Role of leukotriene B4 (LTB4)-LTB4 receptor 1 signaling in post-incisional nociceptive sensitization and local inflammation in mice

Miho Asahara; Nobuko Ito; Yoko Hoshino; Takaharu Sasaki; Takehiko Yokomizo; Motonao Nakamura; Takao Shimizu; Yoshitsugu Yamada

doi:10.1371/journal.pone.0276135

. 2022 Oct 20;17(10):e0276135. doi: 10.1371/journal.pone.0276135

Role of leukotriene B₄ (LTB₄)-LTB₄ receptor 1 signaling in post-incisional nociceptive sensitization and local inflammation in mice

Miho Asahara ¹, Nobuko Ito ^1,^*, Yoko Hoshino ¹, Takaharu Sasaki ², Takehiko Yokomizo ², Motonao Nakamura ³, Takao Shimizu ^4,⁵, Yoshitsugu Yamada ⁶

Editor: Ichiro Manabe⁷

PMCID: PMC9584502 PMID: 36264904

Abstract

Leukotriene B₄ (LTB₄) is a potent lipid mediator involved in the recruitment and activation of neutrophils, which is an important feature of tissue injury and inflammation. The biological effects of LTB₄ are primarily mediated through the high-affinity LTB₄ receptor, BLT1. Postoperative incisional pain is characterized by persistent acute pain at the site of tissue injury and is associated with local inflammation. Here, we compared the role of LTB₄-BLT1 signaling in postoperative incisional pain between BLT1-knockout (BLT1KO) and wild-type (BLT1WT) mice. A planter incision model was developed, and mechanical pain hypersensitivity was determined using the von Frey test before and after incision. Local infiltration of neutrophils and inflammatory monocytes was quantified by flow cytometry. Inflammatory cytokine levels in the incised tissue were also determined. Mechanical pain hypersensitivity was significantly reduced in BLT1KO mice compared to BLT1WT mice at 2, 3, and 4 days after incision. LTB₄ levels in the tissue at the incision site peaked 3 hours after the incision. Infiltrated neutrophils peaked 1 day after the incision in both BLT1KO and BLT1WT mice. The accumulation of inflammatory monocytes increased 1–3 days after the incision and was significantly more reduced in BLT1KO mice than in BLT1WT mice. In BLT1KO mice, Interleukin-1β and Tumor Necrosis Factor-α levels 1 day after the incision were significantly lower than those of BLT1WT mice. Our data suggest that LTB₄ is produced and activates its receptor BLT1 in the very early phase of tissue injury, and that LTB₄-BLT1 signaling exacerbates pain responses by promoting local infiltration of inflammatory monocytes and cytokine production. Thus, LTB₄-BLT1 signaling is a potential target for therapeutic intervention of acute and persistent pain induced by tissue injury.

Introduction

Leukotriene B₄ (LTB₄), a metabolite of arachidonic acid, is a potent proinflammatory lipid mediator that activates and recruits leukocytes to inflamed regions [1]. The potent biological effects of LTB₄ are mediated primarily through LTB₄ receptor 1 (BLT1), which is a high-affinity G protein-coupled receptor for LTB₄ [1, 2]. BLT1 is expressed in several types of leukocytes, which include neutrophils, macrophages and their precursors [2], monocytes [3], differentiated T cells [4, 5] and dendritic cells [6]. We and others have established BLT1-knockout (BLT1KO) mice [7, 8], and previous studies using BLT1KO mice showed that LTB₄-BLT1 signaling is involved in a variety of inflammatory and immune responses, including allergic airway inflammation [4, 7, 9, 10], multiple sclerosis [11], atherosclerosis [3, 12, 13], inflammatory arthritis [14, 15], tumor promotion [16], and psoriasis [17]. Specifically, regarding the inflammatory model, numerous studies have shown that the blockade of LTB₄-BLT1 signaling significantly inhibits neutrophil recruitment and inflammatory conditions [17–19].

In the 1980s, Levine et al. reported that intradermal injection of LTB₄ evokes thermal and mechanical allodynia [20]. Subsequently, several studies demonstrated the involvement of LTB₄-BLT1 signaling in the progression of inflammatory pain. The expression of BLT1 in murine dorsal root ganglion and the spinal cord has been confirmed [21–23]; moreover, BLT1 mRNA levels were increased in rat spinal cord neurons in a spared nerve injury model of neuropathic pain [22]. Our previous study showed that BLT1KO mice exhibited reduced local inflammation and marked attenuation of acute nociception induced by intraplantar formalin injection, which suggested that LTB₄-BLT1 signaling is involved in the mechanism of inflammatory pain [19].

Tissue injury causes the release of various chemical mediators from injured tissues and immune cells, which initiates the inflammatory response and sensitizes peripheral nociceptors [24–28]. In the early phase of inflammation, neutrophils are the most dominant immune cells, which migrate through the vascular endothelium, accumulate in the injured tissue, and stimulate the release of various nociceptive mediators, such as cytokines, chemokines, and other substances [29, 30]. This initial response is followed by the recruitment of monocytes to inflammatory sites and the activation and proliferation of tissue-resident macrophages, which occur several days after tissue injury [31]. Furthermore, inflammatory monocytes and macrophages release nociceptive mediators and expand the neutrophil response. Thus, tissue injury activates various types of immune cells in chronological order, and nociceptive mediators released from these immune cells support and prolong pain responses, as shown in rodent incisional pain models [32–38].

Therefore, in this study, we focused on the pain associated with tissue injury and investigated the role of LTB₄-BLT1 signaling. As a popular tissue injury pain model, we used the hind paw incisional pain model, which is defined as a preclinical animal model of postoperative pain. We also determined the immune cells, and the cytokine production processes that were affected by BLT1 deficiency at the site of tissue injury during the course of incisional pain.

Materials and methods

Animals

BLT1KO mice were generated and bred on a C57BL/6 background as described previously [39]. All mice were maintained on a 12-hour light/dark cycle with ad libitum access to water and food. This study was carried out in accordance with the University of Tokyo’s guidelines for the care and use of laboratory animals. The protocol was approved by the Animal Experimental Committee of the University of Tokyo (P17-025). All surgery was performed under anesthesia, and all efforts were made to minimize suffering.

Plantar incision

The hind paw plantar incision mouse model was created as previously described with minor modifications [28]. Briefly, mice were anesthetized by inhalation of isoflurane via a nose cone. After sterile preparation, a 5-mm longitudinal incision was made through the skin and fascia of the plantar aspect of the right hind paw with a No. 11 scalpel. The underlying plantaris muscle was elevated and incised longitudinally, allowing the muscle origin and insertion to remain intact. After hemostasis with gentle pressure, the skin was opposed with two sutures using 8–0 nylon threads, and the wound was covered with antibiotic ointment. The mice were then allowed to recover in their home cages.

Behavioral testing

Mice were acclimated to testing cages containing a stainless steel mesh for 1hr per day at least 2 days before testing. Mechanical pain hypersensitivity was estimated using von Frey filaments according to the up-down method, as described previously [40]. Briefly, mice were placed individually on wire mesh platforms in clear cylindrical plastic enclosures and allowed to acclimate to the environment for 30 minutes prior to the von Frey test. Von Frey filaments were applied to the middle of the plantar surface of the hind paw. A series of six von Frey filaments (0.7-, 1.6-, 4-, 6-, 20-, and 40-mN forces) were used. Testing was initiated with 20 mN forces. Whenever a positive response occurred, the next weaker von Frey filament was applied. Whenever a negative response occurred, next stronger one was applied. The test was continued, until the response of six stimuli after the first change in response had been obtained or the test reached either end of the spectrum of the von Frey set. Assessments were performed before surgery (baseline), 2 hours after surgery, and on postoperative days 1, 2, 3, 4, 5, and 7.

