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• W4387168341 abstract "Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Abstract Neutrophils are essential for host defense against Staphylococcus aureus (S. aureus). The neuro-repellent, SLIT2, potently inhibits neutrophil chemotaxis, and might, therefore, be expected to impair antibacterial responses. We report here that, unexpectedly, neutrophils exposed to the N-terminal SLIT2 (N-SLIT2) fragment kill extracellular S. aureus more efficiently. N-SLIT2 amplifies reactive oxygen species production in response to the bacteria by activating p38 mitogen-activated protein kinase that in turn phosphorylates NCF1, an essential subunit of the NADPH oxidase complex. N-SLIT2 also enhances the exocytosis of neutrophil secondary granules. In a murine model of S. aureus skin and soft tissue infection (SSTI), local SLIT2 levels fall initially but increase subsequently, peaking at 3 days after infection. Of note, the neutralization of endogenous SLIT2 worsens SSTI. Temporal fluctuations in local SLIT2 levels may promote neutrophil recruitment and retention at the infection site and hasten bacterial clearance by augmenting neutrophil oxidative burst and degranulation. Collectively, these actions of SLIT2 coordinate innate immune responses to limit susceptibility to S. aureus. Editor's evaluation Bhosle and colleagues present valuable findings on the function of the N-terminal fragment of SLIT2 in the amplification of reactive oxygen species production and exocytosis of secretory granules by neutrophils. The authors present solid in vitro and in vivo data supporting this unexpected role for SLIT2. This work advances our knowledge of innate immunity to pathogens. https://doi.org/10.7554/eLife.87392.sa0 Decision letter eLife's review process Introduction Staphylococcus aureus (S. aureus) is a commensal bacterium as well as a skillful, facultative pathogen causing diverse human diseases ranging from localized SSTI to life-threatening disseminated sepsis (Krismer et al., 2017; Lowy, 1998). Neutrophils, the most abundant subset of leukocytes in circulation, form a formidable first-line of defense against S. aureus invasion and spread (Guerra et al., 2017). Accordingly, patients with neutropenia and defective neutrophil functions are highly susceptible to recurrent and more severe S. aureus infections (Buvelot et al., 2017; Donadieu et al., 2011; Neehus et al., 2021). Infections caused by antibiotic-resistant strains of S. aureus have been steadily increasing worldwide, suggesting that strategies to enhance the effective recruitment of neutrophils and their inherent bactericidal properties are needed to combat the morbidity and mortality associated with S. aureus infections (Chambers and Deleo, 2009; Jernigan et al., 2020). A recent transcriptomic study noted that mRNA encoding Slit guidance ligand 2 (SLIT2), a canonical neuro-repellent, is locally upregulated during S. aureus-induced mastitis (Günther et al., 2017). Among the three mammalian SLIT family members, SLIT1 is exclusively expressed in the nervous system (Wu et al., 2001), while SLIT2 and SLIT3 are also detected outside the nervous system (Bhosle et al., 2020; Kim et al., 2018). We and others have previously reported that SLIT2 inhibits directed neutrophil migration in vitro as well as in vivo (Chaturvedi et al., 2013; Tole et al., 2009; Wu et al., 2001; Ye et al., 2010; Zhou et al., 2022). We also showed that the cleaved N-terminal fragment of SLIT2, N-SLIT2, acts via its receptor, Roundabout guidance receptor 1 (ROBO1), to attenuate inflammasome activation in macrophages by inhibiting macropinocytosis (Bhosle et al., 2020). Additionally, we and others previously reported that primary human and murine neutrophils express ROBO1 (Rincón et al., 2018; Tole et al., 2009; Ye et al., 2010), but not ROBO2 (Rincón et al., 2018). Despite the inhibitory actions of N-SLIT2 on chemotaxis and macropinocytosis in innate