Fig 1: NSlit2 induces actin remodeling in macrophages in a ROBO1-dependent manner.a Total RNA was isolated from murine brain and kidney, as well as from RAW264.7 cells and primary murine bone marrow-derived macrophages (BMDM). Robo1 and Robo2 mRNA expression were investigated by RT-PCR. Gapdh was used as a loading control. b A schematic representation of full-length human SLIT2 isoform 1 (accession number- NP_004778.1) protein, and recombinant NSlit2, CSlit2, and Slit2ΔD2 proteins used in this study. c RAW264.7 cells were incubated with vehicle (phosphate-buffered saline), NSlit2, CSlit2, or Slit2ΔD2 for 15 min at 37 °C and allowed to spread on poly-d-lysine-coated coverslips for 1 h. Cells were fixed, permeabilized, and incubated with AF-488-conjugated phalloidin (green) to label polymerized actin. Scale bar, 25 μm. d, e, g, h All data are presented as mean ± standard error of mean (SEM). Comparisons between the groups were made by one-way analysis of variance (ANOVA), followed by post hoc Tukey’s multiple comparison test. n = 50 cells per treatment group per experiment over three independent experiments. d Experiments were performed as in (c) and cell surface area was measured using Volocity 6.3 software. ****p < 0.0001, NSlit2 vs vehicle, CSlit2, or Slit2ΔD2. e Shape factor for cells in (c) using Volocity 6.3 software. ****p < 0.0001, NSlit2 vs vehicle, CSlit2, or Slit2ΔD2. f ROBO1 levels were knocked down in RAW264.7 cells using specific siRNA. Protein levels (band intensity) were quantified using ImageJ software, version 1.51v and normalized to corresponding β-actin levels. n = 3 independent experiments (Supplementary Fig. 1e). Comparison between the two groups was made by unpaired, two-tailed t-test. ***p = 0.0005, scrambled vs Robo1 siRNA. g ROBO1 protein levels were knocked down in RAW264.7 cells. After 72 h, experiments were performed as in (c). Cell surface area was measured as in (d). ****p < 0.0001 for the indicated comparisons and p = 0.9887, NSlit2 vs Slit2ΔD2 in ROBO1 knockdown conditions. (h) Shape factor for cells in (g) was determined as in (e). ****p < 0.0001 for the indicated comparisons and p = 0.4268, NSlit2 vs Slit2ΔD2 in ROBO1 knockdown conditions. Source data for (a, d, e, f–h) are provided as a Source Data file.
Fig 2: 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 3: Blocking endogenous SLIT2 exacerbates tissue injury in S. aureus skin and soft tissue infection (SSTI).(A) Ear skin samples were collected from mock-infected (0) and S. aureus-infected mice at indicated time points, homogenized, and tissue SLIT2 levels were measured using an ELISA. n=6 mice per group. p=0.0290 (Mock infection vs S. aureus 0.5 day), p<0.0001 (Mock infection vs S. aureus 3 days). (B) Representative images of gross pathology of ear tissue from animals treated as described in (Figure 4—figure supplement 1B). (C–D) Samples were collected as described in (B), fixed in formalin, and stained with hematoxylin and eosin. Scale bar = 100 μm (D) Experiments were performed as in (C). The lesions were blindly scored on an ascending scale of severity (0–5). n=6. p=0.0002 (Mock infection vs S. aureus), p<0.0001 (Mock infection vs S. aureus + N-ROBO1), p=0.0060 (S. aureus vs S. aureus + N-ROBO1), p=0.0016 (S. aureus + Ctr IgG 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—source data 1. Figure 4—source data 1.The file contains source data for Figure 4A, D.
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 does not activate PKC signaling in neutrophils.(A) RAW264.7 cells were incubated with vehicle, N-SLIT2 or N-SLIT2ΔD2 for 15 min, and the protein lysates were immunoblotted for phospho-NCF1 (Ser345), and total Neutrophil Cytosolic Factor 1 (NCF1) (Ser345).n=4. (A) representative blot is shown. (B) Experiments were performed as in (A), densitometry was performed, and the ratio of phospho-NCF1/NCF1 was obtained. p=0.0007 (vehicle vs N-SLIT2) and p=0.0008 (N-SLIT2 vs N-SLIT2ΔD2). (C) Representative immunoblot showing lysates from human neutrophils treated with vehicle, N-SLIT2, N-SLIT2ΔD2, or PMA (200 nM) for 15 min and probed with phospho-PKC (Thr497) and β-actin. n=4. (D) Densitometry analysis of phospho-PKC/β-actin ratios for experiments in (C).p<0.0001 (vehicle vs PMA). (E) Representative immunoblot showing lysates from human neutrophils treated with vehicle, N-SLIT2, N-SLIT2ΔD2, or PMA (200 nM) for 15 min and probed with phospho-PKCδ (Tyr311) and total PKCδ. n=4. (F) Densitometry analysis of phospho-PKCδ/PKCδ ratios for experiments in (E).p<0.0001 (vehicle vs PMA). (G) RAW264.7 cells were treated as described in (A) and the protein lysates were immunoblotted for phospho-p38 (Thr180/Tyr182) and total p38. n=4. A representative blot is shown. (H) Densitometry analysis of phospho-p38/p38 ratios for experiments in (E).p<0.0001 (vehicle vs N-SLIT2) and p<0.0001 (N-SLIT2 vs N-SLIT2ΔD2). Mean values ± SEM. ***p<0.001 and ****p<0.0001. The source data are available as Figure 2—figure supplement 1—source data 1 and Figure 2—figure supplement 1—source data 2. Figure 2—figure supplement 1—source data 1.The file contains source data for Figure 2—figure supplement 1B, D, F, H. Figure 2—figure supplement 1—source data 2.The file contains source data for Figure 2—figure supplement 1A, C, E, G.
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