Withdrawal responses following stimulation were measured, and a tactile stimulus that produced a 50% likelihood of withdrawal was determined. Mechanical Threshold = (10^[Xf+kδ])/10,000, where Xf = value (in log units) of the final von Frey filament used; k = tabular values for the pattern of positive/negative responses; and δ = mean difference (in log units) between stimuli (here, 0.38).

Spontaneous pain related behaviors following LTB₄ administration were assessed using guarding pain score (GPS) of the hind paws according to a previously described method [41]. Immediately after injection of LTB₄ or vehicle, mice were placed individually on a stainless-steel mesh floor (openings, 8 X 8 mm) under a clear plastic cage. GPS was calculated on the basis of weight bearing. Both paws of each animal were closely observed during a 1-min period repeated every 5 min for 30 min. Depending on the position in which the paw was found during the scoring period, a score of 0, 1 or 2 was given. 0: both foot full touch 1: partial 2: complete off. The sum of six scores (0 to 12) was used to assess the spontaneous pain following LTB₄ injection to the hind paw. The person performing behavioral tests was blinded to the treatment and genotypes of mice.

LTB₄ administration in the hind paw

LTB₄ (20110, Cayman Chemical, MI, USA) injection for assessments of mechanical responses were performed under light isoflurane anesthesia using 4% (1 min) for induction and 1.5% (1 min) for maintenance during injection. LTB₄ at dose of 10ng, 1ng in 5μl PBS or vehicle was injected into plantar surface of the hind paw subcutaneously using a Hamilton syringe attached to a 30-G needle in a total volume of 5 μl. For spontaneous pain behaviors, injections were performed using gentle restraint without anesthesia. The needle tip was inserted between digits and directed proximally, where drug or vehicle was injected into the middle of the hind paw. To minimize needle trauma, needle tip did not invade the area that mechanical stimuli were applied. The person performing the behavioral experiments was blinded to the drug and dose.

Quantification of LTB₄ concentration in the paw

Plantar tissue samples, including skin and underlying muscle were collected at different time points after plantar incision under deep anesthesia with pentobarbital. Each sample was diluted with a 200-μL solution of methanol: formic acid (100:0.2) by centrifugation (at 100 g for 1 min). The column was washed with 0.1% formic acid solution, 15% methanol containing 0.1% formic acid, and petroleum ether containing 0.1% formic acid. All samples were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described previously [42].

Flow cytometric analysis

Evaluation of leukocyte infiltration was performed as previously described with minor modifications [43, 44]. Plantar tissue samples, including skin and underlying muscle were first dissected, as described in the previous section. The samples were then minced and digested in a mixture of 1 mg/mL collagenase A (Roche, Mannheim, Germany) plus 2.4 U/ml Dispase II (Roche, Mannheim, Germany) in Hanks’ Balanced Salt solution (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C for 2 hours. Following digestion, the cell suspension was washed in Flow Cytometry staining (FACS) buffer (eBioscience, San Diego, CA, USA), filtered through a 70-μm mesh, and resuspended in FACS buffer. The filtrate was suspended in FACS buffer to produce a single-cell suspension and blocked with Fc Receptor blocking reagent (1:10, Miltenyi Biotec, Tokyo, Japan) on ice for 10 min. For surface receptor labeling, cells were incubated with mixtures of the following antibodies in the dark for 30 min on ice: anti-CD11b-PE (1:800, monoclonal, rat, 130-091-240, Miltenyi Biotec, Tokyo, Japan), anti-Ly6G-APC (1:200, monoclonal, rat, 127614, Biolegend, San Diego, CA,USA), and anti-Ly6C-FITC (1:200, monoclonal, rat, 128006, Biolegend, San Diego, CA, USA). Dead cells were gated out according to propidium iodide (Biolegend, San Diego, CA, USA) staining, and isotype controls were used to titrate each antibody to minimize background staining. Flow cytometry was conducted on an Accuri™ C6 flowcytometer (Becton Dickinson and Company, NJ, USA). Compensation was performed at the beginning of each experiment. For all samples, 50,000 cells were analyzed to generate scatter plots. Cell suspensions were gated on forward-scatter (FSC-A) and side-scatter areas (SSC-A) to exclude debris. Cells that were double-positive for CD11b and Ly6G were recognized as neutrophils. Non-neutrophil myeloid cells (CD11b⁺Ly6G^-) were then gated according to Ly6C expression as Ly6C^high monocytes (inflammatory monocytes) or Ly6C^low monocytes (resident monocytes). All data were analyzed using FlowJo software (Becton Dickinson and Company, NJ, USA).

Cytokine analysis

Tissue cytokine levels were measured using a method similar to that described previously [32]. Briefly, plantar tissue samples, including skin and underlying muscle were dissected as described in the previous section and placed immediately in ice-cold 0.9% sodium chloride that contained a cocktail of protease inhibitors (Complete; Roche, Mannheim, Germany). Samples were then homogenized and centrifuged at 12,000 g at 4°C. The supernatant fractions were stored at −80°C until use.

For cytokine analysis, BD^TM Cytometric Bead Array (CBA) Mouse Cytokine Kits (BD Biosciences, San Jose, CA, USA) were used for the simultaneous detection of mouse interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in a single sample. In accordance with manufacturer recommendations, three capture beads with distinct fluorescence intensities, coated with cytokine-specific capture antibodies, were mixed in equal volumes: 50 μL of each sample and 50 μl of Phycoerythrin-conjugated detection antibodies were added to 50 μL of mixed-bead populations. These capture beads were incubated with recombinant standards or tissue samples for 1 hour at room temperature in the dark to form sandwich complexes.

The samples were washed with wash buffer, and the bead pellet was resuspended in 300 μL wash buffer after discarding the supernatant. Samples were measured on a Flow Cytometer (Accuri™ C6) and analyzed using FCAP Array^TM Software. Individual cytokine concentrations were indicated by their fluorescent intensities. Cytokine standards were diluted to facilitate the construction of calibration curves, which were required to determine protein concentrations of the test samples.

Statistical analysis

Statistical analysis was performed using Prism 6.0 (Graph Pad Software, La Jolla, CA, USA) and R statistical software version 4.2.1 (R Project for Statistical Computing). Two-way analyses of variance (ANOVA) and Bonferroni post hoc tests were used to analyze the behavioral tests, infiltrated neutrophils immune cells, and local cytokines. Analysis of local LTB₄ concentration and Guarding pain score were performed using a one-way ANOVA and Bonferroni post hoc tests. The criterion for statistical significance was p < 0.05. Assumptions of normal distribution and homogeneity of variance were confirmed by Shapiro–Wilk and Levene tests, respectively. All data are presented as means ± standard errors of the mean (SEM) unless otherwise noted.

Results

Mechanical pain hypersensitivity after intraplantar incision

To investigate the role of LTB₄ in tissue injury-induced acute pain, we studied behavioral pain responses to a plantar incision in BLT1KO mice and wild-type littermates (BLT1WT). Withdrawal thresholds of the ipsilateral paw decreased in both BLT1KO and BLT1WT mice, and those of the ipsilateral paw were lowest at 2 hours, which was maintained for 7 days after the incision. BLT1KO mice exhibited reduced mechanical pain hypersensitivity as compared with BLT1WT mice in the ipsilateral paw on post-incisional days 2 (p < 0.0001), and day3, 4 (p < 0.05). The 50% paw withdrawal thresholds of the contralateral paw were not affected by the incision (Fig 1).

