Fig 1: Inhibition of endogenous SLIT2 signaling increases tissue neutrophil infiltration, and attenuates tissue-associated ROS.(A) Ear skin samples were collected as described in (Figure 4A) and tissue SLIT3 levels were measured using an ELISA. n=5. p=0.0002 (Mock infection vs S. aureus 0.5 day), p=0.0003 (Mock infection vs S. aureus 1 day), and p=0.0067 (Mock infection vs S. aureus 2 days). (B) In vivo protocol to block endogenous SLIT2 during S. aureus skin and soft tissue infection (SSTI). Ear pinnae were infected with 5 × 106 colony forming units (CFU) of S. aureus or mock-infected (saline injection) as indicated on day 0. On 2nd and 3rd day, local injections of either 7 μg of control (Ctr) IgG or N-Roundabout guidance receptor 1 (ROBO1) were given at the site of infection. Skin samples were collected on the 4th day after infection. (C) Samples were collected as described in (B) and were homogenized and S. aureus CFU were counted by serial dilution. p<0.0001 (S. aureus vs S. aureus + N-ROBO1) and p<0.0001 (S. aureus + Ctr IgG vs S. aureus + N-ROBO1). N=8. (D–E) Animals were treated as described in (B), skin samples fixed in formalin, stained with Ly6G (red) and F4/80 (green) antibodies, and DAPI (blue). Scale bar = 200 μm. (E) Percent neutrophils (Ly6G+ F4/80–) per unit tissue area were calculated using HALO software. N=4. p=0.0475 (Mock infection vs N-ROBO1), p=0.0008 (Mock infection vs S. aureus), p<0.0001 (Mock infection vs S. aureus + N-ROBO1), p=0.0397 (S. aureus vs S. aureus + N-ROBO1), and p=0.0127 (S. aureus + Ctr IgG vs S. aureus + N-ROBO1). (F–G) Animals were treated as described in (B), and skin samples were collected, fixed in formalin, stained with an anti-8-OHdG antibody, and developed with 3,3'-Diaminobenzidine (DAB) staining. (F) Red arrowheads indicate DAB positive regions, Scale bar = 100 μm. (G) Fold changes (compared to mock infection) in % DAB +ve nuclei were calculated using HALO software. N=4. p<0.0001 (Mock infection vs S. aureus), p=0.0332 (Mock infection vs S. aureus + N-ROBO1), and p=0.0001 (S. aureus vs S. aureus + N-ROBO1). Mean values ± SEM. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. The source data are available as Figure 4—figure supplement 1—source data 1. Figure 4—figure supplement 1—source data 1.The file contains source data for Figure 4—figure supplement 1A, C, E, G.
Fig 2: Anti-staphylococcal actions of N-SLIT2 are partially dependent on increases in extracellular ROS production.(A) RAW264.7 macrophages were incubated with vehicle, N-SLIT2 (30 nM) or N-SLIT2ΔD2 (30 nM) for 15 min and then with unoposonized S. aureus expressing GFP (MOI 10) for an additional 45 min. Extracellular bacteria were labeled using donkey anti-human IgG-Cy3. Macrophage plasma membranes were stained using Acti-stain-AF670. At least 100 macrophages per experimental condition were imaged. n=3. The phagocytic efficiency (B) and index (C) were calculated. (D) Experiments were performed as in Figure (1 A). In some experiments, cells were pre-incubated with DPI (10 μM) for 5 min and then incubated with N-SLIT2 (30 nM) in the presence of diphenyleneiodonium chloride (DPI) for an additional 15 min. n=3. Statistical comparisons between N-SLIT2 and DPI + N-SLIT2 groups are p=0.0082 (0.5 hr), p=0.0259 (1 hr), p=0.1592 (1.5 hr), and p=0.4285 (2 hr). (E) Experiments were performed as in Figure (1E) using PMA (200 nM) as a secondary stimulus instead of S. aureus. Maximum isoluminol relative luminescent units (RLU) was compared. n=4. p=0.0006 (vehicle vs PMA), p=0.0065 (PMA vs N-SLIT2 + PMA), and p=0.0092 (N-SLIT2 + PMA vs N-SLIT2ΔD2 + PMA). (F) Murine bone marrow-derived neutrophils were treated as described in Figure (1E). n=5. p=0.0005 (vehicle vs S. aureus), p=0.0064 (S. aureus vs N-SLIT2 + S. aureus), and p=0.0082 (N-SLIT2 + S. aureus vs N-SLIT2ΔD2 + S. aureus). (G) Human neutrophils were treated as described in (F) and active levels of Rac were measured using a Rac1/2/3 calorimetric G-LISA. n=4. p=0.0024 (vehicle vs S. aureus), p=0.0088 (S. aureus vs N-SLIT2 + S. aureus), and p=0.0038 (N-SLIT2 + S. aureus vs N-SLIT2ΔD2 + S. aureus). Mean values ± SEM. *p<0.05, **p<0.01, ***p<0.001. The source data are available as Figure 1—figure supplement 1—source data 1. Figure 1—figure supplement 1—source data 1.The file contains source data for Figure 1—figure supplement 1B–G.
