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  1. Author Response:

    Reviewer #1 (Public Review):

    Roy et al. investigated glucose-induced changes of selected neutrophil functions using neutrophils from diabetic mice and glucose-exposed murine and human neutrophils. They reconfirm earlier findings that glucose renders neutrophils less responsive to fMLF-mediated chemotaxis and show that expression and surface presentation of the corresponding receptor FPR1, a chemotactic receptor that is high in the signaling hierarchy, is downregulated within the first hour of glucose treatment. Similarly, other elements of neutrophil chemotactic responses including the phospholipase PLC and the cytokine MIP-1/CCL3 are also affected, while the expression of the chemokine receptor CCR1 remains unaltered. Interestingly, supplementing the CCFR1-targeting cytokine CCL3 could restore neutrophil chemotactic fitness and wound healing and thus, might be beneficial for diabetic wound management.

    Conclusions are supported by the data but the study, in its current stage, needs further analysis. The findings suggest a more general effect on the neutrophil expression pattern induced by glucose and unfortunately, this is not addressed and mechanistic insights to explain the observed effects are entirely missing. The finding that CCL3 levels are reduced and that external addition brings neutrophil chemotactic response back to normal is of high translational potential.

    We appreciate the reviewer for their thorough evaluation of our manuscript. As the reviewer appreciates, no single manuscript can possibly address all questions, but impactful manuscripts, as we believe our manuscript is, often also open new areas for follow-up research. We acknowledge that our manuscript does not address all the interesting questions that were raised by the reviewers and the editor, but our data do reveal important information regarding the culprit responsible for the impaired chemotaxis responses in diabetic neutrophils, which had been known but ignored for decades (10), and how this impairment adversely impacts the dynamics of neutrophil trafficking into diabetic wound during the acute phase of healing, early after injury. Our data establish a new paradigm that blames inadequate neutrophil response early after injury (not excess neutrophils in chronic diabetic ulcers), for rendering diabetic wounds vulnerable to infection, which in turn contributes to setting the stage for the sustained and non-resolving inflammatory environment as diabetic wounds age and become chronic. We further show that neutrophil depletion in diabetic animals renders diabetic wounds significantly more vulnerable to infection, indicating that as impaired as diabetic neutrophils may be with respect to their bactericidal functions as has been reported (11, 12), they still maintain some antimicrobial functions. Finally, we show that by jumpstarting the neutrophil influx in diabetic wound through the use of CCL3 which engages CCR1, CCR4, and CCR5 auxiliary receptors (13-15), we can reduce infection levels in diabetic wounds by ~2 log orders in a manner that is completely dependent on neutrophil influx into the wound and significantly stimulate healing. As the reviewer is aware, there is only one FDA-approved therapy (Becaplermin) showing modest effectiveness in stimulating wound healing in diabetic wounds (16-21), and there are no treatments to address infection in diabetic wounds, except for the use of systemic antibiotics which are routinely included in the management of diabetic patients with chronic ulcers (22, 23). Antibiotic overuse can have disastrous consequences, leading to the emergence and the spread of antibiotic resistance, cytotoxicity, allergic reactions, and immunological and neurological diseases (24-29). Our data reveal therapeutic potential for CCL3 topical treatment to enhance infection control and stimulate healing in diabetic wounds.

    Reviewer #2 (Public Review):

    Diabetic wound closure remains a major clinical problem, in the sense that in diabetes, skin wounds do not repair on time and may get infected, installing a feed-forward cycle of inflammatory and tissue damage outcomes. This study chiefly demonstrates that this vicious cycle can be broken by ensuring an exuberant and effective neutrophilic response takes place.

    As such the data presented herein are exemplar in demonstrating: i) the substantial non-redundant role of physiological inflammation (that is, a good inflammatory response when needed is a good thing); ii) the fact that tissue inflammation resolves after a proper and effective neutrophil response and iii) a specific receptor target (FPR1) is affected by diabetes (high glucose) and is central to a defective inflammatory response to infective agents.

    All in all I found the experimental model used very helpful in demonstrating this important physio-patholoigical link between: inflammation onset -> inflammation resolution.

