Overriding impaired FPR chemotaxis signaling in diabetic neutrophil stimulates infection control in murine diabetic wound

  1. Ruchi Roy
  2. Janet Zayas
  3. Sunil K Singh
  4. Kaylee Delgado
  5. Stephen J Wood
  6. Mohamed F Mohamed
  7. Dulce M Frausto
  8. Yasmeen A Albalawi
  9. Thea P Price
  10. Ricardo Estupinian
  11. Eileena F Giurini
  12. Timothy M Kuzel
  13. Andrew Zloza
  14. Jochen Reiser
  15. Sasha H Shafikhani  Is a corresponding author
  1. Department of Medicine, Rush University Medical Center, United States
  2. Division of Hematology/Oncology/Cell Therapy, Rush University Medical Center, United States
  3. Department of Microbial Pathogens and Immunity, Rush University Medical Center, United States
  4. Department of Surgery, Division of Surgical Oncology, University of Illinois at Chicago, United States

Abstract

Infection is a major co-morbidity that contributes to impaired healing in diabetic wounds. Although impairments in diabetic neutrophils have been blamed for this co-morbidity, what causes these impairments and whether they can be overcome, remain largely unclear. Diabetic neutrophils, isolated from diabetic individuals, exhibit chemotaxis impairment but this peculiar functional impairment has been largely ignored because it appears to contradict the clinical findings which blame excessive neutrophil influx as a major impediment to healing in chronic diabetic ulcers. Here, we report that exposure to glucose in diabetic range results in impaired chemotaxis signaling through the formyl peptide receptor (FPR) in neutrophils, culminating in reduced chemotaxis and delayed neutrophil trafficking in the wound of Leprdb (db/db) type two diabetic mice, rendering diabetic wound vulnerable to infection. We further show that at least some auxiliary receptors remain functional under diabetic conditions and their engagement by the pro-inflammatory cytokine CCL3, overrides the requirement for FPR signaling and substantially improves infection control by jumpstarting the neutrophil trafficking toward infection, and stimulates healing in diabetic wound. We posit that CCL3 may have therapeutic potential for the treatment of diabetic foot ulcers if it is applied topically after the surgical debridement process which is intended to reset chronic ulcers into acute fresh wounds.

Editor's evaluation

The data demonstrate substantial neutrophil dysfunction of diabetic or glucose-exposed neutrophils and provide potential therapeutic strategies to improve neutrophil fitness and improve healing of diabetic wounds. The reviewers feel that all their points of concern, suggestions, and comments have been dealt adequately with and that the revised manuscript has improved substantially.

https://doi.org/10.7554/eLife.72071.sa0

Introduction

Diabetic foot ulcers are the leading cause of lower extremity amputations in the United States and are responsible for more hospitalizations than any other complication of diabetes (Sen et al., 2009; Brem and Tomic-Canic, 2007; Reiber et al., 1999; Frykberg, 2002; Boulton, 2000). Infection with pathogenic bacteria, such as Pseudomonas aeruginosa, is a major co-morbidity that contributes to impaired healing in diabetic ulcers (Kirketerp-Møller et al., 2008; Gjødsbøl et al., 2006; Dowd et al., 2008; Redel et al., 2013; Goldufsky et al., 2015). Phagocytic leukocytes, particularly neutrophils (PMNs), play a major role defending wounds from invading pathogens (Martin and Leibovich, 2005). Neutrophils are the first inflammatory leukocytes that infiltrate into the wound (Kim et al., 2008). In addition to their antimicrobial functions mediated by phagocytosis, bursts of reactive oxygen species (ROS), antimicrobial (AMP) production, and neutrophil extracellular trap (NET) (Dovi et al., 2004; Brinkmann et al., 2004), they also express various cytokines and chemokines that set the stage for the subsequent inflammatory and non-inflammatory responses, which further contribute to infection control and partake in healing processes (Velnar et al., 2009; Fenteany et al., 2000; Schäfer and Werner, 2008; Martin, 1997; Diegelmann and Evans, 2004). There appears to be a disconnect in that diabetic ulcers suffer from persistent non-resolving inflammation – characterized by increased neutrophils – yet they fail to control infection. Bactericidal functional impairments in diabetic neutrophils (PMNs) is thought to underlie defective infection control in diabetic wound (Repine et al., 1980; Gallacher et al., 1995). What causes these impairments in diabetic neutrophils remains poorly understood, although the impairment severity has been associated with the degree of hyperglycemia (Repine et al., 1980), suggesting that exposure to high-glucose levels may be a contributing factor to these impairments.

In addition to impaired bactericidal functions, diabetic neutrophils – (isolated from the blood of diabetic patients) – also display impaired chemotactic response (Delamaire et al., 1997). This peculiar functional impairment in diabetic neutrophils has not received much attention primarily because it appears to contradict the clinical findings which finds and blames excessive neutrophil response as a major impediment to healing in chronic diabetic ulcers (Wetzler et al., 2000; Bjarnsholt et al., 2008). Driven by this disconnect and the fact that very little is known about neutrophil trafficking into diabetic wounds particularly early after injury and in response to infection, we sought to assess the possible impact of diabetic neutrophil chemotaxis impairment on the dynamics of neutrophil response and impaired infection control in diabetic wounds.

Results

Neutrophil trafficking is delayed in diabetic wounds

We generated full-thickness excisional wounds in Leprdb/db (db/db) type two diabetic mice and their normal littermates C57BL/6, as described (Goldufsky et al., 2015; Wood et al., 2014), and challenged these wounds with PA103 P. aeruginosa bacteria (103 CFU/wound), which we have shown to establish a robust and persistent infection and cause wound damage in diabetic mice (Goldufsky et al., 2015). Consistent with our previous report (Goldufsky et al., 2015), db/db wounds contained 2–4 log orders more bacteria than normal wounds, indicating that diabetic wounds are vulnerable to increased infection with P. aeruginosa (Figure 1—figure supplement 1). We next collected wound tissues on days 1, 3, 6, and 10 post-infection and assessed them for their neutrophil contents by immunohistochemistry (IHC) using the neutrophil marker Ly6G (Wong et al., 2015; Kroin et al., 2016). Surprisingly, diabetic mice exhibited substantially reduced neutrophil influx in wounds early after injury at days 1 and 3 but significantly higher neutrophil contents in day 6 and day 10, as compared to normal wounds (Figure 1a–b). Corroborating these data, myeloperoxidase (MPO) -– (a marker for primarily activated neutrophils Klebanoff, 2005) – was also substantially reduced in diabetic wounds early after injury at days 1 and 3 but significantly higher in day 10 wounds (Figure 1c). Assessment of neutrophil contents in day one normal and diabetic infected wounds by flow cytometry – where neutrophils were identified as CD45+Ly6C/GhiCD11bhi (Kuijpers et al., 1991; Atzeni et al., 2002) – further corroborated the inadequate neutrophil trafficking into diabetic wounds early after injury (Figure 1d and Figure 1—figure supplement 2). These data indicated that neutrophil response – (which is needed to combat infection) – is delayed in diabetic wounds, rendering these wounds vulnerable to infection early after injury.

Figure 1 with 2 supplements see all
Neutrophil response is delayed in infected diabetic wound tissue.

Normal (C57BL/6) and diabetic (db/db) wounds were infected with PA103 (1000 CFU/wound). (a–b) Wound tissues were harvested at indicated timepoints post-infection and assessed for neutrophil contents by histological analysis using anti-Ly6G antibody. (a) Representative regions from underneath the wounds extending in the dermis are shown at ×40 and ×400 magnification (top and bottom, respectively). A representative magnified region is also inserted in the ×400 magnification images. Black scale bar = 500 µm for ×40 magnification and red scale bar = 50 µm for ×400 magnification. (b) The corresponding data were plotted as the Mean ± SEM. (c) Wounds at indicated timepoints were assessed for their MPO contents by ELISA and the tabulated data are shown as the Mean ± SEM. (d) Day 1 infected wound tissues of C57BL/6 and db/db were evaluated for their neutrophil contents by flow cytometry. Corresponding data were plotted as the Mean ± SEM. (N = 4; ns = not significant, *p < 0.05; **p < 0.01; ***p < 0.001 – are comparisons made between C57BL/6 and db/db at indicated timepoints; or #p < 0.05; ##p < 0.01; ###p < 0.001 are comparisons made within each group to day one values, respectively. Statistical analyses between groups were conducted by One-way ANOVA with additional post hoc testing, and pair-wise comparisons between groups were performed or by unpaired Student’s t-test).

Chemotactic response through the FPR is impaired in diabetic neutrophils

Depending on the tissue or the condition, neutrophil trafficking in response to injury and/or infection occurs in multiple waves mediated by ~30 receptors on neutrophils and involves multiple signaling pathways (de Oliveira et al., 2016; Liu et al., 2012; Sadik et al., 2011; Futosi et al., 2013; Ng et al., 2011; Afonso et al., 2012; Chou et al., 2010). However, the initial neutrophil chemotaxis in response to injury or infection involves the activation of G protein–coupled formyl peptide receptor (FPR) by N-formyl peptides, such as fMet-Leu-Phe (fMLF, a.k.a., fMLP), which is released either by injured tissues or by invading bacteria (de Oliveira et al., 2016; Roupé et al., 2010). FPR1 and FPR2 are two FPR implicated in these responses, although FPR1 appears to be the primary FPR in responding to infection, as it has significantly higher affinity for bacterial formyl peptides, whereas FPR2 has a broader range of ligands than FPR1 and has been implicated in the resolution of inflammation in response to pro-resolving agonists, such as Annexin A1 (Ye et al., 2009; Jeong and Bae, 2020; Bena et al., 2012; Yazid et al., 2012; Serhan and Savill, 2005). Activation of FPR then leads to the upregulation and secretion of lipid signals, such as the leukotriene B4 (LTB4), which in turn activate BLT1, (another G-protein-coupled receptor on neutrophils), amplifying neutrophil trafficking by enhancing the signaling through FPR (Afonso et al., 2012). BLT1 activation in neutrophils by LTB4 also results in upregulation and secretion of pro-inflammatory cytokines, particularly IL-1β which in turn induces the expression and secretion of other ligands (i.e. CCL3 and CXCL1) in tissue resident epithelial cells and inflammatory leukocytes, which further amplify neutrophil trafficking and other inflammatory leukocytes including monocytes, by engaging their respective auxiliary receptors, such as CCR1 and CXCR2 (36, 37, 44, 45).

To assess the role of chemotaxis impairment in reduced neutrophil influx into diabetic wounds early after injury, we isolated neutrophils from the blood of normal and diabetic mice and assessed chemotaxis signaling through FPR in response to fMLF. Compared to normal neutrophils isolated from C57BL/6, db/db neutrophils were significantly impaired in their ability to chemotax toward fMLF (Figure 2a). Consistent with reduced signaling through the FPR in response to bacterial fMLF, expression of FPR1 was significantly diminished in db/db neutrophils, as assessed by western blotting (Figure 2b–c). Further corroborating these data, the percentage of FPR1-positive neutrophils was significantly reduced in day one diabetic wounds, after accounting for reduced number of neutrophils in diabetic wounds early after injury by assessing equal number of neutrophils by flow cytometry (Figure 2d).

Figure 2 with 3 supplements see all
Chemotactic response is impaired in diabetic neutrophils through FPR.

(a–b) Neutrophils were isolated from the peripheral blood of C57BL/6 and db/db animals to assess: (a) their ability to chemotax toward 100 nM fMLP, or (b) for the expression of FPR1 by Western blotting. (c) Densitometry values associated with (b) are plotted as Mean ± SEM (N = 4 blood pools/group, each blood pool was from 4 mice). (d) Equal number of neutrophils (isolated from Day 1 C57B and db/db wounds) were assessed for the surface expression of FPR1 on neutrophils by flow cytometry (N = 3 mice/group). (e–f) Purified neutrophils from peripheral blood of non-diabetic individuals (e), or C57BL/6 bone marrow (f), were exposed to media containing glucose in normal range (90 mg/dl) or in diabetic range (200–500 mg/dl) for 1 hr to assess their ability to chemotax toward 100 nM fMLP. Data are plotted as Mean ± SEM. (N > 4). (g–h) Neutrophils from C57BL/6 bone marrow were exposed to glucose in normal range (90 mg/dl) or in diabetic range (300 mg/dl) for 1 hr and assessed for surface expression of FPR1 by flow cytometry. A representative histogram is shown in (g) and the corresponding tabulated data, plotted as Mean ± SEM is shown in (h) (N = 3). (i–j) Murine neutrophils (from C57B bone marrow) were exposed to glucose in normal or diabetic range (90 mg/dl or 300 mg/dl) for 1 hr and assessed for the expression of indicated proteins by Western blotting. Representative Western blots are shown in (i) and corresponding densitometry values, plotted as Mean ± SEM, are shown in (j). (N ≥ 3 independent experiments). (k–m) Murine neutrophils exposed to normal or diabetic glucose, as described for (g–h), were assessed for Cyclic AMP production by ELISA (k), and for mRNA of Fpr1 and Plcγ by RT-PCR (l-m). (N ≥ 3, ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001. Statistical analyses between groups were conducted by One-way ANOVA with additional post hoc testing, and pair-wise comparisons between groups were performed or by unpaired Student’s t-test).

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Various studies have shown direct correlations between plasma glucose levels and prevalence and/or severity of infection in diabetic patients (Rayfield et al., 1982; Latham et al., 2001; Zerr et al., 1997), suggesting that exposure to high glucose levels may be responsible for impaired neutrophil functions in diabetes. Consistent with these reports, short-term and long-term glycemic control in diabetic rats, has been shown to significantly improve their ability to control Staphylococcus aureus infection (Kroin et al., 2015). To assess the impact of high glucose on signaling through the FPR, we purified neutrophils from human blood and C57BL/6 mice bone marrow (Figure 2—figure supplement 1 and Materials and methods), incubated them in media containing glucose in the normal range (90 mg/dl) or in the diabetic range (200–500 mg/dl) for 1 hr, and evaluated their chemotactic responses toward fMLF. Of note, 1 hr exposure to high glucose in diabetic range had no effect on viability of neutrophils.