Fig 1 — The 50% withdrawal thresholds in the ipsilateral and contralateral paws were measured at 2 h and 1, 2, 3, 4, 5, and 7 days after the plantar incision. The withdrawal thresholds of the ipsilateral paw decreased in both BLT1KO and BLT1WT mice. Data are shown as means ± SEM (n = 15–16 per group). A two-way ANOVA with Bonferroni post hoc tests was used for statistical analysis. *p < 0.05, ****p < 0.0001 BLT1KO ipsilateral vs. BLT1WT ipsilateral. The withdrawal thresholds of the contralateral paw were not affected by the plantar incision.

Quantification of LTB₄ concentration in the plantar tissue

We next quantified the level of LTB₄ in the plantar tissue samples collected at different time points after the incision using LC-MS/MS. LTB₄ concentration peaked at 3hr and decreased, but moderate amounts of LTB₄ were also detected on days 1 and day 3 after incision. LTB₄ concentration increased significantly from baseline 3 hours and 1 day after the incision (p < 0.001, p < 0.01; Fig 2).

Fig 2 — Quantitative analysis of LTB₄ concentration at the incisional site in BLT1WT mice was performed using LC-MS/MS. Data are expressed as pg of LTB₄ per mg of tissue and shown as means ± SEM (n = 6–7 per group). LTB₄ concentration increased significantly from baseline 3 hours and 1day after the incision. A one-way ANOVA with Bonferroni post hoc tests was used for statistical analysis. ***p < 0.001, **p < 0.01 vs. pre-incision.

Leukocyte infiltration in the peri-incisional tissue of the footpad

Because LTB₄ is a strong proinflammatory chemoattractant and is related to inflammatory conditions [1], we hypothesized that the infiltration of leukocytes in the peri-incisional site would be reduced in BLT1KO mice. To determine which profile of leukocyte populations in the peri-incision sites were affected by LTB₄-BLT1 signaling, we measured neutrophils (CD11b⁺Ly6G⁺) and non-neutrophil myeloid cells (CD11b⁺Ly6G^-) using flow cytometry. Non-neutrophil myeloid cells were categorized according to differences in the expression of Ly6C as Ly6C^high monocytes (inflammatory monocytes) or non-classic Ly6C^low monocytes [3, 45–48].

Infiltrated neutrophils peaked 1day after the incision, and there was no significant difference between BLT1WT and BLT1KO mice (Fig 3C). Infiltrated Ly6C^high monocytes increased 1–3 days after the incision (Fig 3D). The accumulation of Ly6C^high monocytes was significantly more attenuated in BLT1KO mice than in BLT1WT mice on post-incisional days 1 (p<0.05) and day3 (p<0.01). Ly6C^low monocytes increased from day 3 and remained high until day 7 after the incision, but their levels did not differ significantly between BLT1WT and BLT1KO mice (Fig 3E).

Cytokine analysis

Because cytokines produced in incised tissue are one of the primary mediators of the acute inflammatory response [32], we focused on proinflammatory cytokines in the incised tissue and compared their levels between BLT1KO and BLT1WT mice.

In BLT1WT mice, IL-1β and TNF-α levels increased from 3 hours after the incision and peaked 1 day after the incision. In BLT1KO mice, IL-1β and TNF-α levels were significantly lower than those in BLT1WT mice at day1 (p<0.0001) (Fig 4A and 4B). In contrast, IL-6 peaked 3 hours after the incision, and there was no significant difference between BLT1WT and BLT1KO mice (Fig 4C).

Fig 4 — Cytokine levels in the planter tissue at pre-incision, 3 hours, and 1 and 7 days after the paw incision are shown. A, IL-1β. B, TNF-α. C, IL-6. In BLT1KO mice, IL-1β and TNF-α levels 1 day after the incision were significantly lower than those in BLT1WT mice. No significant difference in IL-6 levels was found between the groups. Data are shown as means ± SEM (n = 5–7 per group). A two-way ANOVA with Bonferroni post hoc tests was used for statistical analysis. ****p < 0.0001 vs. BLT1WT mice.

Painful reactions following LTB₄ administration

To confirm the direct effect of LTB₄ increase to the pain responses, the experiments of LTB₄ injection and behavior tests were performed. Ten ng of LTB₄ injection into the planter surface of hind paw induced significant increase of guarding pain score compared with the injection of vehicle or injection of 1ng LTB₄. (p < 0.01, p < 0.01; Fig 5A). The 50% paw withdrawal threshold were significantly decreased at 20 min and 40 min following LTB₄ injection compared with vehicle injection (p < 0.001, p < 0.01; Fig 5B) and contralateral injection (p < 0.001; Fig 5B). LTB₄ might directly affect peripheral nociceptor and induce pain-related reaction transiently. Cytokine analysis in the paw at 3hr, 1day and 3day after LTB₄ injection were also performed. IL-1β, TNF-α and IL-6 elevations were small and not significantly different from the vehicle injection group (data not shown).

Fig 5 — A, Effect of injection of LTB₄ on Guarding behavior. Guarding pain behavior measured during a 30 min period after injection. The results are presented as mean ± SEM (n = 6–7 per group) One-way ANOVA, with Bonferroni post hoc tests was used for statistical analysis. *p < 0.01 vs. vehicle-injection and # p < 0.01 vs. 1ng LTB₄ injection. B, Effect of injection of LTB₄ on mechanical pain thresholds. The results are presented as mean ± SEM (n = 7 per group). A two-way ANOVA with Bonferroni post hoc tests was used for statistical analysis. ***p < 0.001, **p < 0.01 vs. vehicle-incision and ### p < 0.001 vs. contralateral injection.

Discussion

In the present study, we showed that BLT1KO mice developed attenuated pain behavior in the incisional pain model. We also observed a significant increase in endogenous LTB₄ in the tissue at the incision site during a relatively early phase following the plantar incision. Compared with wild-type mice, BLT1KO mice showed greater reductions in infiltrated inflammatory monocytes and inflammatory cytokines at the incision site, which suggested that LTB₄-BLT1 signaling in monocytes is a key element of persistent pain responses following tissue injury.

Flow cytometrical analysis of local infiltrating leukocytes in the peri-incisional area revealed a marked increase in neutrophils and inflammatory monocytes during the early phase and an increase in resident monocytes/macrophages during the late phase. Although neutrophils were the earliest to show an increase, previous studies have shown that they are not primarily involved in hyperalgesia in the incision model [44, 49]. Several drugs and antibodies that inhibit the infiltration of neutrophils have been shown to attenuate mechanical responses in an incisional pain model; however, they also have an inhibitory effect on monocytes/macrophages [50]. Ghasemlou et al. reported that the genetic depletion of CD11b⁺Ly6G⁻ myeloid cells (primarily monocytes and macrophages) significantly attenuate the pain response in the incisional pain model and revealed that these non-neutrophilic CD11b-positive myeloid cells (monocytes and macrophages) significantly contribute to the pain response at the incisional site [44]. In the present study, inflammatory monocytes were significantly more reduced, in parallel with the attenuation of the pain response, in BLT1KO mice than in BLT1WT mice, which supports the previous report that the infiltration of monocytes is important in the maintenance of incisional pain. BLT1 is expressed in various types of inflammatory cells, and the expression of BLT1 has been confirmed in circulating C-C Motif Chemokine Receptor 2(CCR2)-high inflammatory monocytes and CCR2-low resident monocytes in peripheral blood [51]. Therefore, we suggest that LTB₄-BLT1 signaling contributed to the recruitment of inflammatory monocytes following the incision and the resulting pain response.