    We appreciate reviewer’s thorough evaluation of our manuscript and for their recognition of the importance of our findings. We are also in complete agreement with the reviewer that proper inflammatory responses are absolutely a good thing when needed (i.e., early after injury or in response to infection) and neutrophils also play an important in initiating the resolution of inflammatory responses.

    Reviewer #3 (Public Review):

    In this study, Roy et al., have focussed on investigating what happens in infections associated with impaired healing in diabetic wounds. Specifically, they have identified that a certain type of white blood cell (i.e. a neutrophil) is dysfunctional, leading to a delay in its ability to help fight off the infections. The findings of the study are interesting, and the therapeutic possibility of treating diabetic wounds with CCL3 is novel and is likely to be of interest to the field. However, the very low n numbers used in the study questions the validity and robustness of the data.

    We apologize to the reviewer for this confusion in the figure legends. Nowhere in this manuscript did we rely on N=2 for our analyses and we completely agree with the reviewers that N=2 is not statistically robust. We indicated N=2 for experiments involving RT-PCR but we had repeated these experiments at least two independent times so the actual number is N>4. We have corrected the figure legends accordingly to address the reviewer’s concern.

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  2. Evaluation Summary:

    This work is of interest to scientists studying pathogenesis-associated neutrophil dysfunction. Roy et al. investigated impaired wound healing associated with infected diabetic wounds, a major clinical problem. The data reveal substantial alterations in the functional competence of diabetic or glucose-exposed neutrophils to react to chemotactic signals and provide potential therapeutic strategies to improve neutrophil fitness and improve wound healing. Conclusions are supported by the data but the study, in its current stage, needs further analysis.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    Roy et al. investigated glucose-induced changes of selected neutrophil functions using neutrophils from diabetic mice and glucose-exposed murine and human neutrophils. They reconfirm earlier findings that glucose renders neutrophils less responsive to fMLF-mediated chemotaxis and show that expression and surface presentation of the corresponding receptor FPR1, a chemotactic receptor that is high in the signaling hierarchy, is downregulated within the first hour of glucose treatment. Similarly, other elements of neutrophil chemotactic responses including the phospholipase PLC and the cytokine MIP-1/CCL3 are also affected, while the expression of the chemokine receptor CCR1 remains unaltered. Interestingly, supplementing the CCFR1-targeting cytokine CCL3 could restore neutrophil chemotactic fitness and wound healing and thus, might be beneficial for diabetic wound management.

    Conclusions are supported by the data but the study, in its current stage, needs further analysis. The findings suggest a more general effect on the neutrophil expression pattern induced by glucose and unfortunately, this is not addressed and mechanistic insights to explain the observed effects are entirely missing. The finding that CCL3 levels are reduced and that external addition brings neutrophil chemotactic response back to normal is of high translational potential.

    Was this evaluation helpful?
  4. Reviewer #2 (Public Review):

    Diabetic wound closure remains a major clinical problem, in the sense that in diabetes, skin wounds do not repair on time and may get infected, installing a feed-forward cycle of inflammatory and tissue damage outcomes. This study chiefly demonstrates that this vicious cycle can be broken by ensuring an exuberant and effective neutrophilic response takes place.

    As such the data presented herein are exemplar in demonstrating: i) the substantial non-redundant role of physiological inflammation (that is, a good inflammatory response when needed is a good thing); ii) the fact that tissue inflammation resolves after a proper and effective neutrophil response and iii) a specific receptor target (FPR1) is affected by diabetes (high glucose) and is central to a defective inflammatory response to infective agents.

    All in all I found the experimental model used very helpful in demonstrating this important physio-patholoigical link between: inflammation onset -> inflammation resolution.

    Was this evaluation helpful?
  5. Reviewer #3 (Public Review):

    In this study, Roy et al., have focussed on investigating what happens in infections associated with impaired healing in diabetic wounds. Specifically, they have identified that a certain type of white blood cell (i.e. a neutrophil) is dysfunctional, leading to a delay in its ability to help fight off the infections.
    The findings of the study are interesting, and the therapeutic possibility of treating diabetic wounds with CCL3 is novel and is likely to be of interest to the field. However, the very low n numbers used in the study questions the validity and robustness of the data.

    Was this evaluation helpful?