NLRP3 inflammasome activity is required for wound healing after burns

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NLRP3 inflammasome activity is required for wound healing after burns

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40X XROOHI XD VINAIKD41X X, D42X XABDIKARIM ABDULLAHID43X X, D4X XDALIA BARAYAND45X X, and D46X XMARC G. JESCHKED47X X

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Survival of burn patients is contingent on effective wound healing, a complex process that requires coordinated responses of myeloid cells and inflammatory pathways. NLRP3, which serves as a platform for secretion of proinflammatory cytokines, is implicated as a central regulator of wound healing. However, its role during the acute dermal and epidermal regeneration in the context of burns is unknown. Wild-type (WT) and NLRP3 / mice were exposed to a 30% TBSA scald burn. Gene expression was conducted via real-time polymerase chain reaction. Trichrome staining was used to assess collagen deposition and granulation tissue formation. F4/80 immunostaining compared macrophage infiltration. Flow cytometric analysis was used to characterize skin macrophage distribution and profile. X X NLRP3, IL1β and IL18 expression was upregulated in skin after burn, and these changes were nonexistent in NLRP3 / . NLRP3 / had decreased expression of proinflammatory cytokines, chemokines, inflammatory markers, and growth factors at 3 days (P < 0.05). NLRP3 / burn skin demonstrated significantly less macrophage infiltration and higher expression of anti-inflammatory markers Arg1 and Fizz1 (P < 0.05) compared to WT. Trichrome staining showed decreased collagen deposition compared to WT. We show that NLRP3 is protective in burn wound healing, primarily through production of inflammatory mediators, macrophage recruitment, and polarization to a proinflammatory phenotype. Our findings highlight a central role of NLRP3 in wound healing through regulation of inflammation and macrophage polarization after burns. (Translational Research 2019; &&:&&-&&)

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From the Department of Surgery, Division of Plastic Surgery, University of Toronto, Toronto, Canada; Department of Immunology, University of Toronto, Toronto, Canada; Ross Tilley Burn Centre, Sunnybrook Health Sciences Centre, Toronto, Canada; Sunnybrook Research Institute, Toronto, Canada.

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Submitted for Publication July 30, 2019; received submitted November 25, 2019; accepted for publication November 25, 2019.

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Reprint requests: Marc G. Jeschke, Director Ross Tilley Burn Centre, Sunnybrook Health Sciences Centre, 2075 Bayview Ave., Rm. D704, Toronto, ON, Canada, M4N 3M5. E-mail address: [email protected].

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Ó 2019 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.trsl.2019.11.002

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Brief commentary Roohi Vinaik, et al. Background

Survival of burns requires effective wound healing, and aberrant healing increases morbidity and mortality. The benefit of inflammatory cells and inflammation in wound healing is debated. NLRP3 inflammasome is a central regulator of this inflammatory response, and NLRP3 activation acutely postburn is necessary for immune cell chemotaxis.

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Translational Significance

Glyburide is an anti-hyperglycemic and an NLRP3 activation blocker. Although acute inflammation is necessary for healing, chronic inflammation and NLRP3 activation contribute to development of hyperproliferative conditions. We demonstrate that early as opposed to delayed glyburide administration impairs healing, highlighting the potential beneficial effects of acute inflammation in normal wound healing after burns.

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INTRODUCTION

Wound healing is a complex process that comprises several phases—inflammation, proliferation, granulation tissue formation, and remodeling. It requires the coordinated response of resident skin cells, soluble factors, and infiltrating inflammatory leukocytes.1 Principally, inflammatory cells at the site of injury have a complex and critical role in cutaneous healing. However, the benefit of these inflammatory cells and in general, inflammationD48X X is a controversial topic. Prolonged inflammation leads to the formation of chronic wounds and hyperproliferative conditions such as hypertrophic scars or keloids.1,2 Thus, while acute inflammation may be beneficial, chronic inflammation is detrimental Q3 to appropriate wound healing .X X Emerging evidence suggests the involvement of inflammasomes in both regulating inflammation required for adequate wound closure and in the pathogenesis of a number of conditions, including sepsis and bury injury. Inflammasomes are multiprotein complexes that have a critical role in innate immunity and are activated in response to cellular damage, tissue injury, and infection. In addition to exogenous pathogen-associated molecular patternsD49X X, they are capable of recognizing endogenous, nonDinfectious, 50X X damage-associated molecular patternsD51X X.3 Nucleotide-binding and oligomerization domain, leucine rich repeat and pyrin domain containing 3 (NLRP3) is the most studied

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inflammasome and is the only member that recognizes DAMPs, which are released after thermal injury.4 Upon NLRP3 activation, IL1b and IL18 precursors are processed to generate active cytokines, inducing an inflammatory response. This response has been demonstrated in white adipose tissue of burn patients and in murine excisional wounds, suggesting a putative role in burn wounds.5,6 In the acute inflammatory period, NLRP3 components are expressed by proD52X Xinflammatory macrophages, which are critical for normal cutaneous responses to injury.7 Macrophages present at the site of injury are comprised of 2D53X X groups: resident and infiltrating macrophages. Resident macrophages are present at relatively low levels, and depletion of this population does not have a major effect on macrophage-dependent healing.8 On the contrary, total macrophage depletion impairs neovascularization and granulation tissue formation, indicating the necessity of recruiting circulating monocytes/macrophages to the site of injury.9 Macrophages can be further phenotypically classified based on their polarization state, which depends on a variety of local factors such as cytokine expression. These macrophages can be polarized to either a proD54X Xinflammatory or an anti-inflammatory subtype. ProD5X Xinflammatory (also known as classically activated) macrophages are usually produced upon stimulation with LPS or IFNg. Anti-inflammatory (alternatively activated) macrophages are produced upon stimulation with cytokines such as IL4 or IL10.10 Wound healing relies on a complex balance between pro- and antiinflammatory macrophages. Macrophage populations change from an early pro- to an anti-inflammatory phenotype as healing progresses, and impaired wound healing can be ascribed to dysregulation of macrophage polarization.11 For example, unregulated proinflammatory populations that fail to switch to antiinflammatory, proD56X Xhealing macrophages are a frequent feature of chronic wounds (e.g., diabetic ulcers).12,13 Alternatively, exogenous anti-inflammatory macrophage administration in the acute phase delays wound healing in a mouse diabetic wound model.8 Taken together, evidence indicates that an intricate balance and a tightly regulated polarization transition are necessary for effective wound healing. To our knowledge, the relationship between NLRP3 inflammasome activation and macrophage recruitment and polarization in skin after burns has not been elucidated yet. While there are currently a limited number of studies investigating NLRP3 in burn wound healing, we previously established that NLRP3 regulates macrophage recruitment to the liver and adipose tissue after thermal injury.14 Similarly, we propose that NLRP3 regulates macrophage recruitment and polarization at the site of

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injury (skin), in addition to the production of inflammatory mediators. Since macrophage recruitment and early inflammation are important elements of wound healing, we postulate that NLRP3 has a protective role in burn wounds. Furthermore, inhibition of NLRP3 activation during the acute phase with drugs such as glyburide would impair normal postDburn 57X X cutaneous healing responses.