In our previous report, BLT1KO mice showed significantly reduced neutrophil-derived inflammatory responses, such as edema formation and Myeloperoxidase (MPO) activity, in the footpad induced by intraplantar formalin injection. These results were observed within 1 hour of the formalin injection and revealed that LTB₄-BLT1 signaling is responsible for neutrophil-derived inflammatory responses during the early phase. Given that the incisional pain model is considerably more invasive and persistent than the formalin-induced pain model, we speculate that not only the initial neutrophil infiltration, but also inflammatory monocytes infiltration induced by LTB₄-BLT1 signaling are involved in the pain enhancement observed in the incisional pain model. In contrast, Ghasemlou et al. also reported that inflammatory (CCR²⁺Ly6C^high) monocytes were not responsible for mechanical pain hypersensitivity during incisional pain. In this study, the increase in the Ly6C^high cell subset lasted longer than they reported, suggesting that intensity of inflammation may stronger than their model and the effect of Ly6C^high cell subset appeared more potently. Emerging studies have shown that inflammatory monocytes (Ly6C^high subset) play important roles in a various disease based on tissue damage, including liver injury [52], myocardial infarction [53], and skin injury [54]. In this study, reduction of inflammatory monocytes (Ly6C^high subset) is almost parallel to the inhibition of pain-related responses in BLT1KO mice. A BLT1 antagonist may be effective as an anti-inflammatory analgesic secondary to NSAIDs, with fewer side effects during the early phase of postoperative pain. Additionally, treatments targeting LTB₄-BLT1 signaling in inflammatory monocytes (the Ly6C^high subset) may be useful for decreasing inflammation to inhibit pain.

Infiltrated neutrophils and monocytes/macrophages are both responsible for the release of inflammatory mediators. Inflammatory mediators can directly activate and sensitize peripheral nociceptors. In particular, IL-1β has been shown to be strongly associated with the modulation of acute pain during the early phase following tissue injury [32, 34, 55]; moreover, TNF-α is known to induce peripheral nociceptor sensitization [56]. We confirmed that, in the injured site 24 h after the incision, the increase in IL-1β and TNF-α was more reduced in BLT1KO mice than in BLT1WT mice. Cell subsets infiltrating the paw suggested that cytokines elevated at day1 are primarily produced from inflammatory monocytes. Swirski et al. presented that inflammatory monocyte (Ly6C^high subset) are more enriched in MPO activity than nonclassical monocytes (Ly6C^low subset) and this MPO-rich inflammatory monocytes (Ly6C^high subset) promote inflammation and tissue destruction through the release of proteases and inflammatory cytokines such as TNF-α in various inflammatory settings including infection and injury [57]. Since the cytokine elevation was transient, it remains to be elucidated how these cytokines up-regulation might be involved in the persistence of postoperative pain.

LTB₄ injection induced a transient spontaneous pain response and lowered the mechanical threshold until 40 minutes after injection. These results were consistent with the previous report of rat [20]. It is speculated that peripherally increased LTB₄ induce pain-related responses via Transient Receptor Potential Vanilloid 1(TRPV1) expressed in the afferent sensory nerves of the skin. Several reports have shown that LTB₄ sensitizes primary afferent nociceptors directly without the action of its specific receptor BLT1 [58, 59]. In our model, LTB₄ production at the injured site was transient, and it was difficult to determine whether the direct sensitization of the peripheral nerve by LTB₄ was persistent and induced prolonged incisional pain. Moreover, LTB₄ and the lipid product of lipoxygenase are considered one candidate for the endogenous agonist of the Transient Receptor Potential Vanilloid 1(TRPV1) receptor at the peripheral terminal [60–62]. However, an extremely high concentration of LTB₄ (EC50 = 11.7 μM) is required to sensitize TRPV1 [60]. By contrast, Zinn et al. reported that relatively low concentrations of LTB₄ (100–200 nM) enhance TRPV1-mediating calcium increases in a BLT1-dependent manner by using the primary culture of the dorsal root ganglion, which suggests that BLT1 is involved in TRPV1 activation [58]. However, it was also difficult to determine whether the transiently increased local LTB₄ induced persistent TRPV1 sensitization and maintained incisional pain. Another group reported that high concentration of LTB4 (10 μM) induced calcium increase in primary culture of the dorsal root ganglion [21]. It is unclear how peripherally produced LTB₄ acts its receptor BLT1 expressed in the dorsal root ganglion and mediate pain sensitization.

There is also a possibility that LTB₄-BLT1 signaling in the spinal cord was involved in the pain response after the incision. Spinal glial cells, such as astrocytes and microglia, have been reported to contribute to mechanical pain hypersensitivity in the incisional model [63] and are strong candidates for the source of LTB₄. LTB₄ released from glial cells binds to its high-affinity receptor BLT1 in the neuron to accelerate neuronal activity in the spinal cord. Further analysis is required to clarify whether glial cells release LTB₄ and whether LTB₄-BLT1 signaling in the central terminal is involved in the mechanism underlying incisional pain.

Conclusions

In a post-incisional pain model, we found that the blockade of LTB₄-BLT1 signaling following paw incision attenuated mechanical pain hypersensitivity, reduced local accumulation of inflammatory monocytes, and suppressed the local increase in IL-1β and TNF-α. These results suggest that the LTB₄-BLT1 axis is involved in peripheral sensitization by promoting the recruitment of inflammatory monocytes in the inflamed site and is a promising novel therapeutic target for post-operative pain.

Acknowledgments

We would like to thank Ms. Tokie Totsu (Department of Anesthesiology, Faculty of Medicine, The University of Tokyo) and Ms. Mai Ohba (Department of Biochemistry, Juntendo University School of Medicine) for experimental support.

Data Availability

All relevant data are within the paper.

Funding Statement

This study was supported by Grants in Aid for Scientific Research, Japan Society for Promotion of Science (JSPS) in the form of grants awarded to NI (20K09190) and TY (18H02627 and 21H04798). This study was also supported by AMED in the form of a grant awarded to TY (22wm0425008s0201). The funders had no role in this study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0276135.r001

Decision Letter 0

Thiago Mattar Cunha

1 May 2022

PONE-D-22-08079Role of leukotriene B4 (LTB4)-LTB4 receptor 1 signaling in post-incisional nociceptive sensitization and local inflammationPLOS ONE

Dear Dr. Ito,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please take into consideration the comments made by Reviewer 1 and 2 regarding the discrepancies between your data and data in the literature. The mechanisms by which BLT1 is mediating Incisional pain need to be further addressed specially the role of different pain pathway compartments.

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Reviewer #1: The manuscript by Asahara investigates the contribution of BLT1 in the development of mechanical pain hypersensitivity produced by incision and the related inflammation. The authors show that Blt1 knock-out animals express reduced pain hypersensitivity after incision compared to wild-type animals. They also report that incision produces, in a time-dependent manner, an increase in LTB4 in the plantar tissue. The genetic deletion of Blt1 reduces the infiltration of monocytes and the production of IL-1beta and TNFalpha in the plantar tissue.

