent effect under the primed conditions tested, but CXCL1 induced an increase in ICAM1 binding in Tiam1^–/– neutrophils, although not in wild type ( Figure 7B ).

Active β2 integrins have higher avidity, meaning they cluster within the membrane to facilitate adhesion. We tested β2 integrin avidity by microscopy. Similar to ICAM1 binding, Mn²⁺ treatment increased clustering equally in wild type and Tiam1^–/– neutrophils. Stimulation with fMLP had again no effect, whereas CXCL1 increased β2 integrin clusters and the size of these clusters in Tiam1^–/– neutrophils but not in wild type ( Figures 7C, D ). Together, these data show that Tiam1 limits CXCL1-stimulated β2 integrin affinity and avidity in neutrophils, despite normal β2 integrin cell surface levels. This may explain how Tiam1 limits CXCL1-dependent chemotaxis.

We were intrigued by the increased adhesion and migration under shear flow of Tiam1^–/– neutrophils on ICAM1, by their increased polarity and random migration on pRGD, and by the increased chemotaxis towards CXCL1, because deficiencies in Rac and Rac-GEFs would usually reduce these responses. To test whether these increased cell responses may be a reflection of altered Rac activity, we tested the fMLP-stimulated activation of Rac in wild type and Tiam1^–/– neutrophils.

First, we tested Rac activity in neutrophils in suspension by Pak-CRIB pull down. Both Rac1 and Rac2 were robustly and dose-dependently activated upon fMLP stimulation, as expected, but there was no difference between wild type and Tiam1^–/– cells ( Supplementary Figure 9 ), consistent with the normal ROS production and degranulation in suspension. In neutrophils adhering to ICAM1, fMLP stimulation also induced robust activation of Rac1 and Rac2. However, both these Rac activities were higher in Tiam1^–/– neutrophils than in wild type ( Figure 8A ). Hence, Tiam1 paradoxically limits the chemoattractant-dependent activation of Rac in adherent neutrophils, rather than promoting it as would be expected from a Rac-GEF.

To investigate Rac activity in a different manner, we compared neutrophils from Rac-FRET mice that express a Raichu-Rac FRET reporter for Rac activity to Rac-FRET neutrophils deficient in Tiam1, Dock2 or Prex1/Vav1 (25). Neutrophils were plated onto ICAM1 or pRGD in the presence of fMLP and were live-imaged by ratiometric TIRF-FRET imaging to visualise Rac activity at the basal cell surface which touches the substrate ( Figures 8B, C ; Supplementary Movie 3 ). Rac activity (FRET ratio) was reduced in Rac-FRET Dock2^–/– neutrophils under both conditions, and was either normal or reduced in Rac-FRET Prex1^–/– Vav1^–/– cells ( Figures 8B, C ), as expected (25). In contrast, Rac activity was increased in Rac-FRET Tiam1^–/– cells, both on ICAM1 and pRGD ( Figures 8B, C ), in line with the results obtained by Pak-CRIB pull down assay. The same increased Rac activity was seen when we plated Rac-FRET Tiam1^–/– neutrophils on glass instead of ICAM1 or pRGD ( Supplementary Figure 10A, Movies 4–6 ), and when we used widefield ratiometric FRET imaging of cells on ICAM1 instead of TIRF-FRET in order to image the whole cell ( Supplementary Figure 10B ). Furthermore, Rac activity was also increased when Tiam1^–/– neutrophils were plated on M18/2 anti-CD18 antibody, which activates β2 integrins (49, 50) ( Supplementary Figure 10C ). In contrast, Rac activity was decreased in Rac-FRET Dock2^–/– cells throughout, as expected ( Supplementary Figure 10A–C, Movies 4–6 ). Hence, Tiam1 limits Rac activity in adherent neutrophils, rather than being required for the activation of Rac as would be expected for a Rac-GEF.

Furthermore, we previously established that peak Rac activity oscillates between the leading edge and uropod of migrating neutrophils plated on glass (44). We investigated if Tiam1 may contribute to these waves in a pilot experiment, by determining the localisation of peak Rac activity along the central longitudinal axis of Rac-FRET Tiam1^–/– neutrophils during their migration towards a micropipette containing fMLP. In wild type neutrophils, Rac activity oscillated between leading edge and uropod every 7 s, whereas these waves were shorter (6 s) in Tiam1^–/– cells, although the distance migrated and likelihood to migrate were unaffected under these conditions ( Supplementary Figure 11 ). Moreover, peak Rac activity was lost from the leading edge of Tiam1^–/– neutrophils, with both the amount of time and number of events of peak activity localising within 0.8 μm of the leading edge being reduced, whereas Rac activity localisation at the uropod was normal ( Supplementary Figure 11 ). These result warrant detailed analysis elsewhere in the future, but they do suggest that Tiam1 may regulate Rac activity waves and generate Rac activity at the leading edge during neutrophil migration.

