Histone Methylation Directs Myeloid TLR4 Expression and Regulates Wound Healing following Cutaneous Tissue Injury

This article is featured in In This Issue, p.1647

Wound healing is a complex, but well-orchestrated, biological event with interplay between a number of resident and infiltrating cell types (1, 2). The recruitment of circulating blood myeloid cells to the site of tissue injury plays an important role in tissue repair. These recruited myeloid cells are critical for the regulated inflammatory response that is necessary for progression through the healing cascade. The precise timing of both the initiation and resolution of inflammation is essential for restoring tissue integrity. The first phase of the inflammatory response is destructive to the tissue and promotes clearance of invading pathogens, whereas the second phase is a resolution phase in which tissue repair ensues (3, 4). For this reason, inflammation is an adaptive process that is necessary to maintain tissue homeostasis (5). In the absence of precise, programmed inflammation, pathologic nonhealing ensues. A common characteristic of poorly healing wounds is an impaired initial immune response to injury and/or a sustained period of inflammation. During the first part of the inflammatory phase of wound healing, myeloid cells, particularly monocytes/macrophages, exist in a proinflammatory state in which they release inflammatory cytokines and mediators, recruit leukocytes, and promote tissue and pathogen destruction (6). After this early inflammatory phase, macrophages undergo a phenotype switch and begin secreting anti-inflammatory mediators as well as growth factors to promote tissue repair and wound resolution (7).

Accumulating evidence suggests that epigenetic regulation of gene expression influences immune cell phenotypes (8, 9). At present, a paucity of data exists on epigenetic-based mechanisms that regulate wound myeloid cell plasticity. Mixed-lineage leukemia 1 (MLL1) is a histone methyltransferase with site specificity for lysine 4 on histone 3 (H3K4) (10, 11). H3K4 trimethylation (H3K4me3) of gene promoter regions is associated with active gene expression (12). In mammals, H3K4me3 is controlled by the SET1/MLL family of enzymes (13). Although the role of MLL1 in oncogenesis has been investigated, few studies have examined the role of MLL1 in innate immunity (10, 14, 15). We have recently identified that MLL1 may regulate macrophage cytokine expression; however, the role of MLL1 in regulating upstream signaling pathways remains poorly defined (16).

One receptor-signaling pathway that has been shown to be instrumental in the regulation of innate immunity, specifically macrophages, neutrophils, and dendritic cells, are the TLRs. TLRs are a family of evolutionarily conserved receptors, which have a key role in host defense by regulating both innate and adaptive immune responses (17). TLR2 recognizes the peptidoglycan and lipopeptide in the cell walls of Gram-positive bacteria, whereas TLR4 recognizes LPS, which is an integral component of the outer membranes of Gram-negative bacteria. Importantly, Gram-negative bacteria are common organisms found in diabetic wounds (18). Following ligand binding, TLR4 elicits immune response through coupling with intracellular adapter proteins, including MyD88, MyD88 adaptor–like (Mal), TIR domain–containing adaptor inducing IFN-β (TRIF), and TRIF-related adaptor molecule (TRAM). Ultimately, TLR signaling pathways regulate gene expression of cytokines, costimulatory molecules, and adhesion molecules. Recent studies suggest that TLR4 plays an important role in sterile inflammation, tissue repair, and response to a variety of injuries. For example, studies in animal models have demonstrated that TLR4 plays key roles during inflammation following ischemia/reperfusion injury, neurodegenerative disease, and thermal injury (19–21). Despite the importance of TLR4 in the regulation of cytokines, there remains a paucity of data on the role of TLR4 and MyD88 in cutaneous wound healing. Of the limited literature, it focuses primarily on early wound healing in keratinocytes (22), and thus, the in vivo role of TLR4 in myeloid cells during the course of healing remains unknown.

Given the importance of TLR4 on immune cell function, particularly macrophage function, we investigated the role of MLL1 in regulating TLR4/MyD88 in cutaneous wound healing. In this study, we show that MLL1 (and hence, H3K4me3), directs dynamic Tlr4 expression in wound myeloid cells during the course of normal wound healing. Using TLR4 knockout (Tlr4^−/−) and MyD88 knockout (Myd88^−/−) mice, we demonstrate that TLR4 and MyD88 signaling are critical for the inflammatory response and that the absence of TLR4 or MyD88 decreases the necessary early inflammatory cytokine response and impairs wound healing. Further, using a myeloid-specific TLR4 knockout (Tlr4^f/fLyz2^Cre+), we demonstrate that myeloid-specific TLR4 is necessary for an adequate early inflammatory response in normal wound healing. Finally, early adoptive transfer of wild-type monocytes/macrophages after tissue injury was sufficient to rescue wound healing with restoration of the inflammatory response. Taken together, our findings suggest that MLL1 regulates Tlr4 expression during normal tissue repair and that TLR4/MyD88 signaling plays an integral role in myeloid cell–mediated inflammation during wound repair.