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METHODS Human samples. D58X X

Patients admitted to the Ross Tilley Burn Center at Sunnybrook Hospital (Toronto, Canada) or nonD59X Xburn patients undergoing elective surgery were consented preD60X Xoperatively for tissue collection. Approval for our study was obtained from the Research Ethics Board at Sunnybrook Hospital. Animals and mDodel. 61X X Animal experiments were conducted in accordance and approved by the Sunnybrook Research Institute Animal Care Committee (Toronto, Ontario, Canada). Wild-type C57/B6 (WT) and NLRP3 knockout (NLRP3 / ) male mice (8D62X X 10 weeks old, n = 5 per group) were purchased from Jackson Laboratories (Bar Harbor, ME) and housed at ambient temperature and cared in accordance with the Guide for the Care and Use of Laboratory Animals. All mice were anesthetized with 2.5% isoflurane and shaved along the dorsal spine region. Ringer’s lactate (2 D63X X3 mL) was injected subcutaneously in all treatment mice to protect the spine and buprenorphine (0.05 D64X X0.1 mg/kg body weight) was injected for pain management. A full-thickness, third degree dorsal scald burn encompassing 15% D65X X20% total body surface area (TBSA) and a 15% wound on the ventral surface was induced by immersing the dorsum of the mice in 98˚C water for 10 seconds and the ventral region for 2 seconds. Mice were sacrificed at 3 and 7 days postD6X Xburn. Sham mice (control) underwent identical experimental procedures, with the exception of the burn injury. Dorsal skin tissue was harvested upon sacrifice and stored in D67X X80˚C until analysis. Flow cytometry. D68X X Skin was minced and digested with collagenase (Life Technologies) for 45 minutes at 37˚ C while shaking. The digested cell suspension was filtered and centrifuged at 1800 rpm for 5 minutes. Pelleted cells were resuspended in FACS buffer (HBSS containing 0.5% BSA) and passed through a 40 mM strainer (BD Bioscience) to remove large cellular debris. Cells were stained with monoclonal antibodies on ice for 30 minutes. Samples were than washed and analyzed using BD LSR II Special Order System (BD Biosciences, San Jose, CaliforniaD69X X). Cells were gated on FSC-A and SSC-A, followed by doublet exclusion (FSC-W x FSC-H, SSC-W x SSC-H). The total

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percentage of monocytes/macrophages was identified using the following flurochrome-conjugated antibodies: anti-CD45 (anti-mouse PE-Cyanine7, eBioscience), anti-CD11b (anti-mouse Alexa APCeFluorÒ 780, eBioscience), and anti-F4/80 (antimouse FITC, eBioscience). Anti-inflammatory macrophages were identified using an anti-CD206 antibody (anti-mouse PE, BioLegend) in accordance with the manufacturer’s flow cytometry protocol. The gating strategy for assessing innate immune cell distributions included leukocytes initially gated based on granularity and CD45 (side scatter x CD45), followed by size (forward scatter). Gated cells were stained for cell surface markers for monocytes and macrophages (CD11b+/F4/80+). Histology and immunohistochemistry. D70X X Dorsal skin was collected and immediately fixed in 10% formalin and then maintained in 70% ethanol before paraffin embedding. Subsequently, tissues were sectioned and incubated with F4/80 (Abcam, #100790) for skin macrophages or NLRP3 (Abcam, #214185) followed by DAB staining. For macrophage count, D71X X3 independent samples were imaged at 40X magnification, and F4/80 positive cells were counted twice manually. For Masson’s trichrome staining, paraffin-embedded slides were heated for 30 minutes at 60˚C. The slides were then deparaffinized with citrosol, followed by rehydration through 100% £ 2, 95%, 70%, and washed in distilled water. Slides were placed in Bouin’s solution (26367 01; EMS, Hatfield, PennsylvaniaD72X X) for 1 hour at 56˚C and washed. Hematoxylin stain (HHS16; Sigma, Saint Louis, MissouriD73X X) and Biebrich scarlet-acid fuchsin solution were applied sequentially for 10 minutes. After each stain the slides were washed. Next, slides were differentiated in phosphomolybdic phosphotungstic acid for 15 minutes, and transferred to aniline blue for 5 minutes. All slides were washed in distilled water and then differentiated in 1% acetic acid for 2 minutes. Slides were dehydrated through 95% ethanol and absolute ethanol followed by clearing in citrosol and were mounted with SHUR/Mount xylene-based liquid mounting media (Triangle Biomedical Sciences, Durham, North CarolinaD). 74X X Imaging was performed on a LSM confocal microscope (Zeiss, Germany). Glyburide treatment. D75X X Glyburide 50 mg/kg (SigmaAldrich) was administered intraperitoneally once every 24 hours and continued until sacrifice at 3 and 7 days. The first dose was administered immediately postD76X Xburn, and the final dose was administered 1 hour prior to collection of tissue. For the delayed treatment group, glyburide was administered once daily after 3 days until sacrifice at 7 days after burn. This dose was chosen as it was previously established to be equivalent to the highest human dose.15

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Gene eDxpression 7X X D78X X using real-time polymerase chain reactionD.79X X