Fig 3: N-SLIT2 enhances p38 MAPK-mediated exocytosis of secondary and tertiary granules.(A–D) 100 μl whole blood from human subjects was exposed to different treatments for 15 min at 37 °C, as indicated. The samples were immediately fixed on ice with 1.6% paraformaldehyde (PFA) and surface CD markers labeled. n=5. (A) Gating strategy for human blood neutrophils: Red blood cells and dead cell debris were excluded based on FSC-A × SSC-A. Doublets were excluded based on SSC-A × SSC-W. Neutrophils were gated in whole blood leukocytes using CD16high × SSC-Ahigh. (B) Human neutrophils were exposed to vehicle, N-SLIT2, or N-SLIT2ΔD2 with or without the p38 MAPK inhibitors, SB 203580 (SB; 10 μM) or p38 MAPK Inhibitor IV (i4; 10 μM), or the MEK1/2 inhibitor PD 184161 (PD; 10 μM) for 15 min, followed by exposure to S. aureus (Sa) for another 15 min at 37 °C, as indicated. Geometric mean fluorescence intensity (gMFI) for CD66b (secondary granules) is shown. p=0.0122 (vehicle vs Sa), p<0.0001 (vehicle vs N-SLIT2 + Sa) p=0.0003 (Sa vs N-SLIT2 + Sa), p=0.0006 (N-SLIT2 + Sa vs N-SLIT2ΔD2 + Sa), p<0.0001 (N-SLIT2 + Sa vs N-SLIT2 + Sa + SB), and p<0.0001 (N-SLIT2 + Sa vs N-SLIT2 + Sa + i4). (C) Neutrophils were treated as in (B) and gMFI for CD18 (secondary and tertiary granules) is noted. p=0.0022 (vehicle vs Sa), p<0.0001 (vehicle vs N-SLIT2 + Sa) p<0.0001 (Sa vs N-SLIT2 + Sa), p<0.0001 (N-SLIT2 + Sa vs N-SLIT2ΔD2 + Sa), p<0.0001 (N-SLIT2 + Sa vs N-SLIT2 + Sa + SB), and p<0.0001 (N-SLIT2 + Sa vs N-SLIT2 + Sa + i4). (D) Human neutrophils were exposed to vehicle, N-SLIT2, or N-SLIT2ΔD2 with or without the p38 MAPK inhibitors, SB 203580 (SB; 10 μM) or p38 MAPK Inhibitor IV (i4; 10 μM), or the MEK1/2 inhibitor PD 184161 (PD; 10 μM) for 15 min, followed by exposure to S. aureus (Sa) for another 15 min at 37 °C, as indicated. Primed neutrophils were identified by cell surface labeling CD66bhigh × CD11bhigh and fold changes in % primed neutrophils relative to vehicle treatment are shown. p=0.0246 (vehicle vs Sa), p<0.0001 (vehicle vs N-SLIT2 + Sa) p=0.0002 (Sa vs N-SLIT2 +Sa), p=0.0008 (N-SLIT2 + Sa vs N-SLIT2ΔD2+ Sa), p<0.0001 (N-SLIT2 + Sa vs N-SLIT2 + Sa + SB), and p<0.0001 (N-SLIT2 + Sa vs N-SLIT2 + Sa + i4). (E) Human neutrophils were exposed to vehicle or N-SLIT2 with or without p38 MAPK inhibitors, SB or i4, or the MEK1/2 inhibitor PD for 15 min, then exposed to S. aureus (Sa) for another 15 min at 37 °C, as indicated. Supernatants were collected and secreted LL-37 levels were measured using an ELISA. n=4. p=0.0092 (vehicle vs Sa), p<0.0001 (vehicle vs N-SLIT2 + Sa) p=0.0005 (Sa vs N-SLIT2 + Sa), p<0.0001 (N-SLIT2 + Sa vs N-SLIT2 + Sa + SB), and p<0.0001 (N-SLIT2 + Sa vs N-SLIT2 + Sa + i4). Mean values ± SEM. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. The source data are available as Figure 3—source data 1. Figure 3—source data 1.The file contains source data for Figure 3B–E.
