The chemorepellent, SLIT2, bolsters innate immunity against Staphylococcus aureus
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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 .
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Reply to the reviewers
We sincerely thank the reviewers for their comprehensive and constructive feedback. Below, we submit our revision plan addressing the points raised by the reviewers.
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
The study analyzes the role of SLIT2 in clearance of S. aureus via neutrophils. It suggests that N-SLIT2 play a key role as an amplifier of the ROS response and release of antimicrobial peptides. The manuscript is well written and technologically …
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
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Reply to the reviewers
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
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Reply to the reviewers
We sincerely thank the reviewers for their comprehensive and constructive feedback. Below, we submit our revision plan addressing the points raised by the reviewers.
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
The study analyzes the role of SLIT2 in clearance of S. aureus via neutrophils. It suggests that N-SLIT2 play a key role as an amplifier of the ROS response and release of antimicrobial peptides. The manuscript is well written and technologically sound. However, a few issues need to be addressed that preclude publication of the manuscript:
We thank the reviewer for the positive feedback.
Major comments:
The study analyzes different parameters of neutrophil function. One major effect of neutrophil activation is NETosis. This has not been addressed in the study albeit it is deemed to act in concert with the other immune mechanisms described.
We thank the reviewer for the suggestion. S. aureus is known to promote NET formation as well as to enhance NET degradation to increase bacterial survival in vivo (Meyers, Crescente et al., 2022, Thammavongsa, Missiakas et al., 2013). Several cellular kinases (Erk, Akt, p38) have been implicated in ROS-induced NETosis, but the exact role of p38 signaling in NETosis remains less clear (Douda, Khan et al., 2015). As recommended by the reviewers, we will now investigate whether N-SLIT2 regulates S. aureus-induced NETosis in neutrophils using Sytox Green, a membrane-impermeable nucleic acid label, as previously described (Douda et al., 2015).
Furthermore, the authors discuss a role of SLIT2 in the regulation of neutrophil migration. However, the current data set does not provide sufficient evidence for this. The reviewer suggests that the authors provide migration/chemotaxis assays and/or in vivo data to prove their hypothesis or revise their argumentation.
Several groups, including ours, have previously demonstrated that SLIT2-ROBO1 signaling potently inhibits neutrophil chemotaxis in vitro and in vivo. The* in vivo* models, in which the negative effects of SLIT2 on neutrophil migration have been shown, include mouse models of peritonitis (Tole, Mukovozov et al., 2009), allergic airway inflammation (Ye, Geng et al., 2010), renal ischemia-reperfusion injury (Chaturvedi, Yuen et al., 2013), and cholangiocarcinoma (Zhou, Luo et al., 2022). Additionally, a recent study showed that shRNA-mediated knockdown of SLIT2 resulted in increased neutrophil infiltration into murine tumors further supporting negative regulatory effect of SLIT2 on neutrophil migration (Geraldo, Xu et al., 2021). In the revised version of the manuscript, we will now discuss these important points in the Introduction and Discussion sections.
In our current study, in an effort to selectively examine the effects of SLIT2 on neutrophil function rather than on neutrophil migration, we intentionally administered N-ROBO1 to block endogenous SLIT2 signaling at 48 and 72 hours after inducing skin and soft tissue infection (SSTI) with S. aureus. In this model, the majority of neutrophil influx occurs early on, namely within 24 hours (Prabhakara, Foreman et al., 2013). We observed that blocking endogenous SLIT2 signaling in a murine model of SSTI resulted in enhanced localized infection and injury. We will now use immunohistochemical analysis to measure tissue infiltration of neutrophils (Ly6G+F4/80-) (Chadwick, Macdonald et al., 2021). In addition, as previously described we will also use IHC to evaluate within the tissue 8-hydroxydeoxyguanosine (8-OHdG), an indicator of oxidative damage (Sima, Aboodi et al., 2016). We will compare levels of 8-OHdG to the number of neutrophils in the tissue as a gross indicator of local ROS production by infiltrating neutrophils.
The timeline of SLIT2 expression indicates that environmental conditions could influence the expression of SLIT2. Have the authors analyzed whether SLIT2 expression is affected by low pH or hypoxia? Is there any data indicating what factors regulate SLIT2 expression? In the same line, it would be interesting to know whether SLIT2 immune effects (specifically ROS and LL37 release) are similarly triggered under hypoxic conditions often found in an abscess.