Mice were maintained in the University of Michigan pathogen-free animal facility, and all protocols were approved by and in accordance with the guidelines established by the Institutional Animal Care and Use Committee. Male and female C57BL/6 (Tlr4^+/+), CD45.1, CD45.2, Tlr4^−/−, and Myd88^−/− mice maintained on a normal chow diet (13.5% kcal fat; LabDiet) were purchased at 20 wk from The Jackson Laboratory (Bar Harbor, ME). Mice with the Mll1 or Tlr4 gene deleted in myeloid cells were generated by mating Mll1^f/f (16) or Tlr4^f/f (kind gift from T. Billiar, University of Pittsburgh) mice with LysM-Cre mice (The Jackson Laboratory). Animals underwent all procedures at 20–24 wk of age. Body weights were determined prior to experimentation.

Before wounding, mice were anesthetized, hair was removed with Veet (Reckitt Benckiser), and skin was cleaned with sterile water. Full-thickness back wounds were created by 4-mm punch biopsy with or without wound splinting as previously described (8, 23). Initial wound surface area was recorded, and digital photographs were obtained daily using an Olympus digital camera. Photographs contained an internal scale to allow for standard measurement calibration. Wound area was quantified using ImageJ software (National Institutes of Health, Bethesda, MD) and was expressed as the percentage of original wound size over time.

On day 3 postwounding, whole wounds were excised using a 6-mm punch biopsy. Wound sections were fixed in 10% formalin overnight before embedding in paraffin. Five-micrometer sections were stained with H&E for evaluation of re-epithelialization and with Masson’s Trichrome stain for collagen deposition. Images were captured using Olympus BX43 microscope and Olympus cellSens Dimension software. Percent re-epithelialization was calculated by measuring distance traveled by epithelial tongues on both sides of wound divided by total distance needed for full re-epithelialization.

Wounds were collected from the backs of the mice postmortem following CO₂ asphyxiation using a 6-mm wound biopsy. Sharp scissors were used to excise the full-thickness dermis with a 1–2-mm margin around the wound, ensuring collection of granulation tissue and wounds were placed in RPMI 1640. Wounds were then carefully minced with sharp scissors and digested by incubating in a 50-mg/ml Liberase TM (Roche) and 20-U/ml DNase I (Sigma-Aldrich) solution. Wound cell suspensions were then gently plunged and filtered through a 100-μm filter to yield a single-cell suspension. Cells were then either MACS for CD3⁻, CD19⁻, Ly6G⁻, and CD11b⁺ cells for RNA studies or cultured ex vivo for application of GolgiStop and subsequent staining for intracellular flow cytometry (24).

Wounds were digested as described above. Single-cell suspensions were incubated with FITC-labeled anti-CD3, anti-CD19, and anti-Ly6G (BioLegend), followed by Anti-FITC MicroBeads (Miltenyi Biotec). Flowthrough was then incubated with anti-CD11b MicroBeads (Miltenyi Biotec) to isolate the nonneutrophil, nonlymphocyte, and CD11b⁺ cells. Cells were saved in TRIzol (Invitrogen) for quantitative RT-PCR analyses.

Chromatin immunoprecipitation assay was performed as described previously (9). Briefly, cells fixed in paraformaldehyde were lysed and sonicated to generate 100–300-bp fragments. To immunoprecipitate, samples were incubated in anti-H3K4me3 Ab (Abcam) or isotype control (rabbit polyclonal IgG) (MilliporeSigma) in parallel samples overnight, followed by addition of Protein A Sepharose beads (Thermo Fisher Scientific). Bound DNA was eluted and purified using phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation. Primers were designed using the Ensembl genome browser to search the TLR4 promoter, and then, National Center for Biotechnology Information Primer-BLAST was used to design primers that flank this site.

CD3⁻CD11c⁻CD19⁻Ly6G⁻NK1.1⁻CD11b⁺ single-cell suspensions were isolated by MACS from spleens of Tlr4^−/− and Tlr4^+/+ mice as described above. One million cells were injected i.v. via tail vein into wounded mice within 2 h of wounding. Wound healing was monitored over time, and wound area was calculated using National Institute of Heath ImageJ software (Bethesda, MD). Initial wound size was calculated immediately after wounding, and wound closure was assessed over time as a percent of initial wound area.