Fig 4: N-SLIT2 does not significantly affect NETosis.(A–B) Blood isolated from healthy human donors was exposed to vehicle (0.9% NaCl), or N-SLIT2 or N-SLIT2ΔD2 (total volume: 50 μl) for 45 min. Samples were diluted with Real-time deformability cytometry (RT-DC) buffer (950 μl), RT-DC was performed, and neutrophil deformation (A) and neutrophil area (B) were calculated. n=4 biological replicates. For neutrophil deformation (A), p=0.0955 (vehicle vs N-SLIT2) and p=0.0581 (N-SLIT2 vs N-SLIT2ΔD2) and for the neutrophil area (B), p=0.0676 (vehicle vs N-SLIT2) and p=0.0631 (N-SLIT2 vs N-SLIT2ΔD2). (C–G) Representative histograms are shown for human neutrophils with different treatments as described in Figure 3 to detect cell-surface expression of the following CD markers: (C) CD63, (D) CD66b, (E) CD18, (F) CD16, and (G) CD11b. (H–J) Neutrophils isolated from healthy human donors were incubated with vehicle, S. aureus (MOI 10), N-SLIT2, N-SLIT2ΔD2, alone or in combination as indicated in a 96-well plate and Sytox Green (5 μM) was added to each well. Fluorescence was measured at 15 min intervals for 3 hr and NETotic indices were calculated. n=4 biological replicates. (H) Average of all readings is shown. Shaded regions represent Mean values ± SEM. NETotic indices at 2 hr (I), and 3 hr (J) are also shown. (I) p=0.0004 (vehicle vs S. aureus), p<0.0001 (vehicle vs N-SLIT2 + S. aureus), p=0.0003 (vehicle vs N-SLIT2ΔD2+ S. aureus), p=0.0883 (S. aureus vs N-SLIT2 + S. aureus), and p=0.1096 (N-SLIT2 + S. aureus vs N-SLIT2ΔD2+ S. aureus). (J) p<0.0001 (vehicle vs S. aureus), p<0.0001 (vehicle vs N-SLIT2 + S. aureus), p<0.0001 (vehicle vs N-SLIT2ΔD2+ S. aureus), p=0.0949 (S. aureus vs N-SLIT2 + S. aureus), and p=0.0732 (N-SLIT2 + S. aureus vs N-SLIT2ΔD2+ S. aureus). *p<0.05, ***p<0.001, and ****p<0.0001. The source data are available as Figure 3—figure supplement 1—source data 1. Figure 3—figure supplement 1—source data 1.The file contains source data for Figure 3—figure supplement 1A, B, I, J.
Fig 5: N-SLIT2 augments extracellular reactive oxygen species (ROS) production in response to S. aureus.(A) Neutrophils, isolated from healthy human donors, were incubated with vehicle (HBSS), N-SLIT2 (30 nM) or N-SLIT2ΔD2 (30 nM) for 15 min, followed by exposure to S. aureus (MOI 10) for the indicated times. Extracellular S. aureus counts were determined by serial dilution. n=3. The statistical comparisons between N-SLIT2 and N-SLIT2ΔD2 groups are shown. p=0.0072 (0.5 hr), p=0.0105 (1 hr), p=0.0478 (1.5 hr), and p=0.0852 (2 hr). (B) Human neutrophils were treated with vehicle, N-SLIT2 or N-SLIT2ΔD2 for 15 min and then incubated with unoposonized S. aureus expressing GFP (MOI 10) for an additional 45 min. Extracellular bacteria were labeled using donkey anti-human IgG-Cy3. Neutrophil plasma membranes were labeled using Concanavalin A-AF647. At least 100 neutrophils per treatment were imaged. n=3. The phagocytic efficiency (C) and index (D) were calculated. (E) The experiments were performed as in ‘A’ and extracellular ROS production was measured every 5 min using isoluminol relative luminescent units (RLU). n=4. The averages of four experiments are shown. The timepoint with maximum extracellular ROS (35 min) is marked with a dotted rectangle. (F) Extracellular ROS production corresponding to maximum isoluminol RLU was compared among experimental groups. p=0.0031 (vehicle vs S. aureus), p=0.0099 (S. aureus vs N-SLIT2 + S. aureus), and p=0.0055 (N-SLIT2 + S. aureus vs N-SLIT2ΔD2 + S. aureus). (G) Experiments were performed as described In (E) in parallel incubating N-SLIT2 (30 nM) with N-ROBO1 (NR; 90 nM) for 1 hr at room temperature before adding to the cells. n=4. Averages of all experiments are shown. The timepoint with maximum extracellular ROS (40 min) is marked with a dotted rectangle. (H) The timepoint with maximum isoluminol relative luminescent units (RLU) was compared across experimental groups. p=0.0057 (vehicle vs S. aureus), p=0.0018 (S. aureus vs N-SLIT2 + S. aureus), and p=0.0028 (N-SLIT2 + S. aureus vs N-ROBO1 +N-SLIT2 + S. aureus). Mean values ± SEM. *p<0.05, and **p<0.01. The source data are available as Figure 1—source data 1. Figure 1—source data 1.The file contains source data for Figure 1A, C, D, F, H.
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