We thank the reviewer for raising this important point and for the suggestions. The regulation of SLIT2 levels in tissues is an active area of research. Hypoxia has been reported to increase SLIT2 expression in placental tissue (Liao, Laurent et al., 2012) but this has not been investigated in the context of bacterial infection. In different physiologic and pathophysiologic settings, vascular endothelial cells, including dermal microvascular endothelial cells (DMEC), have been shown to be an important source of SLIT2 (Romano, Manetti et al., 2018, Tavora, Mederer et al., 2020). We will therefore investigate the effects of hypoxia and low pH, conditions founds within bacterial abscesses, on production of SLIT2 by DMEC. DMEC will be infected with S. aureus and grown in normoxic and hypoxic (2% O2) conditions for up to 72 hours, the time-point at which maximal SLIT2 levels were detected in S. aureus-induced SSTI. We will collect cells and cultured supernatant for measurement of levels of Slit2 mRNA and SLIT2 protein at different time points ranging from 0 to 72 hours after infection. We will incubate neutrophils with the conditioned medium from hypoxic DMEC to measure the effect on LL-37 secretion. Finally, we will expose neutrophils to S. Aureus (+/- N-SLIT2) in a medium with pH ranging from 5.5 to 7.4 and then measure the LL-37 secretion as the reviewer suggested (Zhou & Fey, 2020).
Lastly, it is unclear whether SLIT2 binds to a defined target on the neutrophil. This needs to be highlighted in the discussion in respect to future work and ideally resolved experimentally.
We apologize for the confusion. We and others have previously demonstrated that human and murine neutrophils express ROBO1 but not ROBO2, and that ROBO1 is the primary Roundabout receptor which binds N-SLIT2 in immune cells (Rincon, Rocha-Gregg et al., 2018, Tole et al., 2009). We have now included this information in the Introduction section (please see page- 3). In our manuscript we showed experimentally that incubation of N-SLIT2 with the soluble N-terminal fragment of ROBO1 (N-ROBO1), which contains the N-SLIT2 binding Ig1 motif (Morlot, Thielens et al., 2007), blocked the effect of N-SLIT2 on ROS production, thereby confirming that the observed actions of SLIT2 occurred through ROBO1 (Fig. 1G). In the revised version of the manuscript, we will clarify this point.
Reviewer #1 (Significance (Required)):
The manuscript provides insight into a new mechanism regulating neutrophil function in the presence of S. aureus. The study provides evidence that the N-terminus of SLIT2 is involved in these effects and highlights p38-mediated signaling events as molecular targets increasing antibacterial effects in neutrophils. However, some contradictory findings imply that timing of the response is crucial.
Nevertheless, with the molecular mechanisms not fully understood many questions remain and the study adds to the complexity of the anti-staphylococcal immune response. Therefore, the audience for this manuscript requires knowledge on S. aureus-specific host-pathogen interaction and is not suitable for a broad audience as it does not provide information on a generally new mechanism of neutrophil activation or defense.
We thank the reviewer for pointing out the complexity of host-pathogen (neutrophils and S. aureus) interactions. SLIT2 is well-known for its anti-inflammatory properties via its effects on immune cell chemotaxis* in vivo *(Anand, Zhao et al., 2013, Chaturvedi et al., 2013, Geraldo et al., 2021). We demonstrated that SLIT2-ROBO1 signaling inhibits macropinocytosis in macrophages, and consequently, attenuates NOD2-induced inflammasome activation in mice (Bhosle, Mukherjee et al., 2020). Based on these earlier observations, SLIT2 would be anticipated to impair the innate immune response to infection. Unexpectedly, we found that SLIT2 does not impair, but instead enhances the ability of neutrophils to kill S. aureus. Indeed, through different mechanisms SLIT2 has been shown to have widespread anti-microbial properties against not only S. aureus but instead against diverse pathogens, including *Mycobacterium tuberculosis, intestinal pathogens, *H5N1 influenza, and most recently, COVID-19 (Gustafson, Ngai et al., 2022, London, Zhu et al., 2010). Together, these studies highlight the importance of spatiotemporal regulation of SLIT2 levels in tissues during bacterial and viral infection and the direct effects of SLIT2 on modulating host-pathogen interactions.