Single-cell suspensions were collected and washed two times with cold PBS and filtered into a 96-well plate for surface staining. Cells were initially stained with pacific orange LIVE/DEAD Fixable Viability Dye (Thermo Fisher Scientific) and then washed two times with cold PBS. Cells were then resuspended in Flow Buffer (PBS, FBS, NaN3, and HEPES buffer) and Fc receptors were blocked with anti-CD16/32 (BioLegend) prior to surface staining. Biotinylated mAbs used for surface staining included the following: anti-CD3, anti-CD19, anti-CD45.1, anti-CD45.2, anti–Ter-119, anti-NK1.1, anti-CD11b, anti-Ly6G, and anti-Ly6C (BioLegend). Following surface staining, cells were washed twice, and biotinylated Abs were labeled with streptavidin APC/Cy7. Next, cells were either washed and acquired for surface-only flow cytometry or were fixed with 2% formaldehyde and then washed/permeabilized with BD Perm/Wash buffer (BD Biosciences) for intracellular flow cytometry. After permeabilization, intracellular stains included the following: anti–IL-1β (mature IL-1β; BD Biosciences), anti–TNF-α (BioLegend), and inducible NO synthase (Affymetrix). After washing, samples were then acquired on a three-laser NovoCyte Flow Cytometer (ACEA Biosciences). Data were analyzed using FlowJo software version 10.0 (Tree Star) and data were compiled using Prism software (GraphPad). To verify gating and purity, all populations were routinely back-gated.

Bone marrow (BM) cells were collected by flushing mouse femurs and tibias with RPMI 1640. BM-derived macrophages (BMDMs) were cultured as previously detailed (9). On day 6, the cells were replated, and after resting for 24 h, they were incubated with or without LPS (100 ng/ml; Sigma-Aldrich [L2880] purified by phenol extraction <3% impurities) for 2–6 h; after which, cells were placed in TRIzol (Invitrogen) for RNA analysis.

Total RNA extraction was performed using TRIzol (Invitrogen) according to the manufacturer’s instructions. RNA was then reverse transcribed to cDNA using iScript (Bio-Rad Laboratories). PCR was performed with 2× TaqMan PCR Mix using the 7500 Real-Time PCR System. Primers for Il1b (Mm00434228_m10), Tnfa (Mm00443258_m1), Mll1 (Mm01179235_m1), Cd44 (Mm0 1277164-m1), and Tlr4 (Mm00445273_m1) were purchased (Applied Biosystems). For internal control, 18S rRNA was used. Data were then analyzed relative to 18S rRNA (2Δ cycle threshold). All samples were assayed in triplicate. The threshold cycle values were used to plot a standard curve. Data were compiled in Microsoft Excel and presented using Prism software (GraphPad).

Data were analyzed using GraphPad Prism software version 6. We expressed the results as means ± SEM. The statistical significance of differences between two groups was determined using Student t tests for data that passed a normality test; otherwise, a nonparametric Mann–Whitney U test was used. Differences between more than two groups were evaluated by one-way ANOVA, followed by post hoc analysis (Bonferroni test) for data that passed tests for normality and equal variance (Bartlett test); otherwise, a nonparametric Kruskal–Wallis test, followed by Dunn post hoc analysis was used. All p values <0.05 were considered significant.

Increasing evidence suggests that proper wound healing requires the establishment of a regulated inflammatory response mediated by macrophages (8, 25, 26). The mechanisms responsible for macrophage phenotype in wound repair are incompletely understood. TLR4 is a major receptor that initiates a downstream signaling cascade that promotes inflammation, mostly through MyD88-dependent pathway and NF-κB expression. Further, TLR4 has been shown to play a vital role in the innate immune response to various sterile injuries. To examine the role of TLR4 in vivo in myeloid cells during wound repair, C57BL/6 mice were subjected to 4-mm, full-thickness wounds as previously described (8), and myeloid cells (CD11b⁺[CD3⁻CD19⁻Ly6G⁻]) were isolated from wounds by cell sorting at early time points (days 1–5) postinjury. Expression of TLR4 was significantly upregulated in early wound myeloid cells after injury (Fig. 1A).