Total RNA isolated from skin tissue was analyzed by quantitative real-time polymerase chain reaction (RT-PCR). RNA was isolated from tissue and cells using TRIzol-chloroform (Life Technologies) with subsequent purification using the RNeasy Kit (Qiagen) according to the manufacturer’s instructions. RNA (2mg) was transcribed to cDNA using the high-capacity cDNA reverse transcription kit (Applied Biosystems). RNA was extracted from rodent skin tissue using Trizol (Invitrogen, CalifornaD80X X). Reverse transcription were performed with high-capacity cDNA reverse transcription kit (ABI, MassachusettsD81X X). RT-PCR was performed using the Applied Biosystems Step One Plus Real-Time PCR System. Gene expression was expressed relative to b-actin and was determined using the following formula: 2^(-DDCt). Due to inter-species variability, values were expressed as a ratio of burn to sham. Primer sequences used are available upon request. Western blotting. D82X X Protein from human and rodent skin was extracted in RIPA buffer containing phosphatases and proteases inhibitor cocktails (Roche). Protein concentrations were determined by the BCA protein assay kit (Pierce, Mississauga, OntarioD83X X, Canada). Proteins were resolved by SDS-PAGE followed by western blotting using the following antibodies at 1:1000 concentration: Caspase-1 (Cell Signaling, MA), cleaved caspase-1 (Cell Signaling, MA), IL1b (Cell Signaling, MA), cleaved IL1b (Cell Signaling, MA), and GAPDH (Cell Signaling, MA). Species appropriate secondary antibodies conjugated to horseradish peroxidase (BioRad, Mississauga, OntarioD84X X, Canada) were used and proteins visualized by enhanced chemiluminescence using the BioRad ChemiDoc MP Imaging System. Band intensities were detected, normalized and quantified with the ChemiDoc and Image Lab 5.0 software (BioRad Laboratories, Hercules, CaliforniaD85X X). Antibody concentrations are expressed relative to GAPDH. Statistical analysis. All data are represented as mean § SEM. Statistical analysis was performed using student’s tD86X X test, D87X X1 and D8X X2-way ANOVA and Mann Whitney U test to compare groups, where appropriate. All graphs were created using Graphpad Prism 6.0 (San Diego, CaliforniaD)89X X and analyzed statistically using SPSS 20 (IBM Corp., NY, New York), with significance accepted at P D90X X D91X X< 0.05 (*), D92X XP < 0.01 (**), D93X XP < 0.001 (***) and D94X XP < 0.05 (#), D95X XP < 0.01 (##), D96X XP < 0.001 (###), where appropriate. Asterisks refer to a comparison between burn groups and corresponding shams, and hashtags refer to a comparison between NLRP3 / , WT, and treated mice. Data availability. D97X X The data set generated during and/ or analyzed during the current study are available from the corresponding author on reasonable request.

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RESULTS Increased expression of NLRP3 and its activation

First, we asked if NLRP3 is involved in wound healing after a burn. We conducted a time course study of NLRP3 gene expression in human skin from 25 burn patients (17 male, 8 female) with a mean age of 51 § 3.19 years and an average TBSA of 30.3 § 3.29%. Our findings revealed an acute (0D98X X 2 days) increase after burn compared to normal skin (13.4 vsD9X X 1.15, D10X XP < 0.01) (Fig 1A). IL1b, an NLRP3 activation byproduct, followed a similar pattern (Fig 1A). IL1b expression was higher acutely, 3D10X X 6, and up to 7D102X X 10 days after burn compared to normal skin (98.9 vsD103X X 1.65, D104X XPD105X X < 0.05; 72.3 vsD106X X 1.65, D107X XPD108X X < 0.05; 29.5 vsD109X X 1.65, D10X XPD1X X < 0.01). IL18, another NLRP3 activation byproduct, similarly demonstrated significantly higher expression compared to WT at 3D12X X 6 days postD13X Xburn (1.70 vsD14X X 0.43, D15X XPD16X X < 0.05). To confirm our gene expression results, we assessed NLRP3 activation by evaluating the ratio of protein expression of cleaved to pro-caspase-1 and cleaved to pro-IL1b. There is a significant increase in the ratio of cleaved to pro-caspase-1 and cleaved to pro-IL1b at 0D17X X 2 days compared to normal skin (0.92 vsD18X X 0.33, D19X XPD120X X < 0.05; 29.9 vsD12X X0.11, D12X XPD123X X< 0.001), suggestive of NLRP3 activation (Fig 1A, Figure S1A-D, Figure S1F-I). Furthermore, we stained for NLRP3 in skin obtained from patients 0D124X X 2 days after burn, demonstrating that NLRP3 expression is present and more prominent in deeper dermal layers (Fig 1C). Similar to humans, we show here that NLRP3 gene expression is elevated in WT murine skin at 1 and 3 days after burn (23.1 vsD125X X 1.07 at 1 day, D126X XPD127X X < 0.001; 6.76 vsD128X X 1.07 at 3 days, D129X XPD130X X < 0.05) (Fig 1B). We also assessed gene expression levels of IL1b and IL18. Analogous to NLRP3, IL1b expression is elevated at 1 and 3 days, although levels are relatively unchanged at 7 days after burn potentially due to contribution from NLRP3-independent IL1b activation pathways (11.7 vsD13X X 1.05 at 1 day, D132X XPD13X X < 0.0001; 7.29 vsD134X X 1.05 at 3 days, D135X XPD136X X < 0.05; 6.99 vsD137X X 1.05 at 7 days, D138X XPD139X X < 0.05).16-18 IL18 levels are similarly elevated at 1, 3, and 7 days after burn (4.29 vsD140X X 0.59, D14X XPD142X X < 0.05; 8.42 vsD143X X 0.59 at 3 days, D14X XPD145X X < 0.05; 3.50 vsD146X X 0.59 at 7 days, D147X XPD148X X < 0.05). We compare this to NLRP3 / , which demonstrate no changes in gene expression of NLRP3, IL1b, or IL18 after burn (Figure S2AD149X X C). In order to assess for NLRP3 activation, we measured protein levels of cleaved to pro-caspase-1 and cleaved to pro-IL1b. Similar to our human data, we demonstrate an increase in the ratio of cleaved to pro-caspase-1 and cleaved to pro-IL1b early postD150X X burn (1 and 3 days), with no significant difference at 7 days (Fig 1B, Figure S1E, Figure S1J). Immunohistochemistry for NLRP3 in WT and NLRP3 / murine byproducts in human and murine skin after burns.

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Fig 1. NLRP3 is upregulated in human and murine skin after burn. (a) Time course of gene expression of NLRP3, IL1b and IL18 and protein expression of cleaved to pro-caspase-1 and cleaved to pro-IL1b at 0-10 days after burn compared to normal skin. (b) Gene expression of NLRP3, IL1b and IL18 and protein expression of cleaved to pro-caspase-1 and cleaved to pro-IL1b in murine skin after burn. (c) Immunohistochemical staining for NLRP3 at 0 2D1X X days postDburn 2X X in human burn skin indicates more NLRP3 positive cells in the dermis. (d) Staining for NLRP3 at 3 and 7 days after burn in murine skin indicates more NLRP3 positive cells in the dermis of WT at 3 days. Areas of positive staining are marked with an arrow. Values are presented as mean § standard error. Burn versus normal skin *DP 3X X < 0.05; **P D4X X < 0.01; ***P D5X X < 0.001.