Additionally, SLIT2-induced p38 MAPK activation is not limited to innate immune cells. Li et al. reported this week that SLIT2-ROBO1 signaling activates p38 in pancreatic ductal adenocarcinoma cells as well as metastatic tumors (Li, Zhang et al., 2023). In the revised manuscript, we will discuss all of the important points above.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
Summary: The manuscript deals with the role of the neurorepellent SLIT2 in killing of the bacterial pathogen Staphylococcus aureus. The authors show that neutrophils incubated with the N-terminal region of SLIT2 kill S. aureus more efficiently than neutrophils without pre-exposure to N-SLIT2. This effect was due to an increased production of reactive oxygen species by NADPH oxidase complex activation and stimulating exocytosis of antibacterial peptide containing granules. The concept was proven in an animal model of skin and soft tissue infection in mice in which neutralization of endogenous SLIT2 reduced CFU numbers in ear skin and decreased tissue destruction in response to S. aureus infection.
Major comments:
In general the findings and key conclusions are convincingly covered by the results presented in the manuscript. The methods are adequate to allow the conclusions drawn. Data are clearly presented and easy to follow. Statistical methods are appropriate.
We thank the reviewer for the positive feedback. In the present study, we investigated the effects of SLIT2 on NADPH oxidase (NOX – p47phox) priming. Using novel methodology, neutrophil priming was recently shown to be associated with characteristic cytoskeletal changes (Bashant, Vassallo et al., 2019). We are now collaborating with Dr. Nicole Toepfner (Technische Universität Dresden, Dresden) to investigate SLIT2-induced cytoskeletal changes in neutrophils isolated from whole blood using Real-time deformability cytometry (RT-DC). We believe that these novel studies will further enhance the revised manuscript.
Minor comments:
In the Materials and Methods section line 340 a GFP-expressing S. aureus USA300 strain is indicated. What was the exact strain designation, e.g. LAC or JE2, as USA300 is not a strain name (different strains belong to this pulsed-field electrophoresis based classification).
We thank the reviewer for this comment. The strain designation of the GFP-expressing S. aureus we used is USA300 LAC (Flannagan, Kuiack et al., 2018). In the revised version of the manuscript we will include the correct information (please see page- 10).
In the legend of figure 3 the inhibitors are mentioned for part B and E but not C and D.
We apologize for the error. Figure 3 legend has now been corrected in the revised manuscript.
Figure S4 would be nice to have in the main manuscript.
We thank the reviewer for the suggestion. In the revised manuscript we moved original Supplementary Fig. S4B to main Fig. 4B in the manuscript. The schematic from main Fig. 4B is moved to the new Supplementary Fig. 4B. The graphical summary (original Supplementary Fig. S4C) is now presented as new main Figure 5.
Reviewer #2 (Significance (Required)):
The manuscript deals with a novel mechanism of neutrophil activation by SLIT-2, a protein which was originally thought to act in the nervous system but is also expressed in many peripheral tissues. Importantly SLIT-2 may be involved in tumor suppression but also chemotaxis of immune cells. In this manuscript a novel, rather unexpected role of the N-terminal region of SLIT-2 in activation of antibacterial mechanisms of neutrophils was shown. This could be interesting for a broader readership interested in innate immune mechanisms and bacterial infections. Since little is known on the role of SLIT-2 in response to bacterial infections the paper could initiate a number of new studies in this field. This reviewer has experience with S. aureus virulence and resistance mechanisms and animal infection models.
We thank the reviewer for the very positive feedback regarding the appeal of our manuscript to a broad readership. As noted in our response to Reviewer #1 Significance, recent studies suggest that SLIT2 could not only serve as a therapeutic to combat S. aureus, but could have broad anti-microbial activity against a number of pathogens including *Mycobacterium tuberculosis, intestinal pathogens, *H5N1 influenza, and COVID-19 (Borbora, Bhatt et al., 2022, Gustafson et al., 2022, London et al., 2010). We believe that the ability of SLIT2 to combat diverse bacterial and viral infections will even further enhance the appeal of our manuscript to a broad audience. In the revised manuscript we will expand the discussion to include these very important points.