Searching for mechanisms that might underlie the paradoxical increases in Rac and integrin activity, adhesion, polarity, and migration in adherent Tiam1^–/– neutrophils, we hypothesised that other Rac-GEFs likely took over the role of Tiam1 and overcompensated. To test this hypothesis, we used the Rac mutant Rac1^G15A, which stabilises the transient nucleotide-free state that occurs during the activation of Rac and has high affinity for active Rac-GEFs (52). We stimulated wild type and Tiam1^–/– neutrophils adhering to ICAM1 with fMLP, affinity-purified proteins that interact with Rac1^G15A from lysates, and identified them by mass spectrometry. Preliminary analysis identified 501 proteins in at least one sample ( Supplementary Table 1 ). We selected 21 proteins based on the literature, for their ability to regulate small GTPases and/or cytoskeletal dynamics, or to interact with Tiam1, and analysed them further by targeted proteomics. We could compare 14 of these proteins between the genotypes, and 12 in at least two of three experiments ( Figure 9A ). The Rac-and Cdc42-GEF α-Pix (63) appeared to bind Rac1^G15A more strongly in Tiam1^–/– neutrophils than wild type (Wt/Ko ratio = 0.6), as did the Arf-GAP Git2, an interactor of GPCRs and β-Pix (64) (Wt/Ko ratio = 0.5). The stronger association of α-Pix with Rac1^G15A might explain the higher Rac activity in Tiam1^–/– neutrophils. However, α-Pix and Git2 were only detected in one experiment, so we cannot draw firm conclusions. In contrast, the dual Ras- and Rap-GAP Rasa3 (65, 66) consistently interacted with Rac1^G15A more strongly in wild type than Tiam1^–/– neutrophils (3.4-fold). The Arf- and Arl-GEF Psd4 (67, 68) and the adaptor protein 14-3-3ζ/δ, which bridges Tiam1 to β1 integrins (69), followed a similar trend but this did not reach significance due to considerable variability ( Figure 9A ). The other 9 proteins bound Rac1^G15A equally between wild type and Tiam1^–/– . They comprised Cd177, a counter-receptor for Pecam1 that interacts with integrins (70); the diacylglycerol kinase Dgkζ (71); Ezrin, which crosslinks transmembrane and cytoskeletal proteins and interacts with focal adhesion kinase (72); the RhoA- and Cdc42-GAP Graf1 (73); the Sos adaptor Grb2 (74); the adaptor protein Iqgap1 which stabilises GTP-bound Rac1 and Cdc42 (75); the chaperone Rap1gds1 (also known as SmgGDS) which acts as an atypical RhoA and RhoC-GEF (76); the protein kinase Slk, which regulates focal adhesion turnover (77); and the Ras-GAP Tbc1d10c (78). Hence, Tiam1 controls the association of the Ras-GAP Rasa3, and possibly also of α-Pix, Git2, Psd4 and 14-3-3ζ/δ with nucleotide-free Rac.

Finally, we evaluated the total cellular levels of some of the proteins we identified ( Figure 9B ). α-Pix was 3.5-fold higher in wild type neutrophils than Tiam1^–/– , suggesting that Tiam1 promotes α-Pix expression or stability, despite possibly limiting α-Pix binding to Rac1^G15A. In contrast, Git2 was expressed normally. Rasa3 was 2.9-fold higher in wild type neutrophils and Psd4 7.0-fold higher, which likely explains their association with Rac1^G15A. 14-3-3 expression was normal, despite potentially stronger binding to Rac1^G15A in wild type, suggesting Tiam1 may mediate 14-3-3ζ/δ binding to Rac. Tiam2, which was not detected among Rac1^G15A interactors, was expressed 3.7-fold higher in wild type cells, suggesting that this GEF did not compensate in Tiam1^–/– cells. Talin, a master regulator of adhesion that links integrins and focal adhesions to the cytoskeleton (79), and co-purified with Rac1^G15A in some samples, was expressed normally. Finally, Graf1 and Rapgds1 also appeared to be expressed normally, although we did not formally quantify them.

In conclusion, Tiam1 promotes the expression of several regulators of small GTPase signalling and cytoskeletal dynamics, and it mediates the binding of Rasa3, and possibly also Psd4 and 14-3-3ζ/δ to Rac, as well as potentially limiting the association of α-Pix and Git2 with Rac. Although these data do not conclusively explain how Tiam1 limits Rac activity in adhering neutrophils, it seems likely that this extensive deregulation of relevant proteins does contribute. Together, our data show that Tiam1 is an important regulator of neutrophil responses, and particularly but not exclusively of adhesion-dependent responses, but it fulfils this role in a manner that differs fundamentally from those of other neutrophil Rac-GEFs, and indeed differently to what would be expected from any Rac-GEF ( Figure 10 ).