immune cells, administration of N-SLIT2 in vivo does not confer increased susceptibility to bacterial infections (Chaturvedi et al., 2013; London et al., 2010). It remains unknown if and how N-SLIT2 affects neutrophil responses during S. aureus infection. Herein, we show that N-SLIT2 does not inhibit, but rather enhances, the killing of extracellular S. aureus by neutrophils. In the presence of N-SLIT2, human and murine neutrophils respond more robustly to S. aureus by activating p38 mitogen-activated protein kinase (MAPK) signaling, enhancing the production of extracellular reactive oxygen species (ROS) and release of secondary and tertiary granule contents by neutrophils. In a murine model of S. aureus-induced SSTI (Prabhakara et al., 2013), we found that endogenous levels of SLIT2 protein declined significantly early on but rose to peak levels approximately 3 days after infection, and that blocking endogenous SLIT2-ROBO1 signaling at the site of infection enhanced bacterial survival and worsened the infection. Our results suggest that changes in the levels of SLIT2 at local sites of infection may coordinate neutrophil recruitment, retention, and bactericidal responses to effectively and synergistically target S. aureus. Our work identifies SLIT2 as an endogenous regulator of neutrophil number and activity that coordinates immune responses vital to combat disseminated infection of bacterial pathogens. Results N-SLIT2 augments extracellular ROS production in response to S. aureus We first investigated how SLIT2 affects neutrophil-mediated killing of S. aureus. Incubation of primary human neutrophils with bioactive N-SLIT2, but not inactive N-SLIT2ΔD2 which lacks the ROBO1/2-binding D2 LRR domain (Patel et al., 2012), significantly reduced extracellular S. aureus as early as 30 min (Figure 1A). Decreased extracellular bacteria could result from increased internalization via phagocytosis, and/or increased extracellular bacterial killing. Intriguingly, N-SLIT2 did not affect the ability of human neutrophils (Figure 1B–D) or RAW264.7 murine macrophages to phagocytose S. aureus (Figure 1—figure supplement 1A–C). To determine whether N-SLIT2 decreased bacterial survival by stimulating phagocyte ROS production, a process dependent on NADPH oxidase complex (NOX) activity, we incubated neutrophils with the pan-NOX-inhibitor, diphenyleneiodonium chloride (DPI). DPI partially restored extracellular bacterial survival in the presence of N-SLIT2 (Figure 1—figure supplement 1D). Next, we directly examined the effects of N-SLIT2 on extracellular ROS production by human neutrophils exposed to S. aureus. Neutrophils incubated with vehicle control or with N-SLIT2 alone had very low basal levels of ROS. As expected, S. aureus induced significant ROS production that was indistinguishable from that seen in cells incubated with S. aureus and bio-inactive N-SLIT2ΔD2 (Figure 1E–F). Surprisingly, human neutrophils co-incubated with bio-active N-SLIT2 and S. aureus produced significantly more extracellular ROS than S.aureus-exposed neutrophils incubated with vehicle or N-SLIT2ΔD2 (Figure 1E–F). We found that neutrophils exposed to another secondary ROS-inducing stimulus, namely phorbol-12-myristate-13-acetate (PMA), also produced more extracellular ROS in the presence of N-SLIT2 compared to N-SLIT2ΔD2 (Figure 1—figure supplement 1E). Similar observations were also noted for mouse bone marrow-derived neutrophils (BMDN) indicating that the effect of N-SLIT2 to further enhance extracellular ROS production by S. aureus-exposed neutrophils is not species-specific (Figure 1—figure supplement 1F). To determine whether the observed effects of N-SLIT2 occurred through the canonical ROBO1 receptor, we pre-incubated N-SLIT2 with the soluble N-terminal fragment of the ROBO1 receptor (N-ROBO1). N-ROBO1 fully blocked the ability of N-SLIT2 to boost extracellular ROS production in neutrophils exposed to S. aureus (Figure 1G–H). Since