Exposure to high glucose levels caused significant reduction in chemotactic response to fMLF in both human and mouse neutrophils (Figure 2e–f). While neutrophils exposed to normal glucose showed a bell-shaped curve in their chemotaxis response toward fMLF concentrations (0.01–1000 nM) with 100 nM being the optimum concentration, neutrophils exposed to high glucose showed flat chemotaxis response toward these fMLF concentrations, trending toward lower chemotaxis at higher concentrations (Figure 2—figure supplement 1), indicating that high fMLF ligand concentrations cannot rescue chemotaxis signaling through FPR in neutrophils exposed to high glucose. The bell-shaped response to fMLF in normal neutrophils is in line with previous reports showing reduction in neutrophil chemotactic responses to other ligands at high concentrations (Gomez-Cambronero et al., 2003; Burnett et al., 2017). Of note, exposure to high glucose also caused significant reductions in FPR1 surface expression, FPR1 and PLCγ protein levels, as well as cAMP levels (Figure 2g–k), which are all required to mediate FPR-mediated chemotaxis in neutrophils (Afonso et al., 2012; Heit et al., 2002; Hirsch et al., 2000). Corroborating these data, 1 hr exposure to high glucose resulted in significant reductions in the FPR1 and PLCγtranscription as determined by mRNA analysis by RT-PCR (Figure 2l–m).

To assess whether the adverse impact of high glucose on FPR1 expression was transient or sustained, we exposed purified neutrophils to glucose at 90 or 300 mg/dl and assessed the expression of FPR1 by RT-PCR and by western blotting after 1, 2, or 3 hr post exposure. Data indicated that exposure to high glucose significantly reduced the expression of FPR1 both at the transcriptional and translational levels at all timepoints, indicating that exposure to high glucose dampens the expression of FPR1 in a sustained manner (Figure 2—figure supplement 2). Of note, high glucose similarly dampened the expression of FPR2 both at transcriptional and translational levels, indicating that the adverse impact of high glucose is not restricted to FPR1 (Figure 2—figure supplement 3). Collectively, these data indicated that elevated glucose levels in diabetes is responsible for the reduced chemotactic response through FPR in diabetic neutrophils.

Some auxiliary chemotaxis receptors remain functional under diabetic conditions

Although, the initial neutrophil chemotactic response through FPR and the amplification of neutrophil chemotactic responses via other auxiliary receptors are interconnected and occur sequentially in vivo (Liu et al., 2012; Sadik et al., 2011; Futosi et al., 2013; Ng et al., 2011; Afonso et al., 2012; Chou et al., 2010), none of these receptors appear to be essential on their own and their defects can be overcome by engaging other receptors (Chou et al., 2010; Lämmermann et al., 2013; Park et al., 2009). Chronic diabetic ulcers suffer from increased neutrophil contents (Wetzler et al., 2000; Bjarnsholt et al., 2008), indicating that diabetic neutrophils are capable of migrating into the wound, albeit at dysregulated kinetics as our data show (Figure 1). Together, these findings suggested that chemotactic responses of diabetic neutrophils – although impaired through the FPR (Figure 2 and Figure 2—figure supplement 1) – may be functional through one or more auxiliary receptors that mediate the amplification phase of neutrophil trafficking in wound and toward infection.

To evaluate this possibility, we assessed chemotactic responses toward CCL3 in human and mouse neutrophils after 1 hr exposure to glucose at normal or diabetic levels. The reason we focused on CCL3 was because it engages multiple auxiliary receptors, namely CCR1, CCR4, and CCR5 (Ramos et al., 2005; da Silva et al., 2017; Yoshie and Matsushima, 2015). Of note, CCR1 is an important receptor that is implicated in neutrophil trafficking to post-ischemic tissues (Reichel et al., 2006) and ischemia is an important co-morbidity associated with impaired healing in diabetic wound (Brem and Tomic-Canic, 2007; Armstrong et al., 1998). Data indicated that exposure to glucose in the diabetic range did not affect the chemotactic responses toward CCL3 in human or mouse neutrophils (Figure 3a–b), suggesting that these auxiliary receptors are unaffected by high glucose. To corroborate these data, we assessed the impact of high glucose exposure on CCR1 auxiliary receptor. In line with chemotaxis data, CCR1 expression remained unaffected in neutrophils after exposure to high glucose for 1 hr as assessed by Western blotting (Figure 3c–d), by mRNA analysis (Figure 3e), and by surface expression analysis (Figure 3f–g). Further corroborating these data, CCR1 expression was similar in neutrophils isolated from the blood of db/db and C57BL/6 mice (Figure 3h–i), and the percentage of CCR1-positive neutrophils in db/db day 1 wounds were similar to C57BL/6 day 1 wounds, after accounting for the reduced number of leukocytes in day one diabetic wounds by assessing equal number of neutrophils by flow cytometry (Figure 3j). Of note, surface expression of auxiliary receptor CXCR2, (another important auxiliary receptor involved in the amplification of neutrophil response in wound and toward infection [de Oliveira et al., 2016; Brubaker et al., 2013]), on neutrophils and chemotaxis through the CXCR2 in response to CXCL1 (a.k.a. KC) – a known ligand for CXCR2 (Chintakuntlawar and Chodosh, 2009) – were also unaffected by high glucose exposure in neutrophils (Figure 3—figure supplement 1a-c). Collectively, these data suggested that at least CCR1 and CXCR2 auxiliary receptors may remain functional under diabetic conditions.

Figure 3 with 1 supplement see all
CCR1 receptor remains functional under diabetic conditions.

Human (a) or mouse (b) neutrophils were examined for their chemotactic responses toward CCL3 (5 ng/ml) after 1 hr exposure to glucose in normal (90 mg/dl) or diabetic range (200–500 mg/dl). (N > 3). (c–e) Neutrophils isolated from bone marrow of C57BL/6 were exposed to normal glucose (90 mg/dl) or high glucose (300 mg/dl) for 1 hr and assessed for CCR1 expression by western blotting (c–d) and for mRNA transcription analysis by RT-PCR. (N = 5 for western blots and N = 4 for RT-PCR). (f–g) Neutrophils isolated from bone marrow of C57BL/6 were exposed to normal glucose (90 mg/dl) or high glucose (300 mg/dl) for 1 hr and assessed for CCR1 surface expression by flow cytometry. A representative histogram is shown in (f) and the corresponding data, plotted as Mean ± SEM, is shown in (g) (N = 4). (h–i) Neutrophils isolated from peripheral blood of db/db and C57BL/6 mice were assessed for the expression of CCR1 by western blotting. A representative western blot is shown in (h) and the corresponding tabulated values are shown in (i). (N = 4 mice/group). (j) Equal numbers of neutrophils from day 1 C57BL/6 and db/db infected wounds were assessed for CCR1 surface expression by flow cytometry. (N = 3). (Statistical analyses between groups were conducted by One-way ANOVA with additional post hoc testing, and pair-wise comparisons between groups were performed or by unpaired Student’s t-test; ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001).

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Topical treatment with CCL3 bypasses the requirement for FPR signaling and enhances neutrophil trafficking and infection control in diabetic wound

If auxiliary receptors seem to be unaffected under diabetic conditions as our data in Figure 3 and Figure 3—figure supplement 1 indicate, why is neutrophil trafficking so severely diminished in diabetic wounds early after injury (Figure 1). As discussed above, production of ligands (including CCL3) for auxiliary receptors in tissue ultimately depends on FPR activation (Afonso et al., 2012; Chou et al., 2010; Su and Richmond, 2015; Luster et al., 2005). In addition, leukocytes (i.e. neutrophils) are major cellular sources of ligands for auxiliary receptors (including CCL3) (Ridiandries et al., 2018; Tecchio et al., 2014; Sanz and Kubes, 2012). Therefore, reduced neutrophil response in diabetic wounds early after injury (Figure 1) could also adversely affect the production of ligands for auxiliary receptors in diabetic wounds early after injury (including CCL3). Moreover, increased expression of immunosuppressive IL-10 in diabetic wounds early after injury has been shown to lead to significant reduction in toll-like receptor (TLR) signaling in diabetic wounds early after injury (Roy et al., 2021). And TLR signaling has been implicated in the production of ligands (including CCL3) for auxiliary receptors (Kochumon et al., 2020; Ahmad et al., 2019). Taken all these into account, we posited that although auxiliary receptors on neutrophils may remain functional under diabetic condition, they may not be functioning in diabetic wounds early after injury because of inadequate expression of their ligands. We assessed the expression of CCL3 in day one normal and diabetic wounds infected with P. aeruginosa. In line with our hypothesis, CCL3 expression was substantially reduced in day one diabetic wounds, as assessed by mRNA analysis and Western blotting (Figure 4a–c). These data suggested that although auxiliary receptors on neutrophils may remain functional under diabetic condition, they may not be functioning to recruit neutrophils in diabetic wounds early after injury because of inadequate ligands’ production for the auxiliary receptors. If this is the case, augmenting diabetic wounds with CCL3 early after injury should be able to override deficiency in the FPR signaling and enhance neutrophil migration into diabetic wounds.

Figure 4 with 1 supplement see all
CCL3 topical treatment enhances neutrophil response and infection control in diabetic wound.

(a–c) Day 1 wound tissues of C57BL/6 and db/db infected wounds were harvested and assessed for the CCL3 mRNA levels by RT-PCR (a) and by western blotting (b–c), and the data were plotted as the Mean ± SEM, after normalization to 18 S and GAPDH, respectively (N = 6 mice/group for (a) and 4 mice/group for (b–c)). (d-e) db/db diabetic wounds were treated with either PBS or CCL3 (1 μg/wound) and infected with PA103 (1000 CFU/wound). Twenty-four h post-infection, wounds were collected and assessed for their neutrophil contents by histological analysis using anti-Ly6G antibody. (d) Representative wound images at ×40 and ×400 magnification (top and bottom, respectively) are shown. Inserts are representative magnified regions within the ×400 magnification images. (Black scale bar = 500 µm for ×40 magnification and red scale bar = 50 µm for ×400 magnification). (e) Corresponding data associated with (d) are plotted as Mean ± SEM. (N = 4 mice/group) (f) Neutrophil contents of PBS or CCL3-treated db/db infected wounds at day 1 were assessed by flow cytometry (f) or by MPO analysis (g) and the data were plotted as Mean ± SEM. (N > 3 mice/group for (f) and N = 4 mice/group for (g)). (h–i) db/db mice received either α-Ly6G (100 μg/mouse) to cause neutrophil depletion or α-IgG isoform as control, by intraperitoneal (i.p.) injection. Twenty-four hr after injection, α-IgG or α-Ly6G-treated animals were wounded and treated with either PBS or CCL3 and infected with PA103. The impact of neutrophil depletion on the ability of CCL3 treatment to boost infection control in diabetic wound was assessed by MPO analysis (i) and CFU count determination (h & j) in day 1 wounds. Data were plotted as Mean ± SEM. (N = 4 mice/group for (h); N = 3 mice/group for (i); and N > 4 mice/group for (j). ns = not significant, *p < 0.05; **p < 0.01, ***p < 0.001. Statistical analyses between groups were conducted by One-way ANOVA with additional post hoc testing, and pair-wise comparisons between groups were performed or by unpaired Student’s t-test.).

To test our hypothesis, we treated db/db wounds topically with CCL3 (1 µg/wound) prior to infection and assessed its impact on neutrophil response and infection control in diabetic wounds. Consistent with our hypothesis, one-time topical treatment with CCL3 significantly increased neutrophil trafficking in day one diabetic wounds, as assessed by Ly6G histological analysis (Figure 4d–e), by flow cytometry (Figure 4f), and by MPO analysis (Figure 4g). Importantly, CCL3 treatment significantly enhanced the ability of diabetic wounds to control infection, as demonstrated by nearly a two log-order reduction in the number of bacteria contained in the CCL3-treated db/db wounds (Figure 4h).

To assess the dependence enhanced infection control on neutrophils in CCL3-treated diabetic wounds, we depleted db/db mice of neutrophils by anti-Ly6G antibody (Nozawa et al., 2006), 24 hr prior to wounding and assessed the impact of neutrophil depletion on the ability of CCL3-treated db/db wounds to control P. aeruginosa infection. Anti-Ly6G reduced the neutrophil contents in circulation by ~97% and in wound by ~75% (Figure 4i and Figure 4—figure supplement 1a,b). Neutrophil-depletion also resulted in ~2 log-order more bacteria in diabetic wounds, indicating that despite their known bactericidal functional impairments (Repine et al., 1980; Gallacher et al., 1995), diabetic neutrophils still contribute to a degree in infection control in these wounds (Figure 4j). Importantly, neutrophil-depletion abrogated CCL3’s beneficial effects in boosting antimicrobial defenses against P. aeruginosa in diabetic wounds (Figure 4j), indicating that CCL3-induced enhanced infection control in diabetic wound is dependent on its ability to enhance neutrophil response in diabetic wound.

Treatment with CCL3 does not lead to persistent non-resolving inflammation in infected diabetic wounds and stimulates healing

Although, treatment with CCL3 substantially improved diabetic wound’s ability to control infection by enhancing neutrophil response in day one wounds (Figure 4), it remained a possibility that CCL3 treatment could have long-term adverse consequences, as it could lead to heightened inflammatory environment which would be detrimental to the process of tissue repair and healing in diabetic wounds. Afterall, persistent non-resolving inflammation, (as manifested by increases in pro-inflammatory cytokines and neutrophils), is considered a major contributor to healing impairment in diabetic foot ulcers (Wetzler et al., 2000; Bjarnsholt et al., 2008).