Overall, the topic addressing the mechanisms responsible for surgical pain is interesting. The results are clearly presented and the manuscript easy to read. An important issue here is the mechanisms of action of BLT1 that is not clearly investigated making the reading of the manuscript quite confusing. Indeed, the authors suggest a local mechanisms involving BLT1 expressed in immune cells promoting the recruitment of monocytes. However, BLT-1 is also express in sensory neurons of the DRG and the spinal cord, as reported by the authors. Hence, the experiments and the data presented in the manuscript do not allow to conclude on how BLT1 participates in the development of incision-induced pain hypersensitivity. Extensive revision is needed to address such an important issue. For instance, additional experiments evaluating the effects of intraplantar LTB4 injection on pain sensitivity, monocytes infiltration and cytokines production could be performed.

The additional comments should be considered by the authors.

- Additional experiments with the use of a non-reflexive test should be conducted. The sole description of pain-related behaviors via the use of reflexive test is insufficient to conclude on alteration of pain behaviors. For instance, pain score, place preference paradigm, catwalk etc…are methods used to assess pain-related behaviors without the use of a reflexive response.

- The effects of Blt1 deletion seems moderate and transient on the mechanical pain hypersensitivity. One may question on the importance of Blt1 in incision-induced pain-related behaviors, especially from a clinical point of view. The authors should comment.

- To exclude potential compensatory mechanisms in Blt1 Knock-out mice, pharmacological experiments targetting BLT1 should be considered by the authors. For instance, the effects of intraplantar injection of the BLT1 antagonist ONO-4057 could be evaluated in the incisional pain model.

- Have the experiments been performed in a blinded manner ? This should be indicated in the manuscript. If not, the authors should justify.

- It is admitted that the use of the up and down method for the the von frey test allows to evaluate not hyperalgesia but allodynia. Actually, for the description of the results the terms "Mechanical hyperalgesia" should be changed by "mechanical pain hypersensitivity" as the reflexive test does not allow to truly assess hyperalgesia/allodynia in animals.

- Plantar tissue has been collected to perform biochemical and cellular analysis. However, it is unclear what represents this tissue. Does this include muscle and skin or just the plantar muscle? The authors should comment.

Reviewer #2: Miho Asahara and colleagues evaluated LTB4/BLT1 in post-incisional pain.

Major points

The role of BLT1 was evident at days 2, 3 and 4 post-incision (Figure 1). Why LTB4 production was quantitated at 3h and days 1 and 7? Using the same tool, the authors can ascertain the LTB4 production at time points that were observed as important since BLT1 deficiency caused a reduction of mechanical allodynia at days 2, 3 and 4 post-incision (Figure 1). It is possible that the role of LTB4/BLT1 has consequences that goes beyond the time point of LTB4 production. However, the lack of data at those days do not allow reaching a conclusion. Overall, there seems to be an arbitrary selection of time points of analyzes that prevent putting all data in line to evaluate them. Figure 1 checked many time points, figures 2 and 4 verified 3h, 1 and 7 days, and figure 3 verified 3h, 1, 3 and 7 days.

Why LTB4 production was evident at 3h and then significant alteration of pain was observed only at 2, 3 and 4 days later? Note that this is a difference of many days. It would be interesting to check if injection of LTB4 produces a similar profile.

Quite interestingly, inflammatory monocytes were increased at days 1 and 3, and their recruitment was inhibited by BLT1 deficiency. These results suggest a parallel between pain and inflammatory monocyte recruitment. However, the data do not demonstrate whether at all time points with difference in phenotype there is also difference in LTB4 production. This is important to be done and further supports the question above.

Furthermore, which is the cellular target of LTB4? Do Ly6Chigh inflammatory monocytes express BLT1? Is it a direct effect (or lack of effect in the case of BLT1 deficient mouse) of LTB4 in BLT1 expressed by Ly6Chigh inflammatory monocytes? Are Ly6Chigh inflammatory monocytes producing the cytokines?

The discrepancy between the present finding and the previous finding (DOI: 10.1073/pnas.1501372112) should be better explored with experiments. These papers are suggesting different cellular populations are involved in incisional pain. What are Ly6Chigh inflammatory monocytes doing in incisional pain and how LTB4 can affect their function? What is the explanation for the difference at day 1? At this time point there is no significant analgesia by BLT1 deficiency, but there is reduction of Ly6Chigh inflammatory monocytes and IL-1b and TNFa production. However, Ly6Chigh inflammatory monocytes are high at day 3 and we do not know the cytokine profile.

The current data do not allow to conclude if the phenotype of BLT1 deficiency is a result of direct function of LTB4 in neurons or non-neuronal cells or if it depends on indirect effect of tissue resident cells, which produce other molecules that are the actual actors to produce pain and inflammatory cell recruitment or both. LTB4 can exert neuronal and non-neuronal effects that will lead to pain. The neuronal mechanism (DOI: 10.1016/j.molbrainres.2005.02.029) was clearly not investigated in the present study and the authors used a model of pain.

What is the level of LTB4 in the spinal cord? These authors have shown the increase of pCREB in the spinal cord in the formalin test is BLT1 dependent. What happens in the incisional pain model?

In a prior study, the authors used the MPO assay (doi: 10.1186/s12990-015-0010-9). To give a more comparative view of the results and align them with the literature, it is important to have MPO data and show which cells are producing MPO in the present study. Do these cells express the BLT1 receptors? Do inflammatory monocytes (Ly6Chigh) produce MPO in the present experimental condition? Please, see (doi: 10.1172/JCI42304).

Figure 2. The number of samples ranged from 4 to 7. It is a huge variation in the number of samples. Why?

A huge variation also occurred in Figure 4 with n varying from 5 to 9. Why?

There is no mention on repetition of experiments.

There is no mention about data normality and homogeneity.

Figures 1 and 2 can be combined.

Reviewer #3: In this study, the authors have shown that plantar incision-induced mechanical hypersensitivity, accumulation of inflammatory monocytes and the production of IL-1β and TNF-α in the incised area were significantly reduced in BLT1-knockout mice. Although the present results can be partly expected from previous findings, this study provides further insights into the roles of LTB4-BLT1 signaling in postoperative incisional pain. However, there are some points that should be addressed by the authors, as described below.

1) To indicate that this study is basic research using experimental animals, the animal species should be included in the title.

2) The basal 50% paw withdrawal threshold in C57BL/6 mice is usually less than 1 g. Have you made any mistakes in the calculations in up-down method of von Frey filament test? Provide the detailed methods in the test and the calculated method.

3) Significant reduction of mechanical hypersensitivity in BLT1KO mice was still not observed at 2h and 1d, but peaked between 2d and 4d after the plantar incision. By contrast, the accumulation of inflammatory monocytes and the production of IL-1β and TNF-α were already reduced at 1 day after the plantar incision. How do the authors explain this time gap? The authors should discuss this point in Discussion section.

**********

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PLoS One. 2022 Oct 20;17(10):e0276135. doi: 10.1371/journal.pone.0276135.r002

Author response to Decision Letter 0

14 Jul 2022

We appreciate all reviewers for reviewing our manuscript and giving useful comments to improve our manuscript.

We evaluated the effects of LTB4 injection on pain sensitivity (Fig. 5), and we observed transient pain responses. We speculated that peripherally-increased LTB4 induces pain-related responses via both the BLT1 pathway, expressed in inflammatory monocytes, and the TRPV1 pathway, expressed in the afferent sensory nerves of the skin. However, it was unclear whether LTB4 directly acted on TRPV1 because LTB4 disappeared rapidly. We believe that the direct effect of LTB4 on the peripheral nerve endings in the skin will not be long-lasting. Based on our results, we propose that LTB4-induced inflammation, mainly the recruitment of inflammatory monocytes, is more strongly involved than TRPV1 activation of peripheral nerves by LTB4 in the development of incision-induced hypersensitivity.