References:
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Bashant KR, Vassallo A, Herold C, Berner R, Menschner L, Subburayalu J, Kaplan MJ, Summers C, Guck J, Chilvers ER, Toepfner N (2019) Real-time deformability cytometry reveals sequential contraction and expansion during neutrophil priming. J Leukoc Biol 105: 1143-1153
Bhosle VK, Mukherjee T, Huang YW, Patel S, Pang BWF, Liu GY, Glogauer M, Wu JY, Philpott DJ, Grinstein S, Robinson LA (2020) SLIT2/ROBO1-signaling inhibits macropinocytosis by opposing cortical cytoskeletal remodeling. Nat Commun 11: 4112
Borbora SM, Bhatt S, Balaji KN (2022) Mycobacterium tuberculosis infection elevates SLIT2 expression to modulate oxidative stress responses in macrophages. bioRxiv: 2022.10.13.512188
Chadwick JW, Macdonald R, Ali AA, Glogauer M, Magalhaes MA (2021) TNFalpha Signaling Is Increased in Progressing Oral Potentially Malignant Disorders and Regulates Malignant Transformation in an Oral Carcinogenesis Model. Front Oncol 11: 741013
Chaturvedi S, Yuen DA, Bajwa A, Huang YW, Sokollik C, Huang L, Lam GY, Tole S, Liu GY, Pan J, Chan L, Sokolskyy Y, Puthia M, Godaly G, John R, Wang C, Lee WL, Brumell JH, Okusa MD, Robinson LA (2013) Slit2 prevents neutrophil recruitment and renal ischemia-reperfusion injury. J Am Soc Nephrol 24: 1274-87
Douda DN, Khan MA, Grasemann H, Palaniyar N (2015) SK3 channel and mitochondrial ROS mediate NADPH oxidase-independent NETosis induced by calcium influx. Proc Natl Acad Sci U S A 112: 2817-22
Flannagan RS, Kuiack RC, McGavin MJ, Heinrichs DE (2018) Staphylococcus aureus Uses the GraXRS Regulatory System To Sense and Adapt to the Acidified Phagolysosome in Macrophages. mBio 9
Geraldo LH, Xu Y, Jacob L, Pibouin-Fragner L, Rao R, Maissa N, Verreault M, Lemaire N, Knosp C, Lesaffre C, Daubon T, Dejaegher J, Solie L, Rudewicz J, Viel T, Tavitian B, De Vleeschouwer S, Sanson M, Bikfalvi A, Idbaih A et al. (2021) SLIT2/ROBO signaling in tumor-associated microglia and macrophages drives glioblastoma immunosuppression and vascular dysmorphia. J Clin Invest 131
Gustafson D, Ngai M, Wu R, Hou H, Schoffel AC, Erice C, Mandla S, Billia F, Wilson MD, Radisic M, Fan E, Trahtemberg U, Baker A, McIntosh C, Fan CS, Dos Santos CC, Kain KC, Hanneman K, Thavendiranathan P, Fish JE et al. (2022) Cardiovascular signatures of COVID-19 predict mortality and identify barrier stabilizing therapies. EBioMedicine 78: 103982
Li Q, Zhang XX, Hu LP, Ni B, Li DX, Wang X, Jiang SH, Li H, Yang MW, Jiang YS, Xu CJ, Zhang XL, Zhang YL, Huang PQ, Yang Q, Zhou Y, Gu JR, Xiao GG, Sun YW, Li J et al. (2023) Coadaptation fostered by the SLIT2-ROBO1 axis facilitates liver metastasis of pancreatic ductal adenocarcinoma. Nat Commun 14: 861
Liao WX, Laurent LC, Agent S, Hodges J, Chen DB (2012) Human placental expression of SLIT/ROBO signaling cues: effects of preeclampsia and hypoxia. Biol Reprod 86: 111
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Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
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Referee #2
Evidence, reproducibility and clarity
Summary:
The manuscript deals with the role of the neurorepellent SLIT2 in killing of the bacterial pathogen Staphylococcus aureus. The authors show that neutrophils incubated with the N-terminal region of SLIT2 kill S. aureus more efficiently than neutrophils without preexposure to N-SLIT2. This effect was due to an increased production of reactive oxygen species by NADPH oxidase complex activation and stimulating exocytosis of antibacterial peptide containing granules. The concept was proven in an animal model of skin and soft tissue infection in mice in which neutralization of endogenous SLIT2 reduced CFU numbers in ear skin and …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
Summary:
The manuscript deals with the role of the neurorepellent SLIT2 in killing of the bacterial pathogen Staphylococcus aureus. The authors show that neutrophils incubated with the N-terminal region of SLIT2 kill S. aureus more efficiently than neutrophils without preexposure to N-SLIT2. This effect was due to an increased production of reactive oxygen species by NADPH oxidase complex activation and stimulating exocytosis of antibacterial peptide containing granules. The concept was proven in an animal model of skin and soft tissue infection in mice in which neutralization of endogenous SLIT2 reduced CFU numbers in ear skin and decreased tissue destruction in response to S. aureus infection.