We assessed the long-term impact of CCL3 treatment on IL-1β and TNF-α pro-inflammatory cytokines that are found to be elevated in chronic diabetic foot ulcers (Mirza et al., 2013; Yan et al., 2016; Jeffcoate et al., 2005). Data indicated that while IL-1β and TNF-α continued to rise in the mock-treated db/db wounds as the diabetic wounds aged, in the CCL3-treated diabetic wounds, these pro-inflammatory cytokines were significantly higher during the acute phase of healing early after injury but declined substantially in old wounds, particularly at day 10 (Figure 5a–b). In line with these data, neutrophil wound contents (assessed by histological analysis using neutrophil marker Ly6G staining Pizza et al., 2005) were also highly elevated during the acute phase of healing early after injury in the CCL3-treated diabetic wounds but declined significantly as the wounds aged, as compared with the mock-treated diabetic wounds (Figure 5c–d, Figure 5—figure supplement 1).

Figure 5 with 1 supplement see all
Treatment with CCL3 does not lead to persistent inflammation in infected diabetic wounds.

db/db wounds were treated with PBS or CCL3 (1 μg/wound) and infected with PA103 (1000 CFU/wound). (a–b) Wound tissues were collected at indicated timepoints and assessed for their Il-1β (a) and TNF-α (b) contents by ELISA. (N = 4 mice/group). (c–d) The aforementioned PBS and CCL3-treaded and infected diabetic wounds were assessed for their neutrophil contents by histological analysis using neutrophil marker Ly6G staining. (c) Representative images of regions from underneath the wounds extending in the dermis at ×400 magnification are shown. (Red scale bars = 50 μm). Representative full wound images of these staining can be found in Figure 5—figure supplement 1. (d) The corresponding data were plotted as the Mean ± SEM. (N = 4 mice/group, > 9 random fields/wound/mouse. (*) denotes significance between groups while (#) indicates significance within the same group in comparison to day 1 of respective wound groups. ns = not significant; *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05, ##p < 0.01, ###p < 0.001. Statistical analyses between groups were conducted by One-way ANOVA with additional post hoc testing, and pair-wise comparisons between groups were performed or by unpaired Student’s t-test).

Encouragingly, CCL3 treatment also significantly stimulated healing in infected diabetic wounds, as assessed by wound area measurement (Figure 6a–b), while mock-treated diabetic wounds became exacerbated as the result of P. aeruginosa infection as we have previously shown (Goldufsky et al., 2015). Corroborating these results, CCL3-treated infected diabetic wounds were completely re-epithelized and exhibited epidermal thickening as assessed by H&E histological analysis, while mock-treated infected diabetic wounds became exacerbated (Figure 6c–d).

Figure 6 with 1 supplement see all
Treatment with CCL3 stimulates healing in infected diabetic wounds.

(a–d) db/db wounds were either treated with PBS or CCL3 and infected with PA103 (1000 CFU). Wound healing was assessed at indicated timepoints by digital photography (a–b) or by H&E histological analysis of re-epithelialization (c–d). Representative images are shown in (a & c). (Black scale bar = 1 mm, and the wound gap is shown by dotted line). The corresponding data for (a & c) are shown in (b & d) as the Mean ± SEM. (e–f) Day 10 db/db wounds (treated with either PBS or CCL3 and infected with PA103) were assessed for fibroblast, myofibroblast, elastin, and cartilage healing markers by vimentin, α-SMA, Masson’s Trichrome, and elastin staining, respectively. (e) Representative regions from underneath the wounds extending in the dermis are shown at ×400 magnification. (Red scale bar = 50 µm. For the corresponding full wound images at ×40 magnification, see Figure 6—figure supplement 1). (f) The corresponding data are plotted as the Mean ± SEM. (N = 4 mice/group for (a–b); and N = 4 mice/group for (c–f). *p < 0.05, **p < 0.01, ***p < 0.001. Statistical analyses between groups were conducted by One-way ANOVA with additional post hoc testing, and pair-wise comparisons between groups were performed or by unpaired Student’s t-test).

Fibroblasts and myofibroblasts are key players in extracellular matrix production and granulation tissue maturation during the proliferation and the remodeling phases of wound healing (Wilgus et al., 2008; Skalli et al., 1989; Cheng et al., 2016). However, persistent inflammatory environment in diabetic wounds adversely impacts the functions of fibroblast and myofibroblast, culminating in reduced collagen and elastin extracellular matrix deposition and impaired healing in diabetic chronic wounds (Diegelmann and Evans, 2004; Yue et al., 1986; Augustine et al., 2014). P. aeruginosa infection further exacerbates inflammation and reduces collagen deposition in diabetic wounds (Goldufsky et al., 2015). We evaluated the impact of CCL3 treatment on fibroblast, myofibroblast, collagen, and elastin in day 10 diabetic wounds, using their respective markers: Vimentin, α-SMA, Elastin, and Masson’s Trichrome staining (Goldufsky et al., 2015; Wilgus et al., 2008; Hinz, 2006). CCL3-treated wounds showed significant increases in all these healing markers (Figure 6e–f and Figure 6—figure supplement 1). Collectively, these data indicate that diabetic wounds are not destined to develop persistent non-resolving inflammation, provided that the dynamics of neutrophil trafficking is restored in these wounds early after injury.

Discussion

Diabetic wounds are highly susceptible to infection with pathogenic bacteria, such as P. aeruginosa, which in turn drive these wounds toward persistent non-resolving inflammation which contributes to impaired healing (Goldufsky et al., 2015; Wetzler et al., 2000; Bjarnsholt et al., 2008). Here, we demonstrate that early after injury, the diabetic wound exhibits a paradoxical and damaging decrease in essential neutrophil trafficking, which in turn renders diabetic wounds vulnerable to infection. Our data point to impaired signaling through FPR (resulting from exposure to high glucose levels), as an important culprit responsible for the delay in the neutrophil response to injury and infection in diabetic wounds.

It is worth noting that 1 hr exposure to high glucose levels dramatically impaired chemotaxis signaling through the FPR in neutrophils, suggesting that even a short-term rise in serum glucose levels could potentially make non-diabetic people transiently immunocompromised and susceptible to infection. In line with this notion, hyperglycemia during the perioperative and postoperative periods are found to be significant risk factors for surgical site infection (SSI) (Ambiru et al., 2008; Sadoskas et al., 2016), while glycemic control during the perioperative period has been shown to significantly reduce SSI rates both in human and in animals (Kroin et al., 2015; Sadoskas et al., 2016). It remains unclear why exposure to high glucose dampens the expression and signaling through the FPR. We posit that it may involve metabolic changes, resulting from high glucose in neutrophils. We are actively investigating this possibility.

Our data demonstrate that at least the expression and signaling through CCR1 and CXCR2 auxiliary receptors are not adversely affected by high glucose, but they may not be signaling in diabetic wounds early after injury because of insufficient production of their ligands, such as CCL3. What causes the reduction in the expression of the ligands for these auxiliary receptors in diabetic wounds early after injury remains unclear, but we posit that multiple factors could influence this outcome. One contributing factor could be the reduced influx of neutrophils in diabetic wounds early after injury as we demonstrated here. Leukocytes (including neutrophils) are major cellular sources of these ligands (e.g. CCL3) for auxiliary receptors (Ridiandries et al., 2018). Another contributing factor could be the impaired signaling through FPR as our data demonstrated here. FPR activation in neutrophils has been shown to enhance the expression of the ligands in inflammatory and non-inflammatory cells through the production of other pro-inflammatory signaling cues such as IL-1β (Afonso et al., 2012; Chou et al., 2010; Su and Richmond, 2015; Luster et al., 2005). Another contributing factor could be reduced TLR signaling in diabetic wounds early after injury due to increased IL-10 expression and signaling (Roy et al., 2021). TLR signaling has also been implicated in the production of ligands (e.g. CCL3) for these auxiliary receptors (Kochumon et al., 2020; Ahmad et al., 2019).

Importantly, one-time topical treatment with CCL3 substantially boosted antimicrobial defenses without leading to heightened non-resolving inflammation in diabetic wounds. These data indicate that diabetic wounds will not develop persistent non-resolving inflammation provided that the neutrophil responses are restored in them early after injury. This finding is consistent with reports highlighting the pivotal role of neutrophils also in the resolution phase of inflammation (through the production and release of anti-inflammatory and inflammation resolving proteins and bioactive lipids, such as Annexin A1, lipoxins, and protectin D1), to ensure that the inflammatory responses cease safely without compromising tissue’s defenses against invading pathogens, which they accomplish directly (Jones et al., 2016; Sugimoto et al., 2016; Serhan et al., 2008).

Diabetic chronic wounds are locked in persistent non-resolving inflammation (Goldufsky et al., 2015; Wetzler et al., 2000; Bjarnsholt et al., 2008). Intriguingly, our data indicate that exposure to high glucose causes drastic reduction in both FPR1 and FPR2 expression in neutrophils. Given that FPR2 has been implicated in the resolution of inflammation in response to Annexin A1, lipoxin A4, and resolving D1 inflammation pro-resolving agonists (Ye et al., 2009; Jeong and Bae, 2020; Bena et al., 2012; Yazid et al., 2012; Serhan and Savill, 2005), these data suggest that defective signaling through FPR2 in neutrophils may also be a contributing factor to the sustained non-resolving inflammatory environment in chronic diabetic ulcers. Future studies should investigate the role of FPR2 signaling in the resolution of inflammation in acute wound healing and the possibility that defective signaling in the FPR2 may contribute to sustained and non-resolving inflammatory environment in diabetic chronic ulcers.

It remains a possibility that other auxiliary receptors which amplify the neutrophil migration in wounds and toward infection (e.g. CXCR1, BLT1, etc. de Oliveira et al., 2016), may also remain functional under diabetic condition and their engagement with their respective ligands could similarly enhance antimicrobial defenses in diabetic wounds. Future studies should address these possibilities and evaluate how serum glucose level affects the expression and/or the activity of all the ~30 receptors that mediate neutrophil chemotaxis in diabetic individuals and toward infection.

It is encouraging that one-time topical treatment with CCL3 after injury also substantially improved healing in diabetic wounds. However, given that diabetic foot ulcers are already suffering from neutrophilia and heightened inflammation, the therapeutic value of CCL3 treatment may seem questionable. We posit that CCL3 topical treatment may have real therapeutic potential in diabetic wound care, at least in a subset of type two obese diabetic individuals represented by our animal model, if applied topically after the surgical wound debridement process. The purpose of surgical debridement, which is performed as a standard-of-care weekly or biweekly in the clinics, is to convert a chronic non-healing wound environment into an acute healing environment through the removal of necrotic and infected tissue, and the senescent and non-responsive cells (Golinko et al., 2008; Lebrun et al., 2010; Cardinal et al., 2009). Therefore, debrided wound environment is likely to be more similar to day 1 fresh wounds than day 10 chronic wounds in our studies. Future studies are needed to evaluate the therapeutic potential of CCL3 in diabetic wound care.

Materials and methods

Procedures related to animal studies

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We have an approval from the Rush University Medical Center Institutional Animal Care and Use Committee (IACUC No.: 18–037) to conduct research as indicated. All procedures complied strictly with the standards for care and use of animal subjects as stated in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy of Sciences, Bethesda, MD, USA). We obtained 8-week-old C57BL/6 (normal) and their diabetic littermates, C57BLKS-m Leprdb (db/db) mice from the Jackson Laboratories (Bar Harbor, ME). These mice were allowed to acclimate to the environment for 1 week prior to experimentation. Wounding and wound infection were carried out as we described previously (Goldufsky et al., 2015; Wood et al., 2014). Hematoxylin & Eosin (H&E) staining were performed as we described previously (Goldufsky et al., 2015; Kroin et al., 2016). Neutrophil trafficking into wounds was assessed by immunohistochemical (IHC) analysis using Ly6G staining as described previously (Yang et al., 2019). Wound tissues’ contents of myeloperoxidase (MPO) were assessed by ELISA as described (Kroin et al., 2016). CCL3 expression was assessed by RT-PCR, following the protocol we described previously (Wood et al., 2014). To account for reduced neutrophil migration into day 1 diabetic wounds, data were normalized by 18 S RNA levels. We used Pseudomonas aeruginosa PA103 in these studies. This strain has been described previously (Shafikhani and Engel, 2006; Wood et al., 2015b) and we have shown that it causes massive infection and exacerbates wound damage in db/db wounds (Goldufsky et al., 2015). Infection levels in wounds were evaluated by determining the number of bacteria (colony forming unit (CFU)) per gram of wound tissues, as we described (Goldufsky et al., 2015; Kroin et al., 2015).

Histological analyses and wound healing assessment

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Wound healing was assessed by digital photography; by re-epithelialization assessment using H&E staining; by fibroblasts and myofibroblasts tissue content analyses using vimentin and α-SMA; and by elastin and collagen matrix deposition assessment using elastin or Masson’s Trichrome staining, using previously described techniques (Goldufsky et al., 2015; Wood et al., 2014; Roy et al., 2021; Wilgus et al., 2008; Almine et al., 2012; Diegelmann, 2004). The histological data were obtained from N = 4 mice/group and >9 random fields/wound/mouse. The data were presented as Number of counts per field of view (PFV).

Neutrophil isolation from human and mouse

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We have an Institutional Review Board (IRB)- approved protocol in accordance with the Common Rule (45CFR46, December 13, 2001) and any other governing regulations or subparts. This IRB-approved protocol allows us to collect blood samples from non-diabetic volunteers with their consents for these studies. The blood samples were first checked by a glucometer kit (FreeStyle Lite, Blood Glucose Monitoring System) to ensure that blood glucose level is within the normal range, < 100 mg/dl. Next, human neutrophils were purified from blood using the EasySep Human Neutrophil Enrichment Kit (STEMCELL Technologies), according to manufacturer’s protocol.