The additional comments should be considered by the authors.

Unfortunately, because of the shortage of mice, we were unable to perform the non-reflexive test to compare the behaviors of BLT1KO and BLT1WT mice. As suggested by the reviewers, we have evaluated the effects of intraplantar LTB4 injection on pain sensitivity using the guarding pain score, which enabled us to assess spontaneous pain-related behaviors. (Fig. 5)

Although NSAIDs are standard analgesics administered for postoperative pain, their use can present challenges because of side effects, such as aspirin-induced asthma and gastrointestinal damage. A BLT1 antagonist may be effective as an anti-inflammatory analgesic secondary to NSAIDs, with fewer side effects during the early phase of postoperative pain. Additionally, treatments targeting LTB4-BLT1 signaling in inflammatory monocytes (the Ly6Chigh subset) may be useful for decreasing inflammation to inhibit pain. (L346-353)

We could not obtain a sufficient amount of the BLT1 antagonist to perform the experiments because ONO-4057 is not commercially available. However, we hypothesize that intraplantar administration of a BLT1 antagonist would attenuate incisional pain because painful behavior was observed after intraplantar injection of LTB4. Moreover, local BLT1 antagonist administration may have an inhibitory effect on inflammatory cell infiltration and cytokine increase.

- Have the experiments been performed in a blinded manner ? This should be indicated in the manuscript. If not, the authors should justify.

The experiments were performed in a blinded manner. We have described this in the text. (L130 and142)

We changed the term “Mechanical hyperalgesia” by “Mechanical pain hypersensitivity”.

Description of “Plantar tissue” was added to the method section, as “Plantar tissue, including skin and underlying muscle.”

Reviewer #2: Miho Asahara and colleagues evaluated LTB4/BLT1 in post-incisional pain.

Major points

Additional experiments on the quantification of LTB4 concentration revealed that LTB4 was detected on days 1 and 3. As suggested by several reviewers, we have evaluated the effects of LTB4 injection on pain sensitivity, and we observed transient pain responses. We speculate that peripherally-increased LTB4 induces pain-related responses via both the BLT1 pathway, expressed in inflammatory monocytes, and/or the TRPV1 pathway, expressed in the afferent sensory nerves of the skin.

We quantified LTB4 concentration in the plantar tissue on days 1 and 3 after the incision, the results of which are provided in the new Fig. 2. LTB4 concentration peaked at 3 h, and a moderate level of LTB4 was detected not only on day 1 but also on day 3, which indicated that the LTB4 increase affected Ly6Chigh monocyte recruitment and strengthened inflammation on days 1 and 3.

In regard to the reasons why the pain response in BLT1KO mice was not suppressed on day 1, we speculate that the inflammatory reaction was relatively strong on day 1, and stronger monocyte suppression was necessary for pain inhibition during this period.

In general, BLT1 is highly expressed in neutrophils, and LTB4-BLT1 signaling in neutrophils is the primary initial inflammation response. Consequently, a cascade of the accumulation of neutrophils and other inflammatory cells is induced. In our previous report (Ref. 52), the expression of BLT1 was confirmed in CCR2-high inflammatory monocytes, which were identified as Ly6Chigh inflammatory monocytes. We propose that Ly6Chigh inflammatory monocytes are a target of LTB4 and that LTB4-BLT1 signaling in inflammatory monocytes plays an important role in the progression of inflammation.

Furthermore, according to the reference mentioned below (doi: 10.1186/s12990-015-0010-9), MPO-rich Ly6Chigh monocytes promote inflammation and tissue destruction via the release of proteases and inflammatory cytokines, such as TNF-α, in various inflammatory settings.

Previous reports from other group (DOI: 10.1073/pnas.1501372112) have shown that monocytes (nonneutrophil myeloid cells) are more involved than neutrophils in postoperative pain. In our study, we showed that inhibition of monocytes by suppressing BLT1 signaling also contributes to pain control. BLT1-deficiency resulted in the suppression of monocyte infiltration on days 1 and day 3, and may also be involved in the suppression of cytokines on day 1.

Regarding the reason why the pain response in BLT1KO was not suppressed on day 1, we speculate that inflammatory reaction was relatively strong on day 1; thus, the stronger monocyte suppression may be necessary for pain inhibition.

The data of cytokine production at day 3 was added to the new Fig 4.

We focused on the role of inflammatory cells and LTB4-BLT1 signaling in an incisional pain model. It remains unclear how peripherally-produced LTB4 acts on the DRG; however, it is possible that LTB4 activates BLT1 expressed in the small TRPV1-positive neurons of the DRG and mediates pain sensitization. A description of this has been added to the discussion section. (L369-373, L387-388)

What is the level of LTB4 in the spinal cord? These authors have shown the increase of pCREB in the spinal cord in the formalin test is BLT1 dependent. What happens in the incisional pain model?

In this study, the effect of LTB4-BLT1 signaling on central sensitization, such as an increase of pCREB in the dorsal horn of the spinal cord, was not confirmed. We focused on the peripheral mechanisms, determined which types of inflammatory cells are involved, and investigated whether LTB4 is produced at the site of injury and whether inflammatory cell infiltration is affected by the deficiency of the LTB4 receptor BLT1. LTB4 was not detected in the spinal cord on day 3 of the incisional pain model (data not shown). BLT1 expressed in the small neurons of the DRG may have impacted the modulation of incisional pain.

According to the single-cell RNASeq data (http://bis.zju.edu.cn/MCA/search2.html), cluster 17 with high expression of BLT1 is a neutrophil with high MPO and also expresses Ly6C1 and Ly6C2. In addition, cluster 5 macrophages express Ly6C2, BLT1, and MPO. These data indicated that neutrophils and monocytes/macrophages express both BLT1 and MPO, and release MPO via LTB4-BLT1 signaling. Although it was difficult in our study to determine whether the source of MPO is neutrophils or monocytes, MPO release may contribute to inflammation and pain enhancement.

Do inflammatory monocytes (Ly6Chigh) produce MPO in the present experimental condition? Please, see (doi: 10.1172/JCI42304).

Thank you for the information about inflammatory monocytes (Ly6Chigh) and MPO production. The study reported that inflammatory monocytes (Ly6Chigh) express a 10-fold higher level of MPO than non-classical monocytes (Ly6Clow). We have described this in the discussion section. (L360-368)

Figure 2. The number of samples ranged from 4 to 7. It is a huge variation in the number of samples. Why?

A huge variation also occurred in Figure 4 with n varying from 5 to 9. Why?

There is no mention on repetition of experiments.

There is no mention about data normality and homogeneity.

We quantified LTB4 concentration in the paw at the missing time points, and the sample ranges have been corrected. Individual data are shown in Fig. 2, which illustrates the variation of the data of each sample.

We apologize for the incorrect sample size provided in Fig. 4. We have corrected the sample size, and individual data are shown in the figure to illustrate the variation of the data of each sample.

Figures 1 and 2 can be combined.

1) To indicate that this study is basic research using experimental animals, the animal species should be included in the title.

The animal species used in our study have been added to the title.

The details of the methods of assessment and calculation of the mechanical responses are described in the method section. (L104-121)

We quantified LTB4 concentration in the plantar tissue on days 1 and 3 after the incision, and the results have been added to the new Fig. 2. LTB4 concentration peaked at 3 h and was still detectable on day 3, which indicated that a persistent LTB4 increase affects Ly6Chigh monocyte recruitment and strengthens inflammation on days 1 and 3. BLT1-deficiency suppressed monocyte infiltration on days 1 and 3, and may also be involved in the suppression of cytokines on day 1.