activation of Rac GTPases is essential for optimal NOX function in neutrophils (Diebold and Bokoch, 2001; Hordijk, 2006), we next tested the effects of N-SLIT2 on Rac. N-SLIT2 alone failed to activate Rac but instead required a second stimulus, namely S. aureus (Figure 1—figure supplement 1G). Together, these results demonstrate that N-SLIT2 enhances neutrophil-mediated killing of S. aureus, partly by amplifying extracellular ROS production in a ROBO1-dependent manner. Figure 1 with 1 supplement see all Download asset Open asset 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. https://cdn.elifesciences.org/articles/87392/elife-87392-fig1-data1-v1.xlsx Download elife-87392-fig1-data1-v1.xlsx N-SLIT2 primes NOX by p38-mediated phosphorylation of NCF1 We next studied how N-SLIT2 increases extracellular ROS production in neutrophils. We wondered whether N-SLIT2 induces NOX priming and extracellular ROS production by prompting phosphorylation and translocation of Neutrophil Cytosolic Factor 1 (NCF1; p47phox), a key component of the NOX complex, to the plasma membrane (El-Benna et al., 2009; Li et al., 2010). We found that N-SLIT2 increased phosphorylation of the conserved Ser345 residue of NCF1 in neutrophils (Figure 2A–B), as well as in RAW264.7 macrophages (Figure 2—figure supplement 1A–B). As SLIT2-ROBO2 signaling was recently reported to activate NOX function in neurons by activating protein kinase C (PKC) (Terzi et al., 2021), we examined the effects of N-SLIT2 on PKC activation in neutrophils. PKC activity in cells is tightly regulated by its phosphorylation (Freeley et al., 2011). N-SLIT2 did not induce phosphorylation of PKC in neutrophils (Figure 2—figure supplement 1C–F). SLIT2 orthologues have been reported to activate p38 MAPK signaling in Xenopus and C. elegans neurons (Campbell and Okamoto, 2013; Piper et al., 2006). The p38 MAP kinases are also known to phosphorylate NCF1 at the Ser345 residue to mediate NOX priming (Dang et al., 2006). We, therefore, investigated the effects of N-SLIT2 on p38 MAPK signaling in mammalian phagocytes. N-SLIT2 potently induced p38 MAPK activation in both neutrophils (Figure 2C–D) and macrophages (Figure 2—figure supplement 1G–H). Congruent with these results, incubation with two distinct pharmacologic inhibitors of p38 MAPKs blocked the observed N-SLIT2-induced increase in extracellular ROS in neutrophils exposed to S. aureus (Figure 2E–F). We and others previously showed that SLIT2-ROBO1 signaling robustly activates RhoA, which in turn can activate Rho-associated protein kinases (ROCK), in several cell types, including macrophages and cancer cells (Bhosle et al., 2020; Kong et al., 2015). We found that in the presence of the selective ROCK inhibitor, Y-27632, N-SLIT2 failed to activate p38 MAPK in neutrophils (Figure 2G–H). These findings indicate that N-SLIT2 does not activate, but rather primes, the NOX complex in a p38 MAPK-dependent manner in phagocytes to upregulate ROS production in response to injurious biologic and pharmacological secondary stimuli, including S. aureus and PMA, respectively. Additionally, N-SLIT2-induced activation of the RhoA/ROCK pathway is essential for its effect on p38 MAPK signaling in neutrophils. Figure 2 with 1 supplement see all Download asset Open asset N-SLIT2 primes NADPH oxidase complex (NOX) by p38-mediated phosphorylation of Neutrophil Cytosolic Factor 1 (NCF1). (A) Human neutrophils were exposed to vehicle, N-SLIT2 or N-SLIT2ΔD2 for 15 min and the protein lysates were immunoblotted for phospho-NCF1 (Ser345) and total 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. n=4. p=0.0002 (vehicle vs N-SLIT2) and p=0.0004 (N-SLIT2 vs N-SLIT2ΔD2). (C) Experiments were performed as in (A) and immunoblotting performed for phospho-p38 (Thr180/Tyr182) and total p38. n=4. A representative blot is shown. (D) Experiments were performed as in (C), densitometry was performed, and the ratio of phospho-p38 /p38 was obtained. p=0.0006 (vehicle vs N-SLIT2) and p=0.0004 (N-SLIT2 vs N-SLIT2ΔD2). (E) Human neutrophils were incubated with vehicle, S. aureus, N-SLIT2 + S. aureus, N-SLIT2 + S. aureus + SB203580 (SB, 10 μM), or N-SLIT2 + S. aureus + p38 MAPK Inhibitor IV (i4, 10 μM), and extracellular reactive oxygen species (ROS) were measured as described in Figure (1E). n=4. The averages of four experiments are shown. The timepoint with maximum extracellular ROS (40 min) is marked with a dotted rectangle. (F) Experiments were performed as in (E) and extracellular ROS production at 40 min compared among groups. n=4. p=0.0036 (S. aureus vs N-SLIT2 + S. aureus), p<0.0001 (N-SLIT2 + S. aureus vs N-SLIT2 + S. aureus +SB203580), and p<0.0001 (N-SLIT2 + S. aureus vs N-SLIT2 + S. aureus + p38 MAPK Inhibitor IV). (G) Human neutrophils were exposed to vehicle, N-SLIT2 or N-SLIT2 and Y-27632 together for 15 min and immunoblotting was performed for phospho-p38 (Thr180/Tyr182) and total p38. n=4. A representative blot is shown. (H) Experiments were performed as in (G), densitometry was performed, and the ratio of phospho-p38 /p38 was obtained. p=0.0004 (vehicle vs N-SLIT2) and p=0.0007 (N-SLIT2 vs N-SLIT2 + Y-27632). Mean values ± SEM. **p<0.01, ***p<0.001, and ****p<0.0001. The source data are available as Figure 2—source data 1 and Figure 2—source data 2. Figure 2—source data 1 The file contains source data for Figure 2B, D, F, H. https://cdn.elifesciences.org/articles/87392/elife-87392-fig2-data1-v1.xlsx Download elife-87392-fig2-data1-v1.xlsx Figure 2—source data 2 The file contains source data for Figure 2A, C, G. https://cdn.elifesciences.org/articles/87392/elife-87392-fig2-data2-v1.zip Download elife-87392-fig2-data2-v1.zip N-SLIT2 enhances p38 MAPK-mediated exocytosis of secondary and tertiary granules Sequential cytoskeletal changes in neutrophils have been shown to play an important role in their priming (Bashant et al., 2019; Toepfner et al., 2018). We performed Real-time deformability cytometry (RT-DC) and found that exposure of blood to N-SLIT2 reduced neutrophil cell area and deformability although the effect did not reach statistical significance (Figure 3—figure supplement 1A–B; Bashant et al., 2019; Toepfner et al., 2018). Next, since exposure to the NOX inhibitor, DPI, only partially reversed the effects of N-SLIT2 on extracellular S. aureus survival, we pondered whether other anti-microbial functions of neutrophils are also modified by N-SLIT2. In addition to the oxidative burst, degranulation has been shown to play an important role in the bactericidal responses of neutrophils against S. aureus (Ferrante et al., 1989; Van Ziffle and Lowell, 2009). Neutrophils contain four types of granules, each containing different classes of anti-microbial peptides, of which secondary and tertiary granules are most important for eliminating S. aureus (Borregaard and Cowland, 1997; Mollinedo, 2019; Van Ziffle and Lowell, 2009). To investigate the effects of N-SLIT2 on degranulation, we utilized a recently optimized flow cytometry protocol using specific cluster of differentiation (CD) markers of granule exocytosis (Fine et al., 2019). Another advantage of using a flow cytometry-based approach is that it circumvents the in vitro neutrophil isolation step which is known to affect cellular activation (neutrophil gating strategy; Figure 3A). Neither S. aureus alone nor S. aureus together with N-SLIT2 induced primary granule (CD63) secretion (Figure 3—figure supplement 1C). Congruent with published literature (Lu et al., 2014; Schmidt et al., 2012), the bacteria alone stimulated secondary granule (CD66b) exocytosis from neutrophils (Figure 3—figure supplement 1D). This effect was strikingly enhanced by exposure to N-SLIT2 (Figure 3B and Figure 3—figure supplement 1D). Additionally, the actions of N-SLIT2 were completely obliterated by pharmacological inhibition of p38 MAPK signaling using SB 203580 or p38 MAPK Inhibitor IV, but not MEK1/2 signaling