Murine neutrophils were isolated from either peripheral blood (used in Figure 2a–c; Figure 2—figure supplement 1; Figure 3h–i) or bone marrow (Figures 2f–m3b–g, Figure 2—figure supplement 1 and Figure 2—figure supplements 2 and 3; and Figure 3—figure supplement 1) for the studies involving glucose exposure using EasySep Mouse Neutrophil Enrichment Kit (STEMCELL Technologies), as per manufacture’s protocol and as described previously (Wood et al., 2014; Swamydas et al., 2015). Mouse neutrophils involving comparisons between C57BL/6 normal and db/db diabetic neutrophils were isolated from N = 4 blood pools/group, with each blood pool being from 4 mice. This was to obtain enough neutrophils from mouse blood (~0.8 ml of blood/mouse, 3.2 ml total) for analyses to achieve statistical significance.

Neutrophil chemotactic response

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Purified human and murine neutrophils were incubated in (IX HBSS with 2% HSA) containing glucose at indicated concentrations for 1 hr at 37 °C and stained with Calcein AM (5 µg/mL) for 30 min. After washing the cells, the cell migration assay was performed in vitro using 96-well disposable chemotaxis chambers (Cat. No. 106–8, Neuro Probe, Gaithersburg, MD, USA). Neutrophils chemotaxis toward the chemoattractants were performed at indicated concentrations, or PBS (to account for the background neutrophil migration), following the manufacturer’s protocol. Cell migration was assessed by a fluorescence (excitation at 485 nm, emission at 530 nm) plate reader Cytation 3 Cell Imaging Multi-Mode Reader (Biotek Instruments, Inc). The actual chemotaxis values were obtained by subtracting random chemotaxis values (PBS) from the chemotaxis values in response to indicated ligands.

Flow cytometry

Wound tissue digestion and flow cytometric

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C57BL/6 and db/db wound tissues were obtained at indicated timepoints as described (Wood et al., 2014), weighed, and place immediately in cold HBSS (Mediatech, Inc, Manassas, VA). Subcutaneous fat was removed using a scalpel and scissors were used to cut the tissue into small <2 mm pieces. The tissue was enzymatically dissociated in DNAse I (40 µg/ml; Sigma-Aldrich Co., St. Louis, MO) and Collagenase D (1 mg/ml HBSS; Roch Diagnostics, Indianapolis, IN) at 37 °C for 30 min. Cold PBS was used to stop the dissociation process. The tissue was then mechanically dissociated using the gentleMACS octoDissociator (program B; Miletynyi Biotec, Auburn, CA) and passed through 70 µm nylon screens into 50 ml conical tubes. Cells were washed twice with PBS. Resultant single-cell suspensions were stained using the indicated fluorescently labeled antibodies against cell surface markers, according to standard protocols described previously (Kohlhapp et al., 2012; Zloza et al., 2012). All antibodies were purchased from eBioscience, Inc (San Diego, CA). Flow cytometry was performed using a the LSRFortessa cell analyzer (Becton, Dickinson, and Company) and data were analyzed using FlowJo software (Tree Star, Ashland, OR), as previously described (Wood et al., 2014; Hackstein et al., 2012). Briefly, for the gating strategy, Live singlet lymphocytes were identified by gating on forward scatter-area (FSC-A) versus (vs.) side scatter-area (SSC-A), then LIVE/DEAD staining vs. SSC-A, FSC-A vs. FSC-height (H), SSC-A vs. SSC-H, FSC-width (W) vs SSC-W, and CD45 vs SSC-A. T cells, B cells, and NK cells were excluded using antibodies against CD3, CD19, and NK1.1, respectively, all on one channel as a dump gate. Neutrophils were then identified using CD11b vs Ly6G staining, with neutrophils being CD11b high and Ly6G high. FPR1 and CCR1 expression on neutrophils was then analyzed and is presented as percentage of cells (e.g. neutrophils) expressing the respective marker.

Neutrophil depletion in mice

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Neutrophil depletion in mice were performed as described (Nozawa et al., 2006; Bruhn et al., 2016). Briefly, db/db mice received either anti-Ly6G (100 µg/mouse) to cause neutrophil depletion or an IgG isoform control (100 µg/mouse), by intraperitoneal (i.p.) injection. Neutrophil depletion was confirmed by the assessment of neutrophil content in the blood (circulation) by flow cytometry or in wound tissues by MPO analysis.

Western blot analyses

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We performed western immunoblotting on cell lysates or on tissue lysates, using the indicated antibodies as we described previously (Kroin et al., 2016; Shafikhani and Engel, 2006; Mohamed et al., 2021). Equal amounts of proteins (as determined by BCA analysis) were loaded. GAPDH was used as a loading control.

Gene expression analysis by real-time polymerase chain reaction (RT-PCR)

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Gene expression was assessed by RT-PCR as we described (Wood et al., 2014): cDNA was generated using SuperScript III First-Strand Synthesis System cDNA Synthesis Kit (Cat. No. 18080051) from Thermo Fisher, according to manufacturer’s protocol. RT-PCR was then preformed with gene-specific primer pairs mentioned below, using the Applied Biosystems QuantStudio 7 Flex Real-Time PCR System. The data were calculated using the 2−ΔΔCt method and were presented as ratio of transcripts for gene of interest normalized to 18 S or GAPDH. We performed RT-PCR using the indicated primers listed in the ‘Key Resources Table’.

Statistical analysis

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Statistical analyses were performed using GraphPad Prism 6.0 as we described previously (Roy et al., 2021; Wood et al., 2011; Wood et al., 2015a). Comparisons between two groups were performed using Student’s t-test. Comparisons between more than two groups were performed using one-way analysis of variance (one-way ANOVA). To account for error inflation due to multiple testing, the Bonferroni method was used. Data are presented as Mean ± SEM. Statistical significance threshold was set at -values ≤ 0.05.

Appendix 1

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (C57BL/6 J)C57BL/6 JJackson laboratories000664
Strain, strain background (C57BLKS/J)C57BLKS-m Leprdb/dbJackson laboratories000662
AntibodyAnti-Ly-6G/Ly-6C Monoclonal Antibody (RB6-8C5)(Mouse monoclonal)Thermo Fisher ScientificCat# MA1-10401, RRID:AB_11152791For neutrophil depletion (100 µg/mouse)
AntibodyAnti-Mouse (G3A1) mAb IgG1 Isotype Control antibody(Mouse monoclonal)Cell Signaling TechnologiesCat#5415, RRID:AB_10829607For neutrophil depletion (100 µg/mouse)
AntibodyGAPDH antibody (Rabbit polyclonal)ProteintechCat# 1094-I-AP, RRID:AB_2895245WB (1:10000)
AntibodyAnti-Ly6G antibody clone RB6-8C5 (Rat monoclonal)AbcamCat# ab25377, RRID:AB_470492IHC (1:50)
AntibodyAnti-FPR1 antibody(Rabbit polyclonal)NOVUS BiologicalCat# NB100-56473, RRID:AB_838228WB (1:1000)
AntibodyAnti-FPR2/ FPRL1 antibody(Rabbit polyclonal)NOVUS BiologicalsCat# NLS1878, RRID:AB_2294156WB (1:1000)
AntibodyAnti-PLC1 antibody(Rabbit polyclonal)Cell Signaling TechnologyCat# cs2822, RRID:AB_2163702WB (1:1000)
AntibodyAnti-CCR1 antibody(Rabbit polyclonal)AbnovaCat# PAB0176, RRID:AB_1018941WB (1:500)
AntibodyAnti-α-SMA antibody(Rabbit polyclonal)AbcamCat# ab5694, RRID:AB_2223021
AntibodyAnti-vimentin antibody(Rabbit monoclonal)AbcamCat# ab92547, RRID:AB_10562134
AntibodyMouse CCR1 Alexa Fluor 488-conjugated Antibody(Rat monoclonal)NOVUS BiologicalsCat# FAB5986G, RRID:AB_2895246Flow cytometery
AntibodyAlexa Fluor 700 anti-mouse NK-1.1 Antibody(Mouse monoclonal)BioLegendCat# 108729, RRID:AB_2074426Flow cytometery
AntibodyAlexa Fluor 700 anti-mouse CD3ε Antibody(Syrian Hamster monoclonal)BioLegendCat# 152315, RRID:AB_2632712Flow cytometery
AntibodyAlexa Fluor 700 anti-mouse CD19 Antibody(Rat monoclonal)BioLegendCat# 115527, RRID:AB_493734Flow cytometery
AntibodyBV605 Hamster Anti-Mouse CD11c Clone HL3 (RUO)(Hamster monoclonal)BD BiosciencesCat# 563057, RRID:AB_2737978Flow cytometery
AntibodyF4/80 antibody, Cl:A3-1(Rat monoclonal)Bio-RadCat# MCA497PBT, RRID:AB_1102557Flow cytometery Flow cytometery
AntibodyBV650 Hamster Anti-Mouse CD11c Clone HL3(Hamster monoclonal)BD BiosciencesCat# 564079, RRID:AB_2725779Flow cytometery
AntibodyBV711 Rat Anti-Mouse CD45 Clone 30-F11(Rat monoclonal)BD BiosciencesCat# 563709, RRID:AB_2687455Flow cytometery
AntibodyNK1.1 Monoclonal Antibody (PK136), PE, eBioscience(Mouse monoclonal)Thermo Fisher ScientificCat# 12-5941-82, RRID:AB_466050Flow cytometery
AntibodyCD19 Monoclonal Antibody (eBio1D3 (1D3)), PE, eBioscience(Rat monoclonal)Thermo Fisher ScientificCat# 12-0193-82, RRID:AB_657659Flow cytometery
AntibodyCD3e Monoclonal Antibody (145–2 C11), PE, eBioscience(Hamster monoclonal)Thermo Fisher ScientificCat# 12-0031-82, RRID:AB_465496Flow cytometery
AntibodyFPR1 Polyclonal Antibody(abbit polyclonal)Thermo Fisher ScientificCat# PA1-41398, RRID:AB_2247097Flow cytometery
AntibodyGoat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594(Goat polyclonal)Thermo Fisher ScientificCat# A-11037, RRID:AB_2534095Flow cytometery
AntibodyLy6G Monoclonal Antibody (1A8-Ly6g), PE-Cyanine7, eBioscience(Rat monoclonal)Thermo Fisher ScientificCat# 25-9668-82, RRID:AB_2811793Flow cytometery
AntibodyPerCP Cy5.5 CD45 antibody(Rat monoclonal)BD BiosciencesCat# 550994, RRID:AB_394003Flow cytometery
AntibodyAPC Gr1, PE CD11b antibody(Rat monoclonal)BD BiosciencesCat# 553129, RRID:AB_398532Flow cytometery
AntibodyFITC CD69 antibody(Hamster monoclonal)BD BiosciencesCat# 557392, RRID:AB_396675Flow cytometery
AntibodyPECy7 F4/80 antibody(Rat monoclonal)BioLegendCat# 123114, RRID:AB_893478Flow cytometery
Commercial assay or kitLIVE/DEAD Fixable Aqua Dead Cell Stain Kit, for 405 nm excitationThermoFisher ScientificCat# L34966
Sequence-based reagentFPR1_FIntegrated DNA TechnologiesRT-PCR primersGAGCCTAGCCAAGAAGGTAATC
Sequence-based reagentFPR1_RIntegrated DNA TechnologiesRT-PCR primersTCCCTGGTCCAAGTCTACTATT
Sequence-based reagentFPR2_FIntegrated DNA TechnologiesRT-PCR primersTTGTCTCAATCCGATGCTCTATG
Sequence-based reagentFPR2_RIntegrated DNA TechnologiesRT-PCR primersTCAGGGCTCTCTCAAGACTATAA
Sequence-based reagentPlcg1_FIntegrated DNA TechnologiesRT-PCR primersGGTGAGGCCAAATGTGAGATA
Sequence-based reagentPlcg1_RIntegrated DNA TechnologiesRT-PCR primersGGGCAACCAAGAGGAATGA
Sequence-based reagentCcr1_FIntegrated DNA TechnologiesRT-PCR primersGCTATGCAGGGATCATCAGAAT
Sequence-based reagentCcr1_RIntegrated DNA TechnologiesRT-PCR primersGGTCCAGAGGAGGAAGAATAGA
Sequence-based reagentCcl3_FIntegrated DNA TechnologiesRT-PCR primersTCACTGACCTGGAACTGAATG
Sequence-based reagentCcl3_RIntegrated DNA TechnologiesRT-PCR primersCAGCTTATAGGAGATGGAGCTATG
Sequence-based reagentGAPDH_FIntegrated DNA TechnologiesRT-PCR primersTTGGGTTGTACATCCAAGCA
Sequence-based reagentGAPDH_RIntegrated DNA TechnologiesRT-PCR primersCAAGAAACAGGGGAGCTGAG
Sequence-based reagent18 S_FIntegrated DNA TechnologiesRT-PCR primersCACGGACAGGATTGACAGATT
Sequence-based reagent18 S_RIntegrated DNA TechnologiesRT-PCR primersGCCAGAGTCTCGTTCGTTATC
Commercial assay or kitMyeloperoxidase (MPO) Mouse ELISA KitThermo Fisher ScientificCat# EMMPO
Commercial assay or kitIL-1b ELISA kitThermo Fisher ScientificCat# 88-7013-88
Commercial assay or kitTNF- a ELISA kitThermo Fisher ScientificCat# 88-7324-88
Commercial assay or kitCyclic AMP Competitive ELISA KitCayman chemicalCat# 581,001
Commercial assay or kitEasySep Human Monocytes Enrichment KitSTEMCELL TechnologiesCat# 19,359
Commercial assay or kitEasySep Mouse monocytes Enrichment KitSTEMCELL TechnologiesCat# 19,861
Commercial assay or kitSuperScript III First-Strand Synthesis SystemThermo FisherCat# 18080051
Peptide, recombinant proteinCCL3 (recombinant mouse CCL3/MIP-1α protein)R & D SystemsCat# 450-MA
Peptide, recombinant proteinN-formyl-Met-Leu-Phe (fMLP)SigmaCat# 59880-97-6
Peptide, recombinant proteinRecombinant Human CXCL1/GRO alpha ProteinR & D SystemsCat# 275-GR
Peptide, recombinant proteinRecombinant Mouse CXCL1/KC ProteinR & D SystemsCat# 453-KC
Software, algorithmGraphPadGraphPadhttps://graphpad.com/scientific-software/prism/
OtherHematoxylinThermo Fisher ScientificCat# 7,111 L
OtherEosin YThermo Fisher ScientificCat# 7,211 L
OtherBluing ReagentThermo Fisher ScientificCat# 7,301 L
OtherMasson’s Trichrome stainAbcamCat# ab150686
OtherEasySep BufferSTEMCELL TechnologiesCat. No. 20,144
OtherSYBR Green PCR Master MixThermo FisherCat. No. 4309155
OtherCollagenase DSigmaCat# 9001-12-1
OtherCalcein AMThermo Fischer ScientificCat# C1430

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for each experiment.