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skin similarly shows positive dermal expression in WT at 3 days compared to shams, NLRP3 / and WT at 7 days, which is in accordance with our human data (Fig 1D). These results indicate that NLRP3 expression is elevated acutely after burns in both human and murine skin. NLRP3 inflammasome expression is localized to the deeper dermal layers of the wound bed, indicating a potential role at the site of injury. Mice wounds show very similar expression profiles for NLRP3 and its activation byproducts when compared to humans, thus allowing for a translational comparison. In order to elucidate the role of NLRP3 in wound healing, we employed an NLRP3 / D15X X murine model for all of our subsequent studies.

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NLRP3 / D152X X model to assess if lack of NLRP3 would compromise wound healing after burn. We compared healing in WT and NLRP3 / with trichrome staining of excised wounds (Fig 2AD153X X D). When compared NLRP3 / , WT demonstrated greater dermal collagen deposition and keratinization at 3 days after burn, with further improvements at 7 days (Fig 2A, C). Conversely, NLRP3 / exhibited no changes between 3 to 7 days after burn with regards to collagen deposition (Fig 2B, D), suggesting that lack of NLRP3 impairs wound healing. These results indicate that NLRP3 is required for adequate wound closure after burns. We recently showed that lack of NLRP3 results in differential macrophage recruitment to the liver and adipose tissue.14 Since macrophages are necessary for granulation tissue formation and release of inflammatory

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Fig 2. Impaired wound healing in NLRP3 / . Trichrome staining of excised burn wounds in WT at (a) 3 days and (c) 7 days exhibits greater dermal collagen deposition and keratinization compared to NLRP3 / D6X Xat (b) 3 days and (d) 7 days post-burn. “Normal skin” demarcates the edge of the wound, and “burn” denotes the wound.

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Fig 3. Reduced macrophage proportion acutely after burn in NLRP3 / . (a) F4/80 staining for macrophage infiltration in NLRP3 / D7X Xand WT mice shams, 3 days and 7 days after burn is corroborated by (b) PCR analysis, which indicates greater expression of F4/80 in WT at both time points. (c) F4/80-positive cell counts obtained at 40X magnification (n = 3). (d) Flow cytometry for skin macrophage distribution shows greater macrophage infiltration in WT. (e) Flow cytometry analysis for CD206-positive anti-inflammatory macrophages indicates a greater percentage in NLRP3 / at 3 days compared to WT. (f) Expression of pro- and anti-inflammatory markers at 3 days indicates a proDinflammatory 8X X profile in WT and an antiDinflammatory 9X X profile in NLRP3 / , with no significant differences in marker expression between WT and NLRP3 / D10X aX t 7 days. Values are presented as mean § standard error. Burn versus sham *DP 1X X < 0.05; **P D12X X < 0.01; ***P< D13X X 0.001, WT versus NLRP3 / burn # D14X X < 0.05; ##P P D15X X < 0.01; ###P D16X X < 0.001.

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cytokines and growth factors after burns, we next assessed macrophage infiltration to the site of injury. Immunostaining with the macrophage marker F4/80 demonstrates significant dermal macrophage infiltration to the wound site by 3 days in WT compared to NLRP3 / , with more pronounced differences at 7 days

after burn (Fig 3A,C). Moreover, F4/80 gene expression in skin was greater in WT versus NLRP3 / at both time points (9.95 vsD154X X 3.05 at 3 days, D15X XPD156X X < 0.05; 9.50 vsD157X X 4.78 at 7 days, D158X XPD159X X < 0.05) (Fig 3B). Flow cytometry data corroborates our immunostaining and gene expression results, indicating that

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NLRP3 / did indeed have a differential response at 3 and 7 days after burn (Fig 3D). At 3 days after injury, WT have increased macrophage recruitment relative to sham (814 vsD160X X 54 cells, D16X XPD162X X < 0.001) that remains elevated even at 7 days (1120 vsD163X X 54 cells, D164X XPD165X X < 0.001). NLRP3 / also have increased macrophage infiltration at 3 and 7 days, but these values are significantly less than WT (276 vsD16X X 814 cells at 3 days, D167X XPD168X X < 0.01; 470 vsD169X X 1120 cells at 7 days, D170X XPD17X X < 0.001). Altogether, these results demonstrate impaired macrophage recruitment to the site of injury in mice that lack NLRP3. Macrophages have an important role in local inflammation, granulation tissue formation, and infection control and as such, are integral to wound healing.19 Furthermore, studies have previously shown that macrophage polarization is influential in wound healing as well.19 So, we extended our analysis to compare macrophage polarization between WT and NLRP3 / at the site of injury. Increased proDinflammatory 172X X macrophages in the skin in WT after burn. As discussed previously, macrophages

can be subdivided into classically activated, proDinflammatory 173X X macrophages or alternatively activated, anti-inflammatory macrophages. Previous studies have utilized proD174X Xinflammatory markers including IL6, CCL2 and IL1b, and anti-inflammatory markers such as Arg1, IL10, Fizz1, and CD206, amongst others.20,21 While proD175X Xinflammatory macrophages predominate during the acute phase after injury, our flow cytometry analysis indicates a greater percentage of anti-inflammatory CD206-positive macrophages in NLRP3 / at 3 days (71.3% vsD176X X 58.4%, D17X XPD178X X < 0.05) (Fig 3E), with no significant difference in the percentage of CD206-positive macrophages at 7 days. Based on this, we subsequently assessed differences in anti- and pro-inflammatory marker expression between WT and NLRP3 / (Fig 3F). At 3 days, WT display greater expression of proD179X Xinflammatory markers IL6 and CCL2 relative to NLRP3 / D180X X (1.68 vsD18X X 0.49, D182X XPD183X X < 0.05; 3.31 vsD184X X 1.09, PD D185X X 186X X < 0.01). Conversely, NLRP3 / have a higher expression of anti-inflammatory markers Arg1 and Fizz1 at 3 days compared to WT (7.47 vsD187X X 1.13, D18X XPD189X X < 0.001; 6.24 vsD190X X 0.096, D19X XPD192X X < 0.05). However, there are no significant differences in marker gene expression between WT and NLRP3 / D193X X at 7 days. This is consistent with our flow cytometry results, which suggests that WT have a greater proversus anti-inflammatory distribution at 3 days when compared to NLRP3 / (Fig 3E). However, since there are differences in macrophage infiltration between WT and NLRP3 / , we compared relative marker expression between 3 and 7 days within each group (WT and NLRP3 / ). We show that WT have lower expression of antiD194X Xrelative to proD195X Xinflammatory markers at 3 days;

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however, the opposite expression pattern is seen at 7 days. NLRP3 / on the other hand have a greater expression of anti-inflammatory markers at all time points (Figure S3). Under normal conditions, macrophage phenotype changes from an initial pro- to an anti-inflammatory profile.2 Interestingly, transfer with anti-inflammatory macrophages during the first 3 days after wounding impairs healing.8 This is consistent with our results of impaired wound healing and the early presence of anti-inflammatory macrophages in NLRP3 / , indicated by increased relative expression of the M2 markers Arg1 and Fizz1 and a greater proportion of CD206-positive macrophages.