Increasingly, evidence suggests that epigenetic regulation (e.g., DNA methylation or histone modification) of gene expression plays a key role in influencing inflammatory phenotypes (8, 27). In addition, previous studies have suggested that the histone methyltransferase MLL1 drives inflammatory gene expression in myeloid cells (16, 28). To evaluate if the increased TLR4 expression in wound myeloid cells is due to epigenetic regulation of the TLR4 gene, we examined several histone methylation marks associated with gene activation. We found that H3K4me3 was significantly increased on the Tlr4 promoter in wound myeloid cells on day 5 as compared with earlier time points (day 2) (Fig. 1B). The H3K4me3 methylation mark maintains the chromatin in a conformation so specific genes are effectively activated. Because the methyltransferase, MLL1, specifically methylates H3K4 (12, 29), we examined the expression of Mll1 and found it significantly increased on day 5 following tissue injury, which corresponds to the increased TLR4 levels (Fig. 1C). We have previously shown that myeloid-specific deficiency of Mll1 results in impaired cutaneous wound healing (16). To evaluate the ability of MLL1 to regulate Tlr4 expression, we generated mice deficient in Mll1 in cells of the myeloid lineage with lysosomes (monocytes, macrophages, and granulocytes) by using the Cre/lox system. Myeloid-specific depletion of Mll1 was confirmed by examining MACS splenic monocyte/macrophages from Mll1^f/fLyz2^Cre+ mice and littermate controls (Mll1^f/fLyz2^Cre−) (Fig. 1D). To determine whether Mll1 alters Tlr4 expression, myeloid cells were isolated on day 5 postwounding from Mll1^f/fLyz2^Cre+ mice and littermate controls. Mll1-deficient wound myeloid cells demonstrated significant decrease in Tlr4 expression in the Mll1^f/fLyz2^Cre+ compared with littermate controls (Fig. 1E). These data suggest MLL1-derived H3K4me3 methylation may increase Tlr4 gene expression in wound myeloid cells following injury and that this may control, at least in part, the regulated inflammatory response during tissue repair.

Given the dynamic changes in TLR4 expression in wound myeloid cells during healing, we examined if TLR4 is critical for cutaneous wound repair. To determine if the early increase in TLR4 in wound macrophages is necessary for healing, we wounded Tlr4^−/− and control mice and monitored healing daily. Tlr4^−/− mice had impaired healing throughout the entire wound course compared with controls (Fig. 2A, 2B). When we examined wounds with histology, Tlr4^−/− mice demonstrated impaired epithelialization and decreased collagen content compared with controls (Fig. 2C). There was no difference in the expression of other TLRs or the CD44 coreceptor in Tlr4^−/− and control mice (Supplemental Fig. 1A–C). In this study, these findings suggest that upregulation of TLR4 is necessary for normal wound closure.

Because TLR4 is upregulated in wound myeloid cells and TLR4 is associated with NF-κB–mediated initiation of the inflammatory response, we examined the role in vivo of TLR4 in wound myeloid cell–mediated inflammation (30). This is important because it is well established that regulated inflammation is critical for tissue repair (7, 31). To study this first in vitro, BMDMs from Tlr4^−/−and control mice were stimulated with LPS and analyzed for expression of inflammatory genes known to be important in wound healing, including Ilb and Tnfa (8). This demonstrated significantly decreased Il1b and Tnfa expression in the Tlr4^−/− BMDMs compared with controls (Fig. 3A). To determine whether the in vitro findings translate in vivo, we examined both inflammatory gene expression and cytokine production by quantitative PCR (qPCR) and flow cytometry, respectively, on wound myeloid cells (live, lineage⁻, Ly6G⁻, and CD11b⁺) isolated from Tlr4^−/− and control wounds. We examined day 3 postwounding to allow circulating monocytes adequate time to enter the tissues, transform into macrophages or dendritic cells, and assume a functional role (32, 33). Previous studies suggest that recruited inflammatory cell numbers are at their highest levels on day 3 postwounding (34). There was no difference in the CD11b⁺ cells or neutrophils present within wound tissue on day 3 in Tlr4^−/− and control wounds (Supplemental Fig. 2A, 2B). However, examination of inflammatory cytokines known to play a major role in healing demonstrated that Il1b and Tnfa were significantly reduced at both the gene expression and protein level in the Tlr4^−/− wound myeloid cells (Fig. 3B–D). Taken together, these results suggest that TLR4 plays an important role in initiating the early inflammatory response critical for normal healing.

In response to ligand stimulation, TLR4 triggers downstream signaling via both MyD88-dependent and MyD88-independent pathways (35). The MyD88-dependent pathway ultimately leads to the activation of NF-κB and transcription of prominent inflammatory genes (36). To determine if this downstream pathway is instrumental to impaired wound healing in TLR4 deficiency, we wounded Myd88^−/− and control mice and monitored healing daily. Myd88^−/− mice had impaired healing compared with controls throughout the wound course consistent with Tlr4^−/−mice (Fig. 4A). We also examined sorted wound myeloid cells (CD3⁻CD19⁻Ly6G⁻CD11b⁺) and found that Il1b and Tnfa were significantly reduced in Myd88^−/− wound myeloid cells in comparison with controls (Fig. 4B). These findings suggest that upregulation of the TLR4/MyD88 signaling pathway is necessary for early inflammation and normal wound closure.

To confirm the importance of myeloid-specific TLR4 in cutaneous wound healing, we generated mice deficient in Tlr4 in cells of the myeloid lineage with lysosomes (monocytes, macrophages, and granulocytes) by using the Cre/lox system. Myeloid-specific depletion