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Decision letter

  1. Ursula Rescher
    Reviewing Editor; University of Muenster, Germany
  2. Carla V Rothlin
    Senior Editor; Yale School of Medicine, Yale University, United States
  3. Mauro Perretti
    Reviewer; Queen Mary University of London, United Kingdom

Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "Overriding FPR defective chemotaxis signaling in neutrophils stimulates infection control and healing in diabetic wound" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Carla Rothlin as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Mauro Perretti (Reviewer #2).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

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 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-1α/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.

All three reviewers agree that the research area is important, the findings are novel and interesting and of potential therapeutical value. While the study is well planned and the results are logically presented, two of the reviewers feel that key aspects of the study require additional data to fully support the central conclusions and that the results at the current stage provide an only limited increase in knowledge on metabolic regulation of neutrophil functions. We, therefore, encourage the authors to perform the requested experiments to strengthen their claims.

Experimental work:

1) A major point of concern is that the very low and sometimes unclear n values question the validity and robustness of the data. Please provide power analyses to justify the group sizes. Furthermore, the n number needs to be stated fully in every figure legend as well as in the Methods section, in line with eLife policies. The statement that there are 16 mice per neutrophil comparison group is confusing, as the blood of 4 mice has been pooled. Therefore, one pool is more likely n=1, please address this issue. Also, please correct n values in figure legends according to the information given in the Methods ≥ 5 mice/group.

2) The fact that FPR1 mRNA and FPR1 protein rapidly decrease within 1 hour of high glucose treatment is astonishing and no mechanistic explanation is provided. As there are two homologous FPR receptors responding to formylated peptides (although to different levels of sensitivity/specificity) in human as well as in murine neutrophils, the authors should analyze the expression pattern of both FPR1 and FPR2 in diabetic and glucose-exposed human and murine neutrophils. Because no proof is provided that P. aeruginosa bacterial products (or any other FPR1 ligands) acting via FPR1 are responsible for the observed neutrophil recruitment in healthy mice or the lack of in diabetic mice, it should be addressed whether the downregulation is a more common phenomenon and not restricted to FPR1 This question should be addressed, especially as the title emphasizes the importance of FPR1. Is the diminished cell surface pool due to receptor internalization? Please extend the time course and include days 1, 3, 6 and 10 for a more in-depth analysis of neutrophil changes (FPR, CCL3) and effects of topical treatment to enhance the neutrophil response (figure 4) in in diabetic wounds over time. Are the findings specific for neutrophils or do e.g. monocytes respond similarly?

Methodology:

1) Sex differences have been shown to be important for the diabetic wound healing. Please provide whether male or female mice have been used and the rational for choice of sex and age.

2) As both bone marrow-derived neutrophils and blood neutrophils are used, please provide the reasons for the respective choice of the very different compartments and verify that neutrophils derived from these two compartments behave in a similar manner regarding receptor expression and downregulation. Please include the baseline cells counts (especially neutrophils) for the mice used in the study.

3) Please provide and discuss the exact RFU numbers in Figures S3 and Figure 2f.

Discussion:

Please elaborate on the relevance of your specific findings with regard to (1) the mechanistic basis (CCL3 source? Molecular regulation underlying downregulation on the different expression levels?) and their relevance in the more general picture of the link between onset and resolution of inflammation as discussed in e.g. [PMID: 16369558 DOI: 10.1038/ni1276, PMID: 27199985 PMCID: PMC4845539 DOI: 10.3389/fimmu.2016.00160, PMID: 27021499 DOI: 10.1016/j.smim.2016.03.007].

Reviewer #1 (Recommendations for the authors):

In human as well as in murine neutrophils, there are two receptors (FPR1 and FPR2) that react to formylated peptides, although to different levels of sensitivity/specificity. While the human FPR1 is highly sensitive to fMLF, FPR2 is more reactive towards host peptides, and both receptors also detect mitochondrially derived peptides. In mice, the sensitivity toward fMLF is generally low and other formylated peptides exert stronger chemotactic activities via FPRs. Commonly, FPR1 is thought to function in a pro-inflammatory manner, whereas FPR2 activation is linked to the resolution phase. The authors should analyze the expression pattern of both FPR1 and FPR2 in diabetic and glucose-exposed human and murine neutrophils and also include other ligands. Furthermore, the authors assume that Pseudomonas aeruginosa-infected wounds in diabetic mice show slower healing because the neutrophils are FPR1-deficient and therefore less attracted toward the infection site. However, no proof is provided that P. aeruginosa bacterial products (or any other FPR1 ligands) acting via FPR1 are responsible for the observed neutrophil recruitment in healthy mice or the lack of in diabetic mice. This question should be addressed, especially as the title emphasizes the importance of FPR1. It is also conceivable that another (unknown) receptor is downregulated under diabetic conditions.

Reviewer #2 (Recommendations for the authors):

Roy et al., present an interesting manuscript where the molecular and cellular responses are studied in a model of diabetes wound healing, or lack of, and infection. The study is well planned and logically presented.

The most interesting aspect of the presented work, to me, is the clear link between an effective and exuberant inflammatory reaction and resolution with tissue repair. The ability of CCL3 application to restore a proper neutrophil response to the infected wounds and as such ensure the appropriate management of the infection is an excellent proof that 'the beginning programs the end' [PMID: 16369558 DOI: 10.1038/ni1276]. On a similar vein, dissecting how resolution starts is equally challenging and interesting [PMID: 27199985 PMCID: PMC4845539 DOI: 10.3389/fimmu.2016.00160] and well presented here. Finally, the notion that neutrophils are not just BAD is also excellent and the study here provides a very clear evidence for the link between onset and resolution [PMID: 27021499 DOI: 10.1016/j.smim.2016.03.007]. This issue may need to be highlighted in the Discussion section as chiefly demonstrated here.

I have some comments and some unclear issues I would like the Authors to comment or address.

1. FPR1 mRNA (Figure 2)

I was surprised to see the rapid decade in FPR1 mRNA within 1 hour of high glucose. To my knowledge, FPR1 is not an early response gene (like fos and jun) so it will be appropriate for the Authors to perhaps double check this dataset, perhaps running a time course and study what else goes down within 30-60-90-120 min. Would there be mileage in monitoring FPR2 mRNA too as a relevant control?

2. FPR1 protein

The same question can be asked for the rapid downregulation of FPR1 protein from the cell surface of mouse and human neutrophils following incubation with glucose. The data are convincing here, but again very intriguing how this could occur. More speculation in the Discussion would be good. Also, experiments aiming to assess if the receptor internalised (thus do we find higher intracellular levels?) and if so, how, would be appropriate for a study published in eLife.

3. Legends to the Figures

I find the figure legends written in an unclear manner. usually the Panel letter (e.g. A, or B or C…) is at the beginning of the sentence, whereas here is all a bit mixed up. In some cases groups of 3 mice have been used. Is this acceptable? How reproducible are the Western blots?

4. CCL3

Data in Figure 4C seem to indicate that the infiltrating neutrophils are responsible for delivering CCL3 (or MIP-1alpha). Probably some indication of the potential sources of CCL3 in the infected wounds, including the migrated neutrophils, is worthwhile. And relevant and interesting.

Reviewer #3 (Recommendations for the authors):

The manuscript by Roy et al. entitled "Overriding defective FPR chemotaxis signaling in diabetic neutrophil stimulates infection control in diabetic wound", focuses on impaired healing in diabetic wounds. Moreover, the role that neutrophils play and the therapeutic potential of the pro-inflammatory cytokine CCL3 in the treatment of diabetic foot ulcers. Overall, this is an interesting manuscript, but there are several points which should be addressed:

• The n values for this study seem low, n=2 or 2 in some cases. This reviewer would argue the validity and robustness of performing statistical analysis on such low n numbers and the ultimate value of the findings.

• In every figure legend and methods section, the authors need to state fully the n number to provide transparency in their data and in line with eLife policies.

• Methods section: Neutrophil isolation from human and mouse. "Mouse neutrophils involving comparisons between C57B normal and db/db diabetic neutrophils were extracted from N=4 blood pools/group, with each blood pool being from 4 mice: totaling 16 mice per group." This is incorrect. There are not 16 mice per group. If each group has blood pooled from 4 mice, then this is n=1, not 4, therefore n=4, not 16. This needs to be addressed.

• Methods section: Histological analyses and wound healing assessment. "The histological data, (obtained from n≥5 mice/group and >9 random fields/wound/mouse), were normalized per wound surface area". Although the authors state ≥5 mice/group, the figure legends suggest differently. This needs to be corrected.

• What are the baseline cells counts (especially neutrophils) for the mice used in the study. These should be included.

• In some cases, the authors use bone marrow derived neutrophils and in other cases blood borne neutrophils, although it is unclear to this reviewer what determined the choice. As it is known that these compartments are very different, it would add value to the manuscript to show whether neutrophils derived from these two compartments behave differently and express different cell surface receptors. Furthermore, these changes/similarities should be shown over the time course of 1, 3, 6 and 10 days, as this may help to further explain what is causing the delay in neutrophil influx in diabetic wounds over time.

• Although CCL3 expression is reduced in day 1 diabetic wounds, what happens at 3, 6 and 10 days?

• The finding that topical treatment enhances neutrophil response (figure 4) is very interesting. These data are for day 1, what happens over the time course i.e. 3, 6 and 10 days?

• The authors show nicely the involvement of FPR1, but what about the other known FPR that neutrophils express i.e. FPR2? Does this have a compensatory role or is this receptor also dysfunctional. Data should be provided.

• In addition, what happens to FPR1 (and 2) expression over the time course i.e. 3, 6 and 10 days? Does it change?

• Were the mice male or female or a mixture? Please provide rational for choice and whether sex as a biological variable was considered, especially given the fact that gender differences have been shown in recovery to diabetic wounds.

• The authors chose mice at 8 weeks, with 1 week acclimatisation. Given this young adult age, what is the clinical relevance of this choice of age?

• Figure S3: 100nM fMLP: The data for the 90 mg/dl is around 800 RFU, however, in Figure 2f, this value looks to be much higher (around 1200 RFU). Would the authors provide the exact numbers and discuss further.

• Although this student specifically looks at neutrophils, do the authors have any data to suggest whether these finds are neutrophil specific or whether e.g. monocytes, play a role in the later time points?

• C57/B should be written in full: C57Bl/6. 'FPR receptor' should be changed to just 'FPR'. Proof reading would help – grammar and spelling mistakes need correcting.

https://doi.org/10.7554/eLife.72071.sa1

Author response

Essential revisions:

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 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-1α/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.

All three reviewers agree that the research area is important, the findings are novel and interesting and of potential therapeutical value. While the study is well planned and the results are logically presented, two of the reviewers feel that key aspects of the study require additional data to fully support the central conclusions and that the results at the current stage provide an only limited increase in knowledge on metabolic regulation of neutrophil functions. We, therefore, encourage the authors to perform the requested experiments to strengthen their claims.

Experimental work:

1) A major point of concern is that the very low and sometimes unclear n values question the validity and robustness of the data. Please provide power analyses to justify the group sizes. Furthermore, the n number needs to be stated fully in every figure legend as well as in the Methods section, in line with eLife policies. The statement that there are 16 mice per neutrophil comparison group is confusing, as the blood of 4 mice has been pooled. Therefore, one pool is more likely n=1, please address this issue. Also, please correct n values in figure legends according to the information given in the Methods section ≥ 5 mice/group.

First, we would like to thank the reviewers and the editor for their thorough and insightful reviews of our manuscript. Addressing their suggestions have substantially improved our manuscript as we hope the editor and the reviewers will agree. (New data added to revised manuscripts are Figure 5a-b, Figure S4a-c, Figure 2—figure supplement 1 and Figure 2—figure supplement 1). As for responding to query 1, we sincerely apologize to the reviewers and the editor for this confusion in the figure legends. Nowhere in our 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 experiment at least two independent times so the actual numbers are N>4. We also apologize to the reviewers for indicating N=16 in the animal experiments where blood from 4 mice were pooled together to be able to obtain enough neutrophils. We agree with the reviewer that in reality N=4 blood pools per group (generated from 16 mice/group). As requested, we have made the corrections of statistical analyses both in the figure legends and in the Methods section of the revised manuscript. In addition, we have provided the raw data for all the figures so that anyone who wishes to examine the data will be able to do so.