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phage polarization is regulated by local cytokine levels, and studies have established that production of proD198X Xinflammatory cytokines in injured skin has a central role in wound healing. We previously demonstrated that NLRP3 affects expression of other cytokines and immune mediators in addition to IL1b and IL18.14,22 Hence, we subsequently compared expression of several pro-inflammatory cytokines, chemokines, and growth factors in WT and NLRP3 / D19X X skin. As determined earlier, IL1b expression is significantly downregulated in NLRP3 / compared to WT at all time points (Figure S1b). Similarly, IL6 levels in NLRP3 / D20X X are diminished compared to WT at 3 days (0.49 vsD201X X 1.68, PD D20X X 203X X < 0.05) (Fig 4A); however, no difference is seen at 7 days. Interestingly, TNFa expression was elevated in NLRP3 / at all time points (2.42 vsD204X X 0.23 at 3 days, D205X XPD206X X < 0.001; 6.74 vsD207X X 1.52 at 7 days, D208X XPD209X X < 0.05) (Fig 4B). Next, we measured expression of the inflammatory chemokine, CCL2. Similar to IL6, NLRP3 / CCL2 expression is significantly lower than WT at 3 and 7 days (1.09 vsD210X X 3.31, PD D21X X 21X X < 0.05; 2.64 vsD213X X 9.34, PD D214X X 215X X < 0.05) (Fig 4C). Subsequently, we measured s100a8, which serves as an inflammatory marker in several disease states including psoriasis and hypertrophic scars.23,24 S100a8 expression is upregulated in the epidermis in response to skin injury, and lack of s100a8 impairs keratinocyte migration and proliferation.6 Here, s100a8 expression was downregulated in NLRP3 / at 3 days (3.85 vsD216X X 9.20, D217X XPD218X X < 0.05) with no significant difference at 7 days (Fig 4D). We subsequently compared levels of the growth factors VEGF, FGF2, and TGFb between NLRP3 / and WT (Fig 4E D219X XG). VEGF and FGF contribute to endothelial cell proliferation and angiogenesis, which ultimately facilitates synthesis of a new extracellular matrix (ECM) following injury. This is succeeded by FGF and TGFb-mediated fibroblast infiltration and conversion to myofibroblasts, collagen deposition and

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Fig 4. Decreased expression of pro-inflammatory cytokines, chemokines, and growth factors in NLRP3 / D17X sX kin. Gene expression of the inflammatory cytokines (a) IL6 and (b) TNFa, chemokine (c) CCL2, inflammatory mediator (d) s100a8, and growth factors (e) VEGF, (f) FGF2 and (g) TGFb. Values are presented as mean burn/ sham § standard error. WT versus NLRP3 / burn/sham #P D18X X < 0.05; ##P D19X X < 0.01; ###P D20X X < 0.001.

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eventually, scar formation.25 NLRP3 / have decreased expression of VEGF, FGF2, and TGFb at 3 days compared to WT (0.63 vsD20X X 1.86, D21X XPD2X X < 0.01; 0.16 vsD23X X 0.95, D24X XPD25X X < 0.01; 0.76 vsD26X X 1.56, D27X XPD28X X < 0.05). These changes persist at 7 days for VEGF and TGFb (0.58 vsD29X X 1.70, D230X XPD231X X < 0.05; 1.75 vsD23X X 3.10, D23X XPD234X X < 0.01). Taken together, these results suggest that lack of NLRP3 consistently decreases early expression of critical factors that are interconnected and involved in different wound healing steps. Decreased production of proD235X Xinflammatory cytokines and chemokines impairs keratinocyte migration and proliferation and immune cell chemotaxis, underscoring the significance of postD236X X injury inflammation in wound healing.13 Immune cell recruitment (e.g., macrophages) is critical for the production of growth factors and collagen deposition, amongst other functions.26 Here, we demonstrate diminished macrophage infiltration and altered macrophage phenotype in NLRP3 / . Ultimately, these changes culminate in decreased expression of growth factors and poor keratinization and collagen deposition, suggestive of impaired healing in NLRP3 / .

Glyburide treatment during the acute phase impairs wound healing. Glyburide

is a clinically approved drug that also functions as an NLRP3 activation inhibitor. After administering the drug in our murine model, we confirmed that NLRP3 activation byproducts IL1b and IL18 in glyburide treated mice showed significantly lower expression when compared to WT mice (Figure S4). In order to determine if inhibiting NLRP3 during the acute phase results in poor wound healing, we compared the effect of a delayed (3D237X X 7 days after burn) to a daily glyburide treatment (0D238X X 7 days after burn). To compare healing in treated mice to WT, we performed trichrome staining of the excised wounds (Fig 5AD239X X D). We demonstrate minimal dermal collagen deposition at 3 days in treated skin, a feature also seen in NLRP3 / D240X X skin. Additionally, there were no further improvements at 7 days after burn in treated mice, paralleling NLRP3 / . However, delayed treatment mice at 3 days (equivalent to untreated WT) demonstrated greater collagen deposition compared to the treatment group, as expected. Interestingly, skin from the delayed treatment mice at 7 days after burn

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Fig 5. Inhibition of NLRP3 during the acute phase with glyburide impairs wound healing. Similar to NLRP3 / , trichrome staining of excised burn wounds in treated mice at (b) 3 days and (d) 7 days exhibit decreased dermal collagen deposition and keratinization compared to delayed treated mice (a,c). (e) Flow cytometry in glyburide treated skin shows significantly lower levels of macrophages compared to WT and (f) greater CD206-positive macrophages at 3 days. “Normal skin” demarcates the edge of the wound, and “burn” denotes the wound. Values are presented as mean burn/sham § standard error. WT versus NLRP3 / burn #P D21X X < 0.05; ## D2X X < 0.01; ###P P D23X X < 0.001, WT versus glyburide treated burn *DP 24X X < 0.05; **P D25X X < 0.01; ***P D26X X < 0.001.