Regarding the reasons why the pain response in KO mice was not suppressed on day 1, we speculate that the inflammatory reaction was relatively strong on day 1 and, thus, stronger monocyte suppression may have been necessary for pain inhibition during this period.

Attachment

Submitted filename: Responses to the Reviewers .docx

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PLoS One. doi: 10.1371/journal.pone.0276135.r003

Decision Letter 1

Ichiro Manabe

3 Aug 2022

PONE-D-22-08079R1Role of leukotriene B4 (LTB4)-LTB4 receptor 1 signaling in post-incisional nociceptive sensitization and local inflammation in micePLOS ONE

Dear Dr. Ito,

Because of the unavailability of the original editor, I am handling your submission.

Your revised manuscript has been evaluated by three reviewers. Though two of them are more positive, Reviewer #2 raised serious concerns. In particular, as the reviewer pointed out, the results reported in 10.1073/pnas.1501372112 do not support the notion that CCR2+Ly6Chi cells contribute to pain. Accordingly, the mechanistic role of Ly6Chi monocytes in pain in your model needs to be further addressed, and your findings need to be better correlated with the literature.

Please submit your revised manuscript by Sep 17 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
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We look forward to receiving your revised manuscript.

Kind regards,

Ichiro Manabe

Academic Editor

PLOS ONE

Additional Editor Comment:

Because of the unavailability of the original editor, I am handling your submission.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors did an excellent work in addressing my comments and those raised by the different reviewers. The changes made by the authors greatly improve the manuscript. The remaining comments should be considered by the authors

Regarding the LTB4 plantar injection, a suggestion would be to evaluate cytokine concentration in plantar tissue to support the peripheral action of LTB4 on local cytokines.

The expression "mechanical hyperalgesia" is still present in the manuscript (abstract, description of the results, discussion) when the authors refer to von Frey's results. The authors have to change this expression since the up and down method with von Frey allows to assess tactile allodynia (or mechanical pain hypersensitivity as suggested in the first revision).

Reviewer #2: This is the R1 version of the MS of Miho Asahara and colleagues.

Major points:

-In the answer: “Furthermore, according to the reference mentioned below (doi: 10.1186/s12990-015-0010-9), MPO-rich Ly6Chigh monocytes promote inflammation and tissue destruction via the release of proteases and inflammatory cytokines, such as TNF-α, in various inflammatory settings.” The cited reference does not show what was mentioned.

-Previous question was: The discrepancy between the present finding and the previous finding (DOI:10.1073/pnas.1501372112) should be better explored with experiments. These papers are suggesting different cellular populations are involved in incisional pain. What are Ly6Chigh inflammatory monocytes doing in incisional pain and how LTB4 can affect their function? What is the explanation for the difference at day 1? At this time point there is no significant analgesia by BLT1 deficiency, but there is reduction of Ly6Chigh inflammatory monocytes and IL-1b and TNFa production. However, Ly6Chigh inflammatory monocytes are high at day 3 and we do not know the cytokine profile.

Answer was: Previous reports from other group (DOI: 10.1073/pnas.1501372112) have shown that monocytes (nonneutrophil myeloid cells) are more involved than neutrophils in postoperative pain. In our study, we showed that inhibition of monocytes by suppressing BLT1 signaling also contributes to pain control. BLT1-deficiency resulted in the suppression of monocyte infiltration on days 1 and day 3, and may also be involved in the suppression of cytokines on day 1.

Regarding the reason why the pain response in BLT1KO was not suppressed on day 1,

we speculate that inflammatory reaction was relatively strong on day 1; thus, the stronger monocyte suppression may be necessary for pain inhibition.

The data of cytokine production at day 3 was added to the new Fig 4.

Comment: Authors did not answer the question, but rather tried to blur what was questioned. The finding (DOI:10.1073/pnas.1501372112) disproved the contribution of inflammatory (CCR2(+)Ly6C(hi)) monocytes in post-incisional pain. The current Authors are working to demonstrate that Ly6Chigh inflammatory monocytes are contributing to post-incisional pain since BLT1 deficiency reduced their presence in the tissue and pain. In addition to this dismissed explanation of the Authors, Ly6Chigh inflammatory monocytes reduced when there was no reduction of pain, and again, Authors do not give a reasonable explanation, but rather speculate without data.

-As mentioned by Reviewer #1, this MS needs more pain measurements. Significant reduction of allodynia was observed at days 2, 3 and 4. However, LTB4 production was significant only at 3h and day 1. LTB4 activates TRPV1, thus, there is need to evaluate thermal hyperalgesia.

-Another point that might be happening is that the Authors are not looking at the right cell type. Authors must assess CD11b(+)Ly6G(-) myeloid cells as previously demonstrated (DOI:10.1073/pnas.1501372112). This might be the reason why the results are not aligning.

-Figure 4. Two cytokines known to induce pain (TNFa and IL-1b) have their production reduced by BLT1 deficiency at day 1 only, again, when there is no change in pain. I understand that some mediators cause nociceptor sensitization, however, this can be seen within some hours and not skipping one entire day. The time points of measurement after incision might also be influencing the results.

-Authors also mention that they are presenting the individual results to allow verifying the variability but did not answer the question on whether results are normal and homogeneous.

Reviewer #3: The authors have responded appropriately to this reviewer’s comments.

However, the basal 50% paw withdrawal threshold shown in Figure 5 as an additional experiment to determine the effect of intraplantar injection of LTB4 (about 1.5 g) is quite different from that in Figure 1 (about 3.6-3.7 g). The authors should comment to this difference. Is the experimenter performing von Frey filament test who was blinded to the treatment and genotypes of mice one and the same person?

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Cyril Rivat

Reviewer #2: No

Reviewer #3: No

**********

PLoS One. 2022 Oct 20;17(10):e0276135. doi: 10.1371/journal.pone.0276135.r004

Author response to Decision Letter 1

16 Sep 2022

We appreciate all reviewers for reviewing our manuscript and giving useful comments to improve our manuscript again.

Additional Editor Comment:

Because of the unavailability of the original editor, I am handling your submission.

Reviewers' comments:

Reviewer's Responses to Questions

Review Comments to the Author

Regarding the LTB4 plantar injection, a suggestion would be to evaluate cytokine concentration in plantar tissue to support the peripheral action of LTB4 on local cytokines.

Measurements of cytokine concentration in plantar tissue following LTB4 injection were performed previously. However, IL-1β, TNF- α and IL-6 elevation were small and not significantly different from vehicle injection group. (L298-300). The increase in LTB4 may not be directly related to local cytokine production.

We checked again and replaced the term “Mechanical hyperalgesia” by “Mechanical pain hypersensitivity”.

Reviewer #2: This is the R1 version of the MS of Miho Asahara and colleagues.

Major points:

I apologize for citing the wrong reference. The correct reference is doi: 10.1084/jem.20070885.

Answer was: Previous reports from other group (DOI: 10.1073/pnas.1501372112) have shown that monocytes (non-neutrophil myeloid cells) are more involved than neutrophils in postoperative pain. In our study, we showed that inhibition of monocytes by suppressing BLT1 signaling also contributes to pain control. BLT1-deficiency resulted in the suppression of monocyte infiltration on days 1 and day 3, and may also be involved in the suppression of cytokines on day 1.

Regarding the reason why the pain response in BLT1KO was not suppressed on day 1,

we speculate that inflammatory reaction was relatively strong on day 1; thus, the stronger monocyte suppression may be necessary for pain inhibition.