using PD 184161 (Figure 3B). It is noteworthy that N-SLIT2 also augmented surface expression of S. aureus-induced CD18, which is stored in both secondary and tertiary granules, in a p38 MAPK dependent manner (Figure 3C and Figure 3—figure supplement 1E; Borregaard and Cowland, 1997; Mollinedo, 2019). On the other hand, we found no differences in the surface expression of CD16, which is stored in secretory vesicles, in any of the tested conditions, a finding in agreement with the priming-associated CD marker signature described for neutrophils in circulation and tissues (Figure 3—figure supplement 1F; Fine et al., 2019). We next used the surface expression of CD66b and CD11b to calculate the percentage of primed neutrophils (Figure 3—figure supplement 1D and G; Fine et al., 2019). Exposure of blood to N-SLIT2 and S. aureus together significantly increased the fraction of primed neutrophils as compared to S. aureus alone (Figure 3D). Next, we measured the secretion of LL-37, a highly potent cationic anti-staphylococcal peptide that is selectively stored in neutrophil secondary granules in its pro-peptide form (Noore et al., 2013; Sørensen et al., 1997). Incubation of human neutrophils with S. aureus resulted in the release of LL-37, which was further amplified in the presence of N-SLIT2 and blocked by the p38 inhibitors (Figure 3E). Production of neutrophil extracellular traps (NETosis) is known to be modulated by intra- and extra-cellular ROS and can also regulate anti-bacterial responses by neutrophils in vitro and in vivo (Poli and Zanoni, 2023). We investigated the effects of N-SLIT2 on NETosis and found that exposure to N-SLIT2 alone failed to induce NETosis. Treatment of neutrophils with N-SLIT2 and S. aureus together resulted in more NETosis than that with the bacteria alone but this effect was not statistically significant (Figure 3—figure supplement 1H–J). Collectively, our results indicate that SLIT2-induced activation of p38 MAPK augments the production of extracellular ROS, exocytosis of secondary granules, and secretion of the anti-bacterial LL-37 peptide from neutrophils in response to exposure to S. aureus. Figure 3 with 1 supplement see all Download asset Open asset 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. https://cdn.elifesciences.org/articles/87392/elife-87392-fig3-data1-v1.xlsx Download elife-87392-fig3-data1-v1.xlsx Blocking endogenous SLIT2 exacerbates tissue injury in S. aureus SSTI A recent transcriptomic screen has reported that SLIT2 mRNA is upregulated during S. aureus-induced mastitis (Günther et al., 2017). To determine whether this increase is conserved at a protein level during SSTI, we used an established murine model of S. aureus SSTI, which mimics community-acquired human infections (Prabhakara et al., 2013). Surprisingly, we found that local SLIT2 levels were reduced as early as 12 hr after the infection (Figure 4A). Following the initial decrease, the levels of SLIT2 gradually increased to reach a peak at 3 days (Figure 4A). Unlike SLIT2, SLIT3 levels in the infected tissue remained significantly lower than in control mock-infected tissue for the first 2 days (Figure 4—figure supplement 1A). To ascertain the target of SLIT2 in S. aureus SSTI, we used a soluble N-ROBO1 to block endogenous SLIT2 (Bhosle et al., 2020; Geraldo et al., 2021). Based on the observed peak in endogenous SLIT2 levels at Day 3 after S. aureus infection, N-ROBO1 or control IgG was administered on Days 2 and 3 after infection (Figure 4—figure supplement 1B). Strikingly, the bacterial counts were more than twofold higher in N-ROBO1-treated mice as compared to the IgG-treated counterparts (Figure 4—figure supplement 1C). We and others have shown that SLIT2-ROBO1 signaling inhibits chemotactic neutrophil migration in vivo but this has not been examined in the context of an infection so far (Chaturvedi et al., 2013; Tole et al., 2009; Zhou et al., 2022). S. aureus infection in mice who