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had greater collagen deposition compared to mice treated since day 0 after burn. Earlier, we demonstrated that lack of NLRP3 diminishes macrophage recruitment to the site of injury. Flow cytometry results similarly indicate that daily glyburide treatment results in significantly lower macrophage levels compared to WT at 3 days (266 vsD241X X 814 cells, PD D24X X 243X X < 0.01) and 7 days (429 vsD24X X 1120 cells, D245X XPD246X X < 0.001) (Fig 5E). However, delayed glyburide results in a similar pattern of macrophage

infiltration to WT untreated mice. Moreover, our data indicated a greater percentage of CD206-positive anti-inflammatory macrophages in glyburide treated mice at 3 days compared to WT (84.8% vsD247X X 58.4%, D248XPDX 249X X < 0.05) (Fig 5F). Taken together, these results suggest that glyburide treatment resembles NLRP3 / in terms of the underlying mechanistic changes. However, the timing of glyburide administration and therefore, NLRP3 inhibition, is critical to wound healing. Inhibition of NLRP3 after

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the acute phase (delayed glyburide treatment) mimics WT with regards to wound healing and macrophage infiltration. Contrarily, inhibition during the acute phase (daily treatment) resulted in impaired wound healing and macrophage infiltration. Glyburide

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Evidence suggests that glyburide also has a host of anti-inflammatory effects, so we subsequently compared other pro-inflammatory cytokines, chemokines, and growth factor levels in glyburide daily treated and delayed treatment mice to WT and NLRP3 / as discussed previously.27 Similar to our flow cytometry and immunohistochemical results, we anticipated that expression levels for daily treated mice should mimic NLRP3 / and delayed treatment should be similar to WT, highlighting the importance of NLRP3 activation during the acute phase. We initially compared expression of the pro-inflammatory cytokines IL6 and TNFa. There is no significant difference between daily treated and NLRP3 / , and IL6 levels in

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daily treated and NLRP3 / mice are similarly diminished compared to WT at 3 days (0.16 vsD251X X 1.68, D25X XPD253X X < 0.01; 0.49 vsD254X X 1.68, D25X XPD256X X < 0.05) (Fig 6A). Similarly, there is no significant difference between daily treatment and NLRP3 / with regards to TNFa, and both have higher expression of TNFa compared to WT at 7 days (6.48 vsD257X X 1.52, D258X XPD259X X < 0.05; 6.74 vs D260X X1.52, D261X XPD26X X < 0.05); furthermore, they have greater expression compared to delayed treatment (6.48 vsD263X X 1.42, D264X XPD265X X < 0.05; 6.74 vsD26X X 1.42, PD D267X X 268X X < 0.05) (Fig 6B). We subsequently measured expression of CCL2, demonstrating that daily treated groups had similar levels to NLRP3 / . CCL2 levels are significantly lower than WT at 3 (0.96 vsD269X X 3.31, D270X XPD271X X < 0.001 for daily treated; 1.85 vsD27X X 3.31, D273X XPD274X X < 0.01 for NLRP3 / ) and 7 days (4.27 vsD275X X 9.34, D276X XPD27X X < 0.05 for daily treated; 2.64 vsD278X X 9.34, D279X XPD280X X < 0.01 for NLRP3 / ), with a similar pattern seen in delayed treatment mice (Fig 6C). Successively, we measured s100a8 and demonstrated that it is downregulated in daily treated and NLRP3 / mice at 3 days compared to WT (3.81 vsD281X X 9.20, D28X XPD283X X < 0.05 for daily

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Fig 6. Glyburide treatment results in comparable reductions in proDinflammatory 27X X cytokines, chemokines, and growth factors to NLRP3 / . Gene expression of the inflammatory cytokines (a) IL6 and (b) TNFa, chemokine (c) CCL2, inflammatory mediator (d) s100a8, and growth factors (e) VEGF, (f) FGF2 and (g) TGFb. Values are presented as mean § standard error. WT versus NLRP3 / burn #P D28X X < 0.05; ##P D29X X < 0.01; ###P D30X X < 0.001, WT versus glyburide treated burn *DP 31X X < 0.05; **P D32X X < 0.01; ***P< D3X X 0.001, NLRP3 / versus glyburide treated burn ˚DP 34X X < 0.05; ˚˚DP 35X X < 0.01; ˚˚˚DP 36X X < 0.001, daily treated versus delayed treatment P D37X X < 0.05; P D38X X < 0.01; P D39X X < 0.001.

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treated; 3.85 vsD284X X 9.20, D285X XPD286X X < 0.05 for NLRP3 / ) with no difference at 7 days (Fig 6D). Thereafter, we compared levels of the growth factors VEGF, FGF2, and TGFb between treated and WT mice (Fig 6ED287X X G). Treated mice have decreased expression of VEGF (0.095 vsD28X X 1.86, D289X XPD290X X < 0.001) and FGF2 (0.074 vsD291X X 0.95, D29X XPD293X X < 0.001) at 3 days compared to WT with notable differences at 7 days for VEGF and TGFb (0.55 vsD294X X 1.70, D295X XPD296X X < 0.05; 1.42 vsD297X X 3.10, D298X XPD29X X < 0.01). Similar to the aforementioned markers, delayed treatment had a similar expression pattern to WT. In summary, these results suggest that blocking NLRP3 activation after thermal injury impairs production of critical growth factors and inflammatory mediators. Consequently, there is diminished macrophage infiltration to the site of injury in treated mice, and macrophages present are potentially anti-inflammatory. Ultimately, these alterations did indeed culminate in impaired wound healing, which is consistent with our NLRP3 / data. Specifically, we show here that delayed inhibition of NLRP3 after the acute phase does not compromise wound healing, highlighting the cruciality of acute inflammation.