The data of cytokine production at day 3 was added to the new Fig 4.

According to the FACS data of immune cells of planter tissue (DOI:10.1073/pnas.1501372112), Ly6Chigh cell subset peaked on day1 and declined on day3. On the other hand, Ly6Chigh cell subset increased on day1 and peaked on day3 in this study, suggesting that intensity of inflammation may be stronger than their model and the effect of Ly6Chigh cell subset appeared more potently. We have described this in the text (L348-353).

Previous literature reported that CD11b+Ly6G- myeloid cells are required for the mechanical hypersensitivity not for the thermal hypersensitivity that follows incisional wound-induced inflammation (DOI:10.1073/pnas.1501372112). In this study, we also focused on the role of inflammatory cells and LTB4-BLT1 signaling in the mechanical pain hypersensitivity following incision.

CD11b+Ly6G- myeloid cells are assessed by analyzing the cell population after eliminating CD11b+Ly6G+ cells. Cell suspensions were first gated on forward-scatter (FSC-A) and side-scatter (SSC-A) to exclude debris. Neutrophils were then selected as double-positive for CD11b and Ly6G, quantified, and eliminated from further analysis. Non-neutrophil myeloid cells (CD11b+Ly6G-) were then gated based on Ly6C expression as Ly6Chigh monocytes or Ly6Clow monocytes. Representative FACS plots of at 3day time point after incision from WT mice are shown in the below figures (Please see another response to reviewers document). Thus, CD11b+Ly6G+ myeloid cells (Neutrophil) and CD11b+Ly6G-Ly6C+ non-neutrophil myeloid cells (monocyte) were analyzed. In this study, FACS analysis of CD11b+Ly6G+ myeloid cells were shown in Figure 3 A and C. CD11b+Ly6G+ myeloid cells peaked on day1, and no significant difference was observed between BLT1WT and BLT1KO. On the other hand, CD11b+Ly6Chigh subset were significantly reduced in BLT1KO mice than BLT1WT mice on day1 and day3 (Figure 3 B and D).

Ghasemlou et.al. showed that CD11b+Ly6G+ neutrophils do not contribute to the development of incisional pain by CD11b+Ly6G+ cell depletion study. Furthermore, depletion of CD11b+Ly6G- cells were confirmed by CD11b-TK /GCV mice and these treatments showed significant increase of mechanical threshold. (DOI:10.1073/pnas.1501372112). Since most Ly6G- cells (=Ly6C+) disappeared in this cell-depletion treatment, it was difficult to distinguish which subset group (Ly6Clow or Ly6Chigh) was responsible for the incisional pain. They tried to see the effect of depletion of Ly6Chigh and CCR2KO mice were used. In CCR2KO, reduction in all three subsets of Ly6C were shown; Ly6Clow subset displayed a much smaller reduction at 24h than 3d. However, there was no significant difference in incisional pain responses between CCR2WT and CCR2KO.

Their results of the recruitment of CD11b+Ly6G- cells contribute to incisional pain is aligning our results, but whether or not which subset of CD11b+Ly6C+ cells were responsible in incisional pain was remains to be elucidated. In this study, recruitment of the CD11b+Ly6Chigh subset were reduced in BLT1KO and could be involved in the attenuation of pain response.

We consider that the reduction of the two cytokines reflects the suppression of monocyte infiltration by BLT1KO. However, whether a transient early increase of two cytokines is involved in the pain response cannot be resolved in this study. We speculate that the inflammatory reaction was relatively strong on day1, and, thus, stronger monocyte suppression may be necessary for pain inhibition during this period. Furthermore, in this study, neutrophil recruitment did not differ between KO and WT and peaked on day1, suggesting that other inflammatory mediators and chemokines released from neutrophils may sensitize peripheral nociceptors on day1. Description of peak time of neutrophil infiltration was corrected (L252).

-Authors also mention that they are presenting the individual results to allow verifying the variability but did not answer the question on whether results are normal and homogeneous.

Analysis of Normality were performed by Shapiro-Wilk test. Homogeneity of variance was confirmed by the Leven test using R statistical software version 4.2.1(R project for statistical computing). Description was added in the Method section. (L298-300)

Reviewer #3: The authors have responded appropriately to this reviewer’s comments.

The mice used for behavior test in Figure 5 were 8 weeks old, and younger than the mice used in Figure 1(9-14 weeks old). It is possible that the age of the mice used affected the baseline threshold.

The experiments of assessment of pain behavior were performed in a blinded manner to the treatment and genotypes of mice.

Attachment

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PLoS One. doi: 10.1371/journal.pone.0276135.r005

Decision Letter 2

Ichiro Manabe

29 Sep 2022

Role of leukotriene B4 (LTB4)-LTB4 receptor 1 signaling in post-incisional nociceptive sensitization and local inflammation in mice

PONE-D-22-08079R2

Dear Dr. Ito,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Ichiro Manabe

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PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors addressed the remaining issues except that in the abstract lines 29 and 33, the term "Mechanical hyperalgesia" should still be changed.

Reviewer #3: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #3: No

**********

PLoS One. doi: 10.1371/journal.pone.0276135.r006

Acceptance letter

Ichiro Manabe

10 Oct 2022

PONE-D-22-08079R2

Role of leukotriene B4 (LTB4)-LTB4 receptor 1 signaling in post-incisional nociceptive sensitization and local inflammation in mice

Dear Dr. Ito:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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PERMALINK

Role of leukotriene B4 (LTB4)-LTB4 receptor 1 signaling in post-incisional nociceptive sensitization and local inflammation in mice

Miho Asahara

Nobuko Ito

Yoko Hoshino

Takaharu Sasaki

Takehiko Yokomizo

Motonao Nakamura

Takao Shimizu

Yoshitsugu Yamada

Roles

Abstract

Introduction

Materials and methods

Animals

Plantar incision

Behavioral testing

LTB4 administration in the hind paw

Quantification of LTB4 concentration in the paw

Flow cytometric analysis

Cytokine analysis

Statistical analysis

Results

Mechanical pain hypersensitivity after intraplantar incision

Fig 1. Effect of BLT1 blockade on mechanical withdrawal threshold after plantar incision.

Quantification of LTB4 concentration in the plantar tissue

Fig 2. Local LTB4 concentration in the plantar tissue after the incision and pain assessment after local injection of LTB4 in BLT1WT mice.

Leukocyte infiltration in the peri-incisional tissue of the footpad

Fig 3. Leukocyte infiltration in the plantar tissue after the paw incision.

Cytokine analysis

Fig 4. Level of inflammatory cytokines after plantar incision by time course.

Painful reactions following LTB4 administration

Fig 5. Pain assessment after local injection of LTB4 in BLT1WT mice.

Discussion

Conclusions

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Thiago Mattar Cunha

Roles

Author response to Decision Letter 0

Decision Letter 1

Ichiro Manabe

Roles

Author response to Decision Letter 1

Decision Letter 2

Ichiro Manabe

Roles

Acceptance letter

Ichiro Manabe

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Role of leukotriene B₄ (LTB₄)-LTB₄ receptor 1 signaling in post-incisional nociceptive sensitization and local inflammation in mice

LTB₄ administration in the hind paw

Quantification of LTB₄ concentration in the paw

Quantification of LTB₄ concentration in the plantar tissue

Fig 2. Local LTB₄ concentration in the plantar tissue after the incision and pain assessment after local injection of LTB₄ in BLT1WT mice.

Painful reactions following LTB₄ administration

Fig 5. Pain assessment after local injection of LTB₄ in BLT1WT mice.