2) The fact that FPR1 mRNA and FPR1 protein rapidly decrease within 1 hour of high glucose treatment is astonishing and no mechanistic explanation is provided. As there are two homologous FPR receptors responding to formylated peptides (although to different levels of sensitivity/specificity) in human as well as in murine neutrophils, the authors should analyze the expression pattern of both FPR1 and FPR2 in diabetic and glucose-exposed human and murine neutrophils. Because no proof is provided that P. aeruginosa bacterial products (or any other FPR1 ligands) acting via FPR1 are responsible for the observed neutrophil recruitment in healthy mice or the lack of in diabetic mice, it should be addressed whether the downregulation is a more common phenomenon and not restricted to FPR1 This question should be addressed, especially as the title emphasizes the importance of FPR1. Is the diminished cell surface pool due to receptor internalization? Please extend the time course and include days 1, 3, 6 and 10 for a more in-depth analysis of neutrophil changes (FPR, CCL3) and effects of topical treatment to enhance the neutrophil response (figure 4) in in diabetic wounds over time. Are the findings specific for neutrophils or do e.g. monocytes respond similarly?

We thank the reviewers and the editor for their recognition of the importance of our data. As the reviewers and the editor astutely pointed out, (both in human and mouse), FPR1 has a much higher affinity for bacterial formyl peptides than FPR2, whereas, FPR2 has a broader range of ligands than FPR1 and it can also respond to other ligands, such as amyloid peptides, antimicrobial peptides, and lipid mediators (1, 2). In addition, FPR2 receptor has also been implicated in the resolution of inflammation in response to Annexin A1, lipoxin A4, and resolving D1 pro-resolving agonists (3-5). Given that this manuscript was about diabetic neutrophil’s impaired chemotactic response toward infection, we had focused on FPR1 receptor in this manuscript. With that said, we now provide new data to show that exposure to HG also significantly dampens the expression of FPR2 receptor in neutrophils both at the transcriptional and translational levels (please see Figure 2—figure supplement 3). We also provide new data to show that exposure to HG substantially reduces both FPR1 and FPR2 at 1, 2, and 3 hours post exposure (please see Figure 2—figure supplement 2 and Figure 2—figure supplement 3), indicating that the adverse impact of HG on FPR1 expression is not transient and it is sustained. We are in complete agreement with the reviewers and the editor that our manuscript does not provide direct evidence that the recognition of P. aeruginosa by neutrophils is primarily mediated by the FPR1 and because of this reason, we have revised the manuscript to take into account the reviewers and the editor’s suggestion, by de-emphasizing FPR1. Rather we use the term FPR instead.

As for the suggestion to extend the time course of neutrophil characterizations in normal and diabetic wounds to days 3, 6, and 10, in addition to day 1 data that we had provided, we believe that expanding neutrophil characterizations beyond day 1 would go beyond the scope of this manuscript and could also potentially raise other interesting questions that may detract from the main point of this manuscript which is the novel finding that inadequate neutrophil response early after injury, renders diabetic wounds vulnerable to infection. We are very much interested in addressing the questions raised by the editor and the reviewers such as whether the impact of HG on FPR signaling is limited to neutrophils or all leukocytes. Although, we do believe that with respect to infection control, our data clearly show that the neutrophils play the primary role in controlling P. aeruginosa infection in wound (please see Figure 4), therefore, we focused on neutrophils. We also agree that it would also be interesting to assess the dynamics of neutrophil surface expression of chemotaxis receptors and their ligands (such as CCL3) during early and late phases of healing in normal and diabetic wounds. However, we believe that such studies would require a more comprehensive analyses on all ~30 chemotaxis receptors on neutrophils and their respective ligands. In addition, we would also need to characterize the wounds’ environmental factors that can potentially enhance or dampen the expression of these receptors and/or their signaling pathways in neutrophils in wounds at these timepoints. We would also need to validate our results. As the editor and the reviewers appreciate, these studies are beyond the scope of any single manuscript. In fact, we have submitted a grant proposal to conduct these comprehensive studies and also to tease out the mechanism (s) that is responsible for the HG-induced reduction in the FPR receptor signaling in diabetic neutrophils. 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 affects the dynamics of diabetic neutrophil trafficking into wound during the acute phase of healing, early after injury. Our data clearly establish a new paradigm that blames inadequate neutrophil response early after injury for rendering diabetic wounds vulnerable to infection and for setting the stage for the sustained and non-resolving inflammatory environment during the chronic phase as diabetic wounds age (11, 12). We also show that neutrophil depletion in diabetic animals by anti-Ly6G causes diabetic wounds to contain significantly more bacteria, 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 degree of antimicrobial functions under diabetic conditions. Finally, we show that by harnessing these neutrophils in diabetic wounds early after injury by topical treatment with CCL3, which engages CCR1, CCR4, and CCR5 auxiliary receptors (13-15), we can reduce infection levels in diabetic wounds by ~2 log orders and significantly improve healing in diabetic wounds.

As the editor and the reviewers are 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, other than 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). Therefore, our data are also very important as they reveal therapeutic potential for CCL3 topical treatment to enhance infection control and stimulate healing in diabetic wounds. We hope that the reviewers and the editor agree with us that 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.

Methodology:

1) Sex differences have been shown to be important for the diabetic wound healing. Please provide whether male or female mice have been used and the rational for choice of sex and age.

We appreciate and agree with the reviewer #3 that sex is a critical biological variable that needs to be considered in designing any experiment. In studies involving human neutrophils, we isolated neutrophils from both male and female volunteers, and regardless of their sex, these neutrophils behaved similarly with respect to their chemotactic responses toward fMLF and CCL3. In addition, neutrophils isolated from male and female volunteers also exhibited the same reduction in FPR signaling when exposed to HG, suggesting that sex does not affect neutrophil chemotaxis signaling, at least in cell culture. However, since several prior publications have indicated that type 2 diabetic mellitus is more common in males than females [100-103], and because of the budgetary constraints and the high expenses associated with type 2 db/db diabetic mice, we restricted our analysis to male db/db mice.

2) As both bone marrow-derived neutrophils and blood neutrophils are used, please provide the reasons for the respective choice of the very different compartments and verify that neutrophils derived from these two compartments behave in a similar manner regarding receptor expression and downregulation. Please include the baseline cells counts (especially neutrophils) for the mice used in the study.

We appreciate and agree with the reviewers that bone marrow neutrophils and blood neutrophils are not the same but our decision to use neutrophils from these different sources was deliberate because we intended to address different questions using these neutrophils. The initial observation – that showed diabetic neutrophils had impaired chemotaxis – involved neutrophils that were isolated from the blood of diabetic patients (10). Therefore, we isolated neutrophils from the blood of diabetic mice to show that the same chemotaxis impairment was also present in neutrophils isolated from the blood of diabetic mice, as a way to reproduce the human diabetic condition with respect to this impairment. On the other hand, when our goal was to assess the impact of high glucose on chemotaxis and chemotaxis signaling in neutrophils, we isolated neutrophils from the bone marrow of normal mice and exposed them to HG to show that exposure to HG was sufficient to cause similar chemotaxis impairment in normal neutrophils. Of note, bone marrow leukocytes (including neutrophils) in diabetic animals do not appear to have the same functional impairments as peripheral blood leukocytes in diabetic animals or human, as they behave similarly to leukocytes extracted from bone marrow of normal mice (11, 32-34), suggesting that bone marrow leukocytes in diabetic animals are protected from the adverse effects of exposure to HG in the bone marrow compartment. As the reviewers and the editor are aware, neutrophils are produced in bone marrow and released into circulation (30, 31), hence our rationale for using bone marrow as the source of normal neutrophils. Finally, it was also more practical to use bone marrow neutrophils, because we could obtain significantly more neutrophils from bone marrow than blood of these mice. As we had indicated in our manuscript, we had to pool the blood of 4 mice (16 mice/group) to be able to obtain enough neutrophils for a single analysis. As for the request to include the baseline neutrophil counts in our animal studies, we believe that these are not necessary for the following reasons. Neutrophils are rarely found in the skin and are not considered skin-resident cells, but they migrate into the skin in high numbers in response to inflammatory conditions, such as injury and/or infection (35-39). Consistent with these reports, we have also demonstrated that in the absence of infection and injury, neutrophils are not detected in the skin of mice (37). Given that we did not keep the skins of the mice at T0 (time of wounding), we would need to repeat these studies to generate the requested data. We hope that the reviewers and the editor agree with us that including the baseline numbers on neutrophils would not add any new information or change any of the figures or their interpretations and thus repeating these studies is not warranted, particularly because both co-first authors on this manuscript have left the lab.

3) Please provide and discuss the exact RFU numbers in Figures S3 and Figure 2f.

As the reviewer appreciates, many factors can cause intrinsic variations in data, including the operator and/or source of reagents. In our experience, even using the fluorescent dye from the same vial and at same concentration could lead to different intensities and RFU numbers in replicate experiments, particularly if the experiments were performed by different individuals, which was the case for Figure 2f and Figure 2—figure supplement 1. The important point is that in both experiments (which they had their own appropriate controls), we did obtain similar significant results showing that neutrophils treated with normal glucose level (90 mg/dl) displayed significantly higher chemotaxis as compared to neutrophils treated with HG. As requested by the reviewer, we provide the raw data and the exact RFUs for Figure 2f (100nM fMLF) and Figure 2—figure supplement 1 (at indicated fMLF concentrations). We have included a file called “Source data” that contain the raw data used in all the figures should the reviewers wish to evaluate them.

Discussion:

Please elaborate on the relevance of your specific findings with regard to (1) the mechanistic basis (CCL3 source? Molecular regulation underlying downregulation on the different expression levels?) and their relevance in the more general picture of the link between onset and resolution of inflammation as discussed in e.g. [PMID: 16369558 DOI: 10.1038/ni1276, PMID: 27199985 PMCID: PMC4845539 DOI: 10.3389/fimmu.2016.00160, PMID: 27021499 DOI: 10.1016/j.smim.2016.03.007].

We thank the reviewer for his/her knowledge of the field and for bringing to our attention these articles, particularly Jones, et al. article (2016, Seminars in Immunology), which discusses the role of neutrophils in inflammation resolution. We have revised the Discussion section to address the reviewer’s request. Regarding the mechanistic basis for CCL3 cellular sources and the reduction in CCL3 expression in diabetic wounds early after in jury, we posit that multiple factors could contribute to the reduction in CCL3 in diabetic wound early after injury. As we discussed in the introduction section, production of ligands (including CCL3) for auxiliary receptors in tissue ultimately depends on FPR activation in neutrophils, resulting in the production of IL-1b which induces the production of these auxiliary ligands from inflammatory and non-inflammatory cells (40-43). Therefore, reduced signaling through FPR, would be expected to adversely affect the expression of the ligands (including CCL3) for auxiliary receptors in diabetic wound early after injury. In addition, leukocytes (including neutrophils) are major cellular sources of CCL3 (44). Therefore, we speculate that reduction in CCL3 expression in diabetic wound early after injury is at least in part due to reduced neutrophil influx in these wounds, as we show in this manuscript (Figure 1). Moreover, we just published a paper in the Journal of Investigative Dermatology (45), showing that dysregulation in the expression of immunosuppressive IL-10 (high IL-10 expression in diabetic wounds early after injury but low IL-10 expression in old diabetic wounds) leads to significant reduction in toll-like receptor (TLR) signaling and culminates in significant reduction in the production of pro-inflammatory cytokines in diabetic wounds early after injury. Given that TLR signaling has also been implicated in the production of ligands (including CCL3) for auxiliary receptors expression (46, 47), we posit that reduced TLR signaling could also contribute to reduced CCL3 expression in diabetic wound early after injury. As the editor and the reviewers appreciate, substantial amount of work is needed to teas out the contributions of these factors in the dynamics of CCL3 and other ligands for the auxiliary receptors, which is beyond the scope of this manuscript. Nevertheless, we have added a section in the Discussion section of the revised manuscript to discuss these possibilities.

As for the link between our data (Figure 4) and the onset inflammatory responses in the CCL3-treated wounds, we expected this outcome, given that CCL3 is an important pro-inflammatory cytokine which has been shown to recruit neutrophils by engaging multiple auxiliary receptors, such as CCR1, CCR4, and CCR5 (40, 43, 48-50). As for the resolution of inflammatory responses in the CCL3-treated diabetic wounds during the late stages of healing in diabetic wounds, it could be due of reduction in infection and bacterial burden in CCL3-treated diabetic wounds as we have shown here (Figure 4h-j), thus reducing the need for neutrophils to combat infection; but it also could be due to the production and release of anti-inflammatory and inflammation resolving proteins and bioactive lipids by recruited neutrophils, such as Annexin A1, lipoxins (e.g., LXA4), and protectin D1 (reviewed in (51)). In addition, uptake of neutrophil apoptotic bodies by macrophages through efferocytosis has also been shown to induce the M2 anti-inflammatory macrophage differentiation which further resolve inflammation. We have added a section in the Discussion section of the revised manuscript regarding this.

Reviewer #1 (Recommendations for the authors):

In human as well as in murine neutrophils, there are two receptors (FPR1 and FPR2) that react to formylated peptides, although to different levels of sensitivity/specificity. While the human FPR1 is highly sensitive to fMLF, FPR2 is more reactive towards host peptides, and both receptors also detect mitochondrially derived peptides. In mice, the sensitivity toward fMLF is generally low and other formylated peptides exert stronger chemotactic activities via FPRs. Commonly, FPR1 is thought to function in a pro-inflammatory manner, whereas FPR2 activation is linked to the resolution phase. The authors should analyze the expression pattern of both FPR1 and FPR2 in diabetic and glucose-exposed human and murine neutrophils and also include other ligands. Furthermore, the authors assume that Pseudomonas aeruginosa-infected wounds in diabetic mice show slower healing because the neutrophils are FPR1-deficient and therefore less attracted toward the infection site. However, no proof is provided that P. aeruginosa bacterial products (or any other FPR1 ligands) acting via FPR1 are responsible for the observed neutrophil recruitment in healthy mice or the lack of in diabetic mice. This question should be addressed, especially as the title emphasizes the importance of FPR1. It is also conceivable that another (unknown) receptor is downregulated under diabetic conditions.