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DISCUSSION

Skin serves as a protection against microbial invasion and thermal dysregulation. Damage to the skin barrier by thermal injury initiates an immediate inflammatory response that is critical for wound healing. This postD30X Xburn inflammation is regulated by NLRP3 inflammasome, which is one of the key mediators of inflammation in tissue injury and infection. As we demonstrate in this study, NLRP3 gene expression is upregulated during the postD301X Xburn acute phase (1 and 3 days) in murine skin and from 0-2 days in human skin. Gene expression of the inflammatory cytokines IL1b and IL18, which are NLRP3 activation byproducts, is also upregulated at these time points. Additionally, protein expression of cleaved to pro-caspase-1 and cleaved to pro-IL1b also increases at these time points, providing further evidence of NLRP3 activation. Certain changes persist into the second phase of healing (7 days postD302X Xburn in mice, 7D30X X 10 days for human IL1b and 3D304X X 6 days for human IL18), highlighting the overlap between the different stages of wound healing and the pervasive effects of the early inflammatory response. Indeed, we show that lack of NLRP3 and its inflammatory byproducts impairs postD305X Xburn healing, indicated by decreased keratinization and collagen deposition at the site of injury. After generation of an initial inflammatory response, the next stage of healing involves recruiting circulating

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monocytes to the wound bed, which later differentiate into mature macrophages.28 Interestingly, we demonstrate that this is dependent on NLRP3 gene status.14 In mice that lack NLRP3, there is decreased expression of inflammatory mediators and importantly, chemokines such as CCL2. This corresponds with decreased macrophage recruitment to skin 3 days postDburn, 306X X which is sustained at 7 days. Conversely, we see increased macrophage recruitment to the site of injury in mice that express the NLRP3 gene, as expected. As highlighted earlier, macrophages can be either pro-inflammatory or anti-inflammatory. We show here that macrophages in the first 3 days after burn injury demonstrate greater expression of IL6 and CCL2 relative to Arg1 and Fizz1, suggesting a greater distribution of the pro-inflammatory subtype. Interestingly, NLRP3 / have greater Arg1 and Fizz1 expression at 3 days relative to IL6 and CCL2, which is indicative of a greater distribution of anti-inflammatory macrophages. Although prolonged pro-inflammatory macrophage activation is deleterious and is a potential cause of chronic wounds, macrophage depletion and transfer with anti-inflammatory macrophages within the first 3 days after wounding is not beneficial; rather, it may impair healing in a diabetic mouse model.8 Our data is consistent with these findings, and NLRP3 / mice that express more anti-inflammatory macrophages in this phase have impaired wound healing. Throughout the stages of wound healing, macrophage infiltration and polarization is critical as macrophages enable tissue repair by initiating the release of cytokines and growth factors such as VEGF, FGF2 and TGFb.29 These factors in turn stimulate angiogenesis and induce fibroblast and keratinocyte migration into the wound bed when the early inflammatory period subsides (2 D307X X10 days after wounding).30 We demonstrate that at 7 days, relative expression of anti-inflammatory markers is greater than proD308X Xinflammatory, potentially suggesting that the macrophages present are wound healing, anti-inflammatory macrophages. This phenotypic switch is coupled with elevated VEGF and TGFb expression at 7 days, which is not seen in NLRP3 / . Taken together, these results show that NLRP3 is necessary for macrophage recruitment after injury and for the production of pro-inflammatory cytokines, chemokines and growth factors. These in turn prompt migration of skin cells into the wound bed and activate the proliferative phase of healing, gradually phasing into the anti-inflammatory remodeling phase seen at 2 D309X X3 weeks.31 It follows then that blocking NLRP3 activation with drugs such as glyburide should have wound impairing effects analogous to the NLRP3 / D310X X model. With respect to inflammatory cytokine,

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chemokine and growth factor production, we show that postD31X Xburn glyburide treatment has similar outcomes to knocking out NLRP3. Treated mice also have decreased recruitment of macrophages to the site of injury, with a predominance of anti-inflammatory rather than proDinflammatory 312X X macrophages at 3 days. Furthermore, trichrome staining of excised burn wounds indicates impaired wound healing in glyburide treated mice, evidenced by poor collagen deposition compared to WT even at 7 days postD31X Xburn. Interestingly, treating with glyburide after the acute phase (delayed treatment) abrogates these changes and results in similar wound healing, macrophage recruitment, and gene expression profiles to WT mice, highlighting the importance of acute inflammatory changes in postDburn 314X X responses. However, there are some limitations in the present study. Firstly, this is primarily a rodent study, although it has wider clinical applications as well. For example, glyburide may be administered in the clinical setting as a blood glucose regulator in burn patients in addition to patients with type II diabetes. However, it is important to note that although it regulates NLRP3 activation via inhibition of ATP-sensitive K+ channels, glyburide is not specific for NLRP3. Rather, studies have shown that glyburide can inhibit the P2 £ 7 receptor and the ABC transporter ABC1A.32 Commercially available MCC950 or JC124 are alternative options and are specific NLRP3 inhibitors. While we utilized glyburide due to its translational aspect, further confirmation using NLRP3-specific inhibitors would be of interest. Another important caveat is that while our data demonstrates that early glyburide administration is detrimental, it does not provide evidence that delayed treatment is beneficial per se. Rather, delayed treatment has an analogous outcome to WT. Based on the data provided herein, we can currently only conclude that delayed treatment is less detrimental compared to early treatment. We primarily utilized this model as a further indication that acute inflammation is necessary for normal wound healing. In this study, we establish a link between postD315X Xburn inflammation and wound healing. To our knowledge, this is the first study connecting NLRP3 inflammasome, macrophage recruitment and polarization, and wound healing in burns. Here, we highlight the importance of the inflammatory response in promoting postD316Xburn X recovery. Specifically, we demonstrate the necessity of acute inflammation, which is activated in response to microbial invasion or tissue injury, versus chronic hyperinflammation. Hyperinflammation is often seen in various conditions such as cancer, diabetes, and burns and is a maladaptive response that contributes to poor patient outcomes.33 Extending our

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murine results to humans, our data suggests that drugs that mitigate inflammation (e.g. glyburide) should not be prescribed during the acute phase. However, regulating inflammation beyond the acute phase is an important therapeutic strategy. Potentially, delayed inhibition could prevent the development of hyperproliferative conditions without compromising wound healing. However, this is speculative, and we cannot exclude the fact that regulating NLRP3 during the acute phase may be necessary in these cases. Therefore, while we postulate that delayed inhibition of inflammation with glyburide may be beneficial in hyperproliferative conditions, early partial NLRP3 inhibition is a potential alternative. Although previous work has shown increased postDburn 317X X mortality in NLRP3 / , partial inhibition by heterozygous knockouts or lower doses of glyburide administered throughout the postburn phase may be beneficial in severe burn models.14 However, further murine studies investigating partial NLRP3 inhibition and human studies utilizing delayed or low dose glyburide and wound healing outcomes are needed at this point.

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ACKNOWLEDGDMENTS 319X X

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We would like to thank Dr. Mile Stanojcic for his advice and assistance. This work was supported by grants from the Canadian Institutes of Health Research (#123336), the Canada Foundation for InnovationX X Lead- Q5 er’s Opportunity Fund (#25407) and National Institutes of Health (2R01GM087285-05A1). All authors have read the journal’s policy on disclosure of conflicts of interest and authorship agreement. There are no conflicts of interest.