Major comments:
In general the findings and key conclusions are convincingly covered by the results presented in the manuscript. The methods are adequate to allow the conclusions drawn. Data are clearly presented and easy to follow. Statistical methods are appropriate.
Minor comments:
In the Materials and methods section line 340 a GFP-expressing S. aureus USA300 strain is indicated. What was the exact strain designation, e.g. LAC or JE2, as USA300 is not a strain name (different strains belong to this pulsed-field electrophoresis based classification). In the legend of figure 3 the inhibitors are mentioned for part B and E but not C and D. Figure S4 would be nice to have in the main manuscript.
Significance
The manuscript deals with a novel mechanism of neutrophil activation by SLIT-2, a protein which was originally thought to act in the nervous system but is also expressed in many peripheral tissues. Importantly SLIT-2 may be involved in tumor suppression but also chemotaxis of immune cells. In this manuscript a novel, rather unexpected role of the N-terminal region of SLIT-2 in activation of antibacterial mechanisms of neutrophils was shown. This could be interesting for a broader readership interested in innate immune mechanisms and bacterial infections. Since little is known on the role of SLIT-2 in response to bacterial infections the paper could initiate a number of new studies in this field.
This reviewer has experience with S. aureus virulence and resistance mechanisms and animal infection models.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
The study analyzes the role of SLIT2 in clearance of S. aureus via neutrophils. I suggests that N-SLIT2 play a key role as an amplifier of the ROS response and release of antimicrobial peptides. The manuscript is well written and technologically sound. However, a few issues need to be addressed that preclude publication of the manuscript:
Major comments
The study analyzes different parameters of neutrophil function. One major effect of neutrophil activation is NETosis. This has not been addressed in the study albeit it is deemed to act in concert with the other immune mechanisms described.
Furthermore, the authors discuss a role of …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
The study analyzes the role of SLIT2 in clearance of S. aureus via neutrophils. I suggests that N-SLIT2 play a key role as an amplifier of the ROS response and release of antimicrobial peptides. The manuscript is well written and technologically sound. However, a few issues need to be addressed that preclude publication of the manuscript:
Major comments
The study analyzes different parameters of neutrophil function. One major effect of neutrophil activation is NETosis. This has not been addressed in the study albeit it is deemed to act in concert with the other immune mechanisms described.
Furthermore, the authors discuss a role of SLIT2 in the regulation of neutrophil migration. However, the current data set does not provide sufficient evidence for this. The reviewer suggests that the auhtors provide migration/chemotaxis assays and/or in vivo data to prove their hypothesis or revise their argumentation. The timeline of SLIT2 expression indicates that environmental conditions could influence the expression of SLIT2. Have the authors analyzed whether SLIT2 expression is affected by low pH or hypoxia? Is there any data indicating what factors regulate SLIT2 expression?
In the same line, it would be interesting to know whether SLIT2 immune effects (specifically ROS and LL37 release) are similarly triggered under hypoxic conditions often found in an abscess? Lastly, it is unclear whether SLIT2 binds to a defined target on the neutrophil. This needs to be highlighted in the discussion in respect to future work and ideally resolved experimentally.
Significance
The manuscript provides inisght into a new mechanism regulating neutrophil function in the presence of S. aureus. The study provides evidence that the N-terminus of SLIT2 is involved in these effects and highlights p38-mediated signaling events as molecular targets increasing antibacterial effects in neutrophils. However, some contradictory findings imply that timing of the response is crucial. Nevertheless, with the molecular mechanisms not fully understood many questions remain and the study adds to the complexity of the antistaphyloccocal immune response. Therefore, the audience forthis manuscript requires knowledge on S. aureus-specific host-pathogen interaction and is not suitable for a broad audience as it does not provide information on a generally new mechanism of neutrophil activation or defense.
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