We appreciate reviewer’s knowledge of the field and precisely because of the differences between FPR1 and FPR2, as noted by the reviewer, we had focused our analysis on FPR1, given that this manuscript involves the role of impaired chemotaxis in diabetic neutrophils in response to Pseudomonas aeruginosa infection. Per reviewer’s request, we now provide new data which show that HG significantly dampens the expression of both FPR1 and FPR2 (please see Figure 2—figure supplement 2 and Figure 2—figure supplement 3). These data are interesting as they suggest that reduced FPR1 signaling may be an important factor in reduced neutrophil trafficking into diabetic wounds early after injury. In contrast, reduced signaling through FPR2 may be an important contributing factor in the persistent non-resolving inflammatory environment in old and chronic diabetic wounds, which contain a lot of neutrophis, as have been reported (52-54) and confirmed by our data (Figure 1). We acknowledge that we do not provide direct evidence that P. aeruginosa is primarily recognized by FPR1, nor do we believe that FPR1 is the only receptor responsible for P. aeruginosa recognition by innate immune responses in wounds. However, as the reviewer is aware, FPR signaling plays a crucial role in the initial neutrophil wave toward injury and infection (40, 42, 55-59), and it is therefore logical to posit that defective signaling through FPR in diabetic wound plays an important role in reduced neutrophil influx in diabetic wound early after injury as we show in this manuscript. We also agree with the reviewer that it would be important to assess the importance of FPR1 in the recognition and control of P. aeruginosa infection in normal and diabetic mice but we believe that such comprehensive studies would require substantial amount of additional work, which as the reviewer appreciates would go beyond the scope of this manuscript.

Reviewer indicated “In mice, the sensitivity toward fMLF is generally low and other formylated peptides exert stronger chemotactic activities via FPRs”. We apologize to the reviewer if we have misinterpreted the reviewer’s comments which suggest to us that bacterial fMLF recognition by FPR may not play a meaningful physiological role during infection. However, if our interpretation of the reviewer’s comment is correct, we respectfully disagree with the reviewer. FPR knockout mice are highly vulnerable to bacterial infection, even in animal models that do not involve wounds or surgery (60, 61), suggesting that bacterial recognition (most likely bacterial formyl peptides) by FPR is likely to play an important role in combating infection.

Reviewer #2 (Recommendations for the authors):

Roy et al., present an interesting manuscript where the molecular and cellular responses are studied in a model of diabetes wound healing, or lack of, and infection. The study is well planned and logically presented.

The most interesting aspect of the presented work, to me, is the clear link between an effective and exuberant inflammatory reaction and resolution with tissue repair. The ability of CCL3 application to restore a proper neutrophil response to the infected wounds and as such ensure the appropriate management of the infection is an excellent proof that 'the beginning programs the end' [PMID: 16369558 DOI: 10.1038/ni1276]. On a similar vein, dissecting how resolution starts is equally challenging and interesting [PMID: 27199985 PMCID: PMC4845539 DOI: 10.3389/fimmu.2016.00160] and well presented here. Finally, the notion that neutrophils are not just BAD is also excellent and the study here provides a very clear evidence for the link between onset and resolution [PMID: 27021499 DOI: 10.1016/j.smim.2016.03.007]. This issue may need to be highlighted in the Discussion section as chiefly demonstrated here.

I have some comments and some unclear issues I would like the Authors to comment or address.

1. FPR1 mRNA (Figure 2)

I was surprised to see the rapid decade in FPR1 mRNA within 1 hour of high glucose. To my knowledge, FPR1 is not an early response gene (like fos and jun) so it will be appropriate for the Authors to perhaps double check this dataset, perhaps running a time course and study what else goes down within 30-60-90-120 min. Would there be mileage in monitoring FPR2 mRNA too as a relevant control?

We appreciate the concern raised by the reviewer about the quick reduction in FPR1 at transcriptional and translation level after exposure to HG for 1h. We were also surprised by these results. We have done a follow-up assessment as requested by the reviewer. We confirm our initial data and show that exposure to HG (for 60, 90, and 180 minutes) causes substantial reduction in both FPR1 and FPR2 mRNA (as assessed by RT-PCR) and protein levels (as assessed by Western blotting) (Please see Figure 2—figure supplement 2 and Figure 2—figure supplement 3). We have submitted a grant proposal to determine the mechanism underlying HG-induced repression of FPR expression. We believe that it involves changes in the metabolic state of the neutrophils, caused by exposure to HG.

2. FPR1 protein

The same question can be asked for the rapid downregulation of FPR1 protein from the cell surface of mouse and human neutrophils following incubation with glucose. The data are convincing here, but again very intriguing how this could occur. More speculation in the Discussion would be good. Also, experiments aiming to assess if the receptor internalised (thus do we find higher intracellular levels?) and if so, how, would be appropriate for a study published in eLife.

We appreciate the reviewer’s astute comments regarding the rapid decline in the surface expression of FPR receptor in neutrophils in response to exposure to HG. We do not know the answer to this but it is possible that it may be merely a reflection of ~90% reduction in the mRNA levels of FPR1, combined with normal FPR1 cellular turnovers. As the reviewer is aware, most cellular receptors are subject to internalization and cycling (62-64). It is also possible that exposure to HG additionally accelerates FPR1 surface protein internalization and/or protein degradation and/or turnovers. More detailed studies are needed to tease out these possibilities and to determine the mechanism underlying HG-induced reduction in FPR surface receptor.

3. Legends to the Figures

I find the figure legends written in an unclear manner. usually the Panel letter (e.g. A, or B or C…) is at the beginning of the sentence, whereas here is all a bit mixed up. In some cases groups of 3 mice have been used. Is this acceptable? How reproducible are the Western blots?

We apologize to the reviewer for the confusions in the figure legends. Our purpose for writing the figure legends the way we did was to reduce the length of the figure legends. For example, if 5 out of 7 panels in a figure shared the same procedure for wounding, treatment, and/or infection but differed only on data analyses (i.e., Western blot, RT-PCR, and flow cytometry, etc), we indicated panel letters (a-e) in the beginning prior to providing s brief description of the protocol, followed by specific letters to indicate the type of analyses after. As for the n (numbers used), we had performed these Western blotting experiments two to three independent times, each time in duplicates or we had used 3- 4 mice per group each time. As the reviewer appreciates, the purpose of “n” is to reach enough statistical power to reach significance and the minimum number of trials per group should be at least N=3. We are asked by our IACUC director at Rush to prevent the unnecessary use of animals in our studies where possible. In addition, these animals (particularly db/db mice) are very expensive further necessitating the prudent use of animals in our studies. Of note, these Western blot experiments are very reproducible. Please see the new data in Figure 2—figure supplement 2 and Figure 2—figure supplement 3 in which the impact of HG on FPR1 and FPR2 expression was assessed.

4. CCL3

Data in Figure 4C seem to indicate that the infiltrating neutrophils are responsible for delivering CCL3 (or MIP-1alpha). Probably some indication of the potential sources of CCL3 in the infected wounds, including the migrated neutrophils, is worthwhile. And relevant and interesting.

We appreciate the reviewer’s astute comments. We believe several factors may be contributing. We have added the following paragraph in the Discussion section to address the source of the ligands (including CCL3/MIP-1a) for auxiliary receptors in infected wounds.

“Our data demonstrate that at least the expression and signaling through CCR1 and CXCR2 auxiliary receptors are not adversely affected by high glucose, but they may not be signaling in diabetic wounds early after injury because of insufficient production of their ligands, such as CCL3. […] TLR signaling has also been implicated in the production of ligands (e.g., CCL3) for these auxiliary receptors (46, 47).”

Reviewer #3 (Recommendations for the authors):

The manuscript by Roy et al. entitled "Overriding defective FPR chemotaxis signaling in diabetic neutrophil stimulates infection control in diabetic wound", focuses on impaired healing in diabetic wounds. Moreover, the role that neutrophils play and the therapeutic potential of the pro-inflammatory cytokine CCL3 in the treatment of diabetic foot ulcers. Overall, this is an interesting manuscript, but there are several points which should be addressed:

• The n values for this study seem low, n=2 or 2 in some cases. This reviewer would argue the validity and robustness of performing statistical analysis on such low n numbers and the ultimate value of the findings.

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.

• In every figure legend and methods section, the authors need to state fully the n number to provide transparency in their data and in line with eLife policies.

We have done as requested by the reviewer.

• Methods section: Neutrophil isolation from human and mouse. "Mouse neutrophils involving comparisons between C57B normal and db/db diabetic neutrophils were extracted from N=4 blood pools/group, with each blood pool being from 4 mice: totaling 16 mice per group." This is incorrect. There are not 16 mice per group. If each group has blood pooled from 4 mice, then this is n=1, not 4, therefore n=4, not 16. This needs to be addressed.

We apologize to the reviewer for indicating N=16 in the animal experiments where 4 blood pools (each blood pool was blood from 4 mice combined together) were used. We agree with the reviewer that in reality N=4 blood pools per group (generated from 16 mice). We have made the corrections, regarding our statistical analyses both in the figure legends and in the Methods section of the revised manuscript.

• Methods section: Histological analyses and wound healing assessment. "The histological data, (obtained from n≥5 mice/group and >9 random fields/wound/mouse), were normalized per wound surface area". Although the authors state ≥5 mice/group, the figure legends suggest differently. This needs to be corrected.

We have revised the legend to clarify this issue. In these analyses, the number of mice per group was indeed N>5. However, we had analyzed at least 9 random fields per wound per mouse.

• What are the baseline cells counts (especially neutrophils) for the mice used in the study. These should be included.

We appreciate the reviewer’s comment in this regard, but we respectfully disagree with the reviewer that the baseline numbers are necessary for the following reasons. Neutrophils are rarely found in skin and are not considered skin-resident cells, but they migrate into the skin in high numbers in response to inflammatory conditions, such as injury and infection (35-39). We have also demonstrated that in the absence of infection and injury, we do not detect neutrophils in the skin of mice (37). For these reasons, we left out the baseline neutrophil numbers at T0, and only focused on day 1, 3, 6, and 10 wounds to assess the dynamics of neutrophil responses during the acute (early) and chronic (late) stages of healing in in normal and diabetic wounds. In order to produce the baseline numbers, we would need to repeat these studies as we did not maintain day 0 skin wounds from these studies. As the reviewer appreciates, diabetic animals are very expensive and in lieu of the reasons we discussed above, we do not believe that these data are necessary or add anything new to the manuscript.

• In some cases, the authors use bone marrow derived neutrophils and in other cases blood borne neutrophils, although it is unclear to this reviewer what determined the choice. As it is known that these compartments are very different, it would add value to the manuscript to show whether neutrophils derived from these two compartments behave differently and express different cell surface receptors. Furthermore, these changes/similarities should be shown over the time course of 1, 3, 6 and 10 days, as this may help to further explain what is causing the delay in neutrophil influx in diabetic wounds over time.

We appreciate and agree with the reviewer that neutrophils extracted from bone marrow, and blood are not the same. However, our decision to use neutrophils from these different sources was deliberate because we intended to address different questions using these neutrophils. The initial observation (showing impaired neutrophil chemotaxis in diabetic human) involved neutrophils that were isolated from the blood of diabetic patients (10). Therefore, we used isolated neutrophils from the blood of diabetic mice to show that the same chemotaxis impairment was also present in neutrophils isolated from the blood of diabetic mice, as a way to reproduce the human diabetic condition with respect to this impairment. On the other hand, when we wished to assess the impact of HG on chemotaxis and chemotaxis signaling in neutrophils, we isolated neutrophils from the bone marrow of normal mice and exposed them to HG to show that exposure to HG was sufficient to cause similar chemotaxis impairment in normal neutrophils. We used bone marrow neutrophils to remove any potential confounding factors that could potentially influence neutrophil’s chemotaxis behavior. As the reviewers and the editor are aware, neutrophils are produced in bone marrow and released into circulation (30, 31), thus the choice of bone marrow as the source of normal neutrophils. Of note, bone marrow leukocytes (including neutrophils) in diabetic animals do not appear to have the same functional impairments as peripheral blood leukocytes in diabetic animals or human (11, 32-34), suggesting that leukocytes in the bone marrow of diabetic mice are protected from the adverse effects of exposure to HG in the bone marrow compartment. Finally, it was also more practical to use bone marrow neutrophils because we could obtain more neutrophils from this compartment. As we had indicated in our manuscript, we had to pool the blood of 4 mice (16 mice/group) to be able to obtain enough neutrophils for a single analysis.

• Although CCL3 expression is reduced in day 1 diabetic wounds, what happens at 3, 6 and 10 days?