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RV performed experiments and wrote portions of the manuscript; AA wrote portions of the manuscript; DB wrote portions of the manuscript; MJ guided experiments and wrote portions of the manuscript.

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COMPETING FINANCIAL D321X X D32X X INTERESTS STATEMENT

The authors have no competing financial interests to declare.

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Supplementary material associated with this article can be found in the online version at doi:10.1016/j. trsl.2019.11.002.

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REFERENCES

1. Eming SA, Krieg T, Davidson JM. Inflammation in wound repair: molecular and cellular mechanisms. J Investig Dermatol 2007;127:514–25. 2. Yousuf Y, Amini-Nik S. The role of myeloid lineage cells on skin healing and skin regeneration. J Tissue Sci Eng 2017;8. 3. Shao B-Z, Zhe-Qi X, Bin-Ze H, Ding-Feng S. NLRP3 inflammasome and its inhibitors: a review. Front Pharmacol 2015;6:262. 4. Zambetti LP, Laudisi F, Licandro G, Ricciardi-Castagnoli P, Mortellaro A. The rhapsody of NLRPs: master players of inflammation. . .and a lot more. Immunol Res 2012;53:78–90. 5. Stanojcic M, et al. Leukocyte infiltration and activation of the NLRP3 Inflammasome in white adipose tissue following thermal injury. Crit Care Med 2014;42:1357–64. 6. Ito H, Kanbe A, Sakai H, Seishima M. Activation of NLRP3 signalling accelerates skin wound healing. Experimental Dermatology 2018;27:80–6. 7. Weinheimer-Haus EW, Mirza RE, Koh TJ. Nod-like receptor protein-3 inflammasome plays and important role during early stages of wound healing. PLos One 2015;10:e0119106. 8. Jetten N, Roumans N, Gijbels MJ, et al. Wound administration of M2-polarized macrophages does not improve murine cutaneous healing responses. PLoS One 2014;9:e102994. 9. Mirza R, DiPietro LA, Koh TJ. Selective and specific macrophage ablation is detrimental to wound healing in mice. Am J Patho 2009;175:2454–62. 10. Atri C, Guerfali FZ, Laouini D. Role of human macrophage polarization in inflammation during infectious diseases. Int J Mol Sci 2018;19:1801. 11. Novak ML, Koh TJ. Macrophage phenotypes during tissue repair. J Leukoc Biol 2013;93:875–81. 12. Okizaki S, et al. Suppressed recruitment of alternatively activated macrophages reduces TGF-b1 and impairs wound healing in streptozotocin-induced diabetic mice. Biomed Pharmacother 2015;70:317–25. 13. Sindrilaru A, et al. An unrestrained proinflammatory M1 macrophage population induced by iron impairs wound healing in humans and mice. J Clin Investig 2011;121:985–97. 14. Vinaik R, Stanojcic M, Jeschke MG. NLRP3 inflammasome modulates post-burn lipolysis and hepatic fat infiltration via fatty acid synthase. Scientific Reports 2018;8:15197. 15. Koh GCKW, et al. Glyburide reduces bacterial dissemination in a mouse model of melioidosis. PLoS One 2013;7:e2500. 16. Satoh T, Kambe N, Matsue H. NLRP3 activation induces ASCdependent programmed necrotic cell death, which leads to neutrophilic inflammation. Cell Death Dis 2013;4:644.

Translational Research && 2019

17. Lukens JR, et al. Critical role for inflammasome-independent IL1b production in osteomyelitis. PNAS 2014;111:1066–71. 18. Gaidt MM, et al. Human monocytes engage an alternative inflammasome pathway. Immunity 2016;44:833–46. 19. Krzyszczyk P, Schloss R, Palmer A, Berthiaume F. The role of macrophages in acute and chronic wound healing and interventions to promote pro-wound healing phenotypes. Front Physiol 2018;9:419. 20. Jablonski KA, et al. Novel markers to delineate murine M1 and M2 macrophages. PLoS One 2015;10:e0145342. 21. Liu C, et al. Targeting the Shift from M1 to M2 macrophages in experimental autoimmune encephalomyelitis mice treated with fasudil. PLoS One 2013;8:e54841. 22. Artlett CM, Thacker JD. Molecular activation of the NLRP3 Inflammasome in fibrosis: common threads linking divergent fibrogenic diseases. Antioxid Redox Signal 2015;22:1162–75. 23. Funes SC, Rios M, Escobar-Vera J, Kalergis AM. Implications of macrophage polarization in autoimmunity. Immunology 2018;154:186–95. 24. Zhong A, et al. S100A8 and S100A9 are induced by decreased hydration in the epidermis and promote fibroblast activation and fibrosis in the Dermis. Am J Pathol 2016;186:109–22. 25. Barrientos S, Stojadinovic O, Golinko MS, Brem H, TomicCanic M. Growth factors and cytokines in wound healing. Wound Repair Regen 2008;16:585–601. 26. Artlett CM. Inflammasomes in wound healing and fibrosis. JD32X X PatholD324X 2X 013;229:157–67. 27. Wynn TA, Barron L. Macrophages: master regulators of inflammation and fibrosis. Semin Liver Dis 2010;30:245–57. 28. Rodero MP, Khosrotehrani K. Skin wound healing modulation by macrophages. Int J Clin Exp Pathol 2010;3:643–53. 29. Ogle ME, Segar CE, Sridhar S, Botchwey EA. Monocytes and macrophages in tissue repair: implications for immunoregenerative biomaterial design. Exp Biol Med 2016;241:1084–97. 30. Gurtner GC, Werner S, Barrandon Y, Longaker MT. Wound repair and regeneration. Nature 2008;453:314–21. 31. Wynn TA, Vannella KM. Macrophages in tissue repair, regeneration, and fibrosis. Immunity 2016;44:450–62. 32. Zhang G, Lin X, Zhang S, Xiu H, Pan C, Cui W. A protective role of glibenclamide in inflammation-associated injury. Mediators Inflamm 2017;2017:3578702. 33. Lamkanfi, M, Mueller, JL, Vitari, AC, Misaghi, S, Fedorova, A, Deshayes, K, Lee, WP, Hoffman, HM, & Dixit, VM. Glyburide inhibits the Cryopyrin/Nalp3 inflammasome. J Cell Biol; 187(1): 61-70. 34. Liu Y-Z, Wang Y-X, Jiang C-L. Inflammation: the common pathway of stress-related diseases. Front Hum Neurosci 2017;11:316.

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