received control IgG treatment or no other treatment resulted in microabscess formation with immune cell infiltration (Figure 4B–D). In N-ROBO1-treated mice, S. aureus infection caused much more extensive tissue injury characterized by diffuse, rather than localized, inflammation (Figure 4B–D). Interestingly, in the absence of bacterial infection, neutralization of endogenous SLIT2 augmented immune cell infiltration in the skin but did not result in local tissue damage in the form of acanthosis (Figure 4C). Next, we directly examined tissue neutrophil infiltration in SSTI using immunohistochemical (IHC) staining (Ly6G+F4/80- cells) (Chadwick et al., 2021). In the absence of SSTI, N-ROBO1 treatment alone was sufficient to increase neutrophil infiltration (Figure 4—figure supplement 1D–E). In line with our earlier findings using H&E, animals administered S. aureus and N-ROBO1 exhibited significantly more local neutrophil infiltration in infected tissue as compared to those administered S. aureus alone or S. aureus with control IgG (Figure 4—figure supplement 1D–E). Finally, we used IHC to measure 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels as a marker of free radical-induced oxidative tissue injury (Sima et al., 2016). N-ROBO1-treated mice had an almost 50% reduction in the 8-OHdG-positive lesion area as compared to animals which received either S. aureus alone or S. aureus and control IgG (Figure 4—figure supplement 1F–G). Together, our findings suggest that immediately after infection the rapid decrease in local levels of SLIT2 promotes infiltration of neutrophils into the site of infection. The later increase in SLIT2 levels may serve dual functions: local retention of neutrophils due to SLIT2’s chemorepellent actions, and direct effects on neutrophils to enhance ROS production and anti-staphylococcal killing responses. Figure 4 with 1 supplement see all Download asset Open asset 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. https://cdn.elifesciences.org/articles/87392/elife-87392-fig4-data1-v1.xlsx Download elife-87392-fig4-data1-v1.xlsx Dermal microvascular endothelial cells are a source of SLIT2 during S. aureus infection We next investigated the potential cellular source of SLIT2 during SSTI. Under specific non-infectious conditions, dermal microvascular endothelial cells (DMEC) and fibroblasts have been previously reported to secrete SLIT2 (Pilling et al., 2014; Romano et al., 2018). To test this possibility, we used a human dermal microvascular endothelial cell line, HMEC-1 (Ades et al., 1992). Infection of HMEC-1 with S. aureus was sufficient to reduce SLIT2 production at 12 hr at the level of mRNA as well as protein (Figure 5A–B). However, the effect was reversed at 48 hr, at which time infected cells produced more SLIT2 than their non-infected counterparts (Figure 5A–B). We next examined how other factors which could be found in a Staphylococcal abscess, namely hypoxia and low pH (acidosis), (Costa and Horswill, 2022; Zhang et al., 2022), modulate SLIT2 production by vascular endothelial cells. Lowering the pH of the medium to 6.6 did not change SLIT2 mRNA in HMEC-1 cells (Figure 5—figure supplement 1A). In contrast, growing the cells in a hypoxic (1% O2) environment replicated the effects of S. aureus infection on SLIT2 production (Figure 5C–D). Finally, we tested the ability of conditioned media from HMEC-1 cells to regulate LL-37 secretion by neutrophils. Conditioned media from HMEC-1 cells taken 48 hr after bacterial infecti" @default.
• W4387168341 created "2023-09-30" @default.
• W4387168341 date "2023-03-20" @default.
• W4387168341 modified "2023-09-30" @default.
• W4387168341 title "Decision letter: The chemorepellent, SLIT2, bolsters innate immunity against Staphylococcus aureus" @default.
• W4387168341 doi "https://doi.org/10.7554/elife.87392.sa1" @default.
• W4387168341 hasPublicationYear "2023" @default.
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