Although we agree with the reviewer that it would be interesting to evaluate the expression of CCL3 at later timepoints, we respectfully believe that this information is not necessary and could possibly detract from the main point of this manuscript for the following reason. The point of this manuscript was why diabetic neutrophils were not trafficking into wounds early after injury if their auxiliary receptors (i.e., CCR1 and CXCR2) remain functional under diabetic condition. This was the reason for us evaluating and showing that CCL3 expression (ligand for CCR1) was significantly diminished in diabetic wounds early after injury. CCL3 is one of many pro-inflammatory cytokines that can recruit neutrophils by engaging ~30 auxiliary receptors (40, 42, 55-59). We used CCL3 to jumpstart the neutrophil response in diabetic wound early after injury because it engages multiple auxiliary receptors on neutrophils, namely CCR1, CCR4, and CCR5 (13-15). As our data show, early after injury diabetic wound environment suffers from inadequate neutrophil response whereas diabetic wounds become progressively more inflamed and contain more neutrophils as they age (Figure 1) and (65). If we assess CCL3 and find it to be elevated in old diabetic wounds (which we think will be the case), then it would raise the question as to what causes CCL3 expression to be elevated in old wounds. On the other hand, if CCL3 expression remains low in the old wounds, it would raise another question as to what is responsible for recruiting neutrophils into old diabetic wounds. We are very much interested in determining the molecular causes responsible for transitioning diabetic wounds from anti-inflammatory (early after injury) into pro-inflammatory (old wounds), but we believe that more comprehensive studies are needed to assess diabetic wound environments during acute and chronic phases of healing and determine and validate the factors (e.g., CCL3 and other pro or anti-inflammatory cytokines) that may be regulated or regulate this transition in diabetic wounds. As the reviewer appreciates such assessments go beyond the scope of this manuscript.

• The finding that topical treatment enhances neutrophil response (figure 4) is very interesting. These data are for day 1, what happens over the time course i.e. 3, 6 and 10 days?

We have done these studies (Please see Figure 5 in the revised manuscript). Our data indicate that as compared to mock-treated diabetic wounds, CCL3-treated diabetic wounds contain significantly more neutrophils at day 1 and 3 but significantly fewer neutrophils at day 6 and day 10, indicating that diabetic wounds are not destined to develop persistent non-resolving inflammation, provided that we jumpstart the neutrophil responses in them early after injury.

• The authors show nicely the involvement of FPR1, but what about the other known FPR that neutrophils express i.e. FPR2? Does this have a compensatory role or is this receptor also dysfunctional. Data should be provided.

We appreciate the reviewers astute comments regarding FPR2. We had originally focused on FPR1 in this manuscript because FPR1 has a much higher affinity for bacterial formyl peptides than FPR2, whereas FPR2 has a broader range of ligands than FPR1 (1, 2). In addition, FPR1 has been implicated in pro-inflammatory responses whereas FPR2 has also been implicated in resolution of inflammation in response to Annexin A1, lipoxin A4, and resolving D1 inflammation pro-resolving agonists (1-5), and because this manuscript was about diabetic neutrophil’s impaired chemotactic response toward infection during the pro-inflammatory phase of acute infection and healing. With that said, we now provide new data (please see Figure 2—figure supplement 2 and Figure 2—figure supplement 3) to show that the expression of both FPR1 and FPR2 are substantially dampened in neutrophils after exposure to HG for 1, 2, and 3 hours.

• In addition, what happens to FPR1 (and 2) expression over the time course i.e. 3, 6 and 10 days? Does it change?

As the reviewer appreciates and as we discussed above, we believe that expanding neutrophil characterizations with respect to FPR and auxiliary receptors and their respective ligands beyond day 1 would go beyond the scope of this manuscript. The point of this manuscript was the novel finding that inadequate neutrophil response early after injury is rendering diabetic wound vulnerable to infection. We believe a comprehensive assessments of all 30 chemotaxis receptors (i.e., FPR1, FPR2, CCR1, CCR4, CCR5, CXCR1, CXCR2, etc.) on neutrophils and their putative ligands (CCL3, CCL5, CXCL1, CXCL2, etc) in normal and diabetic wounds would be needed to shed light on the dynamics of neutrophil responses in normal wound and impaired neutrophil responses in diabetic wounds. In fact, we have submitted a grant proposal to do these studies.

• Were the mice male or female or a mixture? Please provide rational for choice and whether sex as a biological variable was considered, especially given the fact that gender differences have been shown in recovery to diabetic wounds.

We appreciate and agree with the reviewer that sex is a critical biological variable that needs to be considered in designing any experiment. However, since several prior publications have indicated that type 2 diabetic mellitus is more common in males than females [100-103], and because of the budgetary constraints, we restricted our analysis to male db/db (type 2 obese) diabetic mice. Of note, in studies involving human neutrophils, neutrophils were isolated from both male and female volunteers, and regardless of their sex, these neutrophils behaved similarly with respect to their chemotactic responses toward fMLF and CCL3 and the adverse impact of HG on FPR expression and signaling, suggesting that sex does not affect neutrophil chemotaxis behavior or signaling at least in cell culture. Of course, we acknowledge that in vivo, the situation may be different. Therefore, a follow-up study to evaluate the impact of sex on neutrophil’s behavior and function is needed to address this important question.

• The authors chose mice at 8 weeks, with 1 week acclimatisation. Given this young adult age, what is the clinical relevance of this choice of age?

We had previously shown that db/db mice are diabetic at this age (as manifested by their high serum glucose) (54, 65). Unfortunately, the serum glucose levels decline in db/db animals and theses mice short life span because these mice develop ketosis after a few months (66-68). Therefore, we chose this age. We appreciate that type 2 diabetes is more common in middle-aged and elderly people whereas, type 1 diabetes is more common in younger people. However, the rate of type 2 diabetes in young people in USA is increasing substantially due to increasing obesity rates ((69) and according to the Center for Disease Control and Prevention), which db/db mice model at this age represents.

• Figure S3: 100nM fMLP: The data for the 90 mg/dl is around 800 RFU, however, in Figure 2f, this value looks to be much higher (around 1200 RFU). Would the authors provide the exact numbers and discuss further.

As the reviewer appreciates, many factors can cause intrinsic variations in data, including the operator and/or source of reagents. In our experience, even using the fluorescent dye from the same vial and at same concentration can sometimes lead to different intensities and RFU numbers in replicate experiments, particularly if the experiments are performed by different individuals, which was the case for Figure 2f and Figure S3. The important point is that in both experiments, we did obtain similar significant trends as neutrophils treated with normal glucose level (90 mg/dl) displayed significantly higher chemotaxis as compared to neutrophils treated with HG. As requested by the reviewer, we provide the raw data and the exact RFUs for Figure 2f (100nM fMLF) and Figure 2—figure supplement 1 (at indicated fMLF concentrations).

• Although this student specifically looks at neutrophils, do the authors have any data to suggest whether these finds are neutrophil specific or whether e.g. monocytes, play a role in the later time points?

We had focused on neutrophils because the original observation involved chemotaxis impairment in neutrophils isolated from diabetic patients (10) and because Pseudomonas aeruginosa infection control in CCL3-treated diabetic wound was primarily dependent on neutrophils as our data show (Figure 4h-j). We appreciate the reviewer’s comment and agree with the reviewer that it would be interesting to assess whether the same impairment in signaling through FPR also affects other leukocytes. We believe it does and have submitted a grant proposal to expand our findings to other leukocytes. However, as the reviewer appreciates, these studies go beyond the scope of this manuscript.

• C57/B should be written in full: C57Bl/6. 'FPR receptor' should be changed to just 'FPR'. Proof reading would help – grammar and spelling mistakes need correcting.

We thank the reviewer for his/her suggestions and have made the corrections in the revised manuscript.

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https://doi.org/10.7554/eLife.72071.sa2

Article and author information

Author details

  1. Ruchi Roy

    1. Department of Medicine, Rush University Medical Center, Chicago, United States
    2. Division of Hematology/Oncology/Cell Therapy, Rush University Medical Center, Chicago, United States
    Contribution
    Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review and editing
    Contributed equally with
    Janet Zayas
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8298-1321
  2. Janet Zayas

    1. Department of Medicine, Rush University Medical Center, Chicago, United States
    2. Division of Hematology/Oncology/Cell Therapy, Rush University Medical Center, Chicago, United States
    3. Department of Microbial Pathogens and Immunity, Rush University Medical Center, Chicago, United States
    Contribution
    Investigation, Methodology, Writing – original draft, Writing – review and editing
    Contributed equally with
    Ruchi Roy
    Competing interests
    No competing interests declared
  3. Sunil K Singh

    Department of Surgery, Division of Surgical Oncology, University of Illinois at Chicago, Chicago, United States
    Contribution
    Data curation, Formal analysis, Investigation, Validation
    Competing interests
    No competing interests declared
  4. Kaylee Delgado

    1. Department of Medicine, Rush University Medical Center, Chicago, United States
    2. Division of Hematology/Oncology/Cell Therapy, Rush University Medical Center, Chicago, United States
    Contribution
    Formal analysis, Investigation
    Competing interests
    No competing interests declared
  5. Stephen J Wood

    Department of Microbial Pathogens and Immunity, Rush University Medical Center, Chicago, United States
    Contribution
    Methodology
    Competing interests
    No competing interests declared
  6. Mohamed F Mohamed

    1. Department of Medicine, Rush University Medical Center, Chicago, United States
    2. Division of Hematology/Oncology/Cell Therapy, Rush University Medical Center, Chicago, United States
    Contribution
    Methodology
    Competing interests
    No competing interests declared
  7. Dulce M Frausto

    1. Department of Medicine, Rush University Medical Center, Chicago, United States
    2. Division of Hematology/Oncology/Cell Therapy, Rush University Medical Center, Chicago, United States
    Contribution
    Methodology
    Competing interests
    No competing interests declared
  8. Yasmeen A Albalawi

    Department of Microbial Pathogens and Immunity, Rush University Medical Center, Chicago, United States
    Contribution
    Other: Contributed to blood draw and sample preparation
    Competing interests
    No competing interests declared
  9. Thea P Price

    Department of Surgery, Division of Surgical Oncology, University of Illinois at Chicago, Chicago, United States
    Contribution
    Other: Contributed to blood draw and sample preparation
    Competing interests
    No competing interests declared
  10. Ricardo Estupinian

    1. Department of Medicine, Rush University Medical Center, Chicago, United States
    2. Division of Hematology/Oncology/Cell Therapy, Rush University Medical Center, Chicago, United States
    Contribution
    Methodology
    Competing interests
    No competing interests declared
  11. Eileena F Giurini

    1. Department of Medicine, Rush University Medical Center, Chicago, United States
    2. Division of Hematology/Oncology/Cell Therapy, Rush University Medical Center, Chicago, United States
    Contribution
    Methodology
    Competing interests
    No competing interests declared
  12. Timothy M Kuzel

    1. Department of Medicine, Rush University Medical Center, Chicago, United States
    2. Division of Hematology/Oncology/Cell Therapy, Rush University Medical Center, Chicago, United States
    Contribution
    Conceptualization, Resources
    Competing interests
    No competing interests declared
  13. Andrew Zloza

    1. Department of Medicine, Rush University Medical Center, Chicago, United States
    2. Division of Hematology/Oncology/Cell Therapy, Rush University Medical Center, Chicago, United States
    Contribution
    Methodology
    Competing interests
    No competing interests declared
  14. Jochen Reiser

    Department of Medicine, Rush University Medical Center, Chicago, United States
    Contribution
    Conceptualization, Resources
    Competing interests
    No competing interests declared
  15. Sasha H Shafikhani

    1. Department of Medicine, Rush University Medical Center, Chicago, United States
    2. Division of Hematology/Oncology/Cell Therapy, Rush University Medical Center, Chicago, United States
    3. Department of Microbial Pathogens and Immunity, Rush University Medical Center, Chicago, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing – original draft, Writing – review and editing
    For correspondence
    Sasha_Shafikhani@rush.edu
    Competing interests
    Rush University Medical Center has filed a patent (International Application Number: PCT/US19/41112). Dr. Sasha Shafikhani is the listed inventor on this application
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1755-9997

Funding

National Institutes of Health (RO1DK107713)

  • Sasha H Shafikhani

National Institutes of Health (R01AI150668)

  • Sasha H Shafikhani

National Institutes of Health (F31DK118797)

  • Janet Zayas

National Institutes of Health (GM109421)

  • Janet Zayas

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We are thankful to Dr. Lena Al-Harthi and Dr. Celeste Napier for the use their equipment. We also would like to thank Mr. Jeffrey Martinson for his help with the flow cytometry and the rest of Shafikhani lab for their valued opinions on these studies. This work was supported by the National Institutes of Health (NIH) grant RO1DK107713 to (SHS), R01AI150668 to (SHS), F31DK118797 to (JZ), and the NIH PhD institutional training grant GM109421.

Ethics

Human subjects: We have an Institutional Review Board (IRB)- approved protocol in accordance with the Common Rule (45CFR46, December 13, 2001) and any other governing regulations or subparts. This IRB-approved protocol allows us to collect blood samples from non-diabetic volunteers with their consents for these studies.

We have an approval from the Rush University Medical Center Institutional Animal Care and Use Committee (IACUC No.: 18-037) to conduct research as indicated. All procedures complied strictly with the standards for care and use of animal subjects as stated in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy of Sciences, Bethesda, MD, USA).

Senior Editor

  1. Carla V Rothlin, Yale School of Medicine, Yale University, United States

Reviewing Editor

  1. Ursula Rescher, University of Muenster, Germany

Reviewer

  1. Mauro Perretti, Queen Mary University of London, United Kingdom

Publication history

  1. Received: July 9, 2021
  2. Preprint posted: September 11, 2021 (view preprint)
  3. Accepted: February 1, 2022
  4. Accepted Manuscript published: February 3, 2022 (version 1)
  5. Version of Record published: February 15, 2022 (version 2)

Copyright

© 2022, Roy et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Ruchi Roy
  2. Janet Zayas
  3. Sunil K Singh
  4. Kaylee Delgado
  5. Stephen J Wood
  6. Mohamed F Mohamed
  7. Dulce M Frausto
  8. Yasmeen A Albalawi
  9. Thea P Price
  10. Ricardo Estupinian
  11. Eileena F Giurini
  12. Timothy M Kuzel
  13. Andrew Zloza
  14. Jochen Reiser
  15. Sasha H Shafikhani
(2022)
Overriding impaired FPR chemotaxis signaling in diabetic neutrophil stimulates infection control in murine diabetic wound
eLife 11:e72071.
https://doi.org/10.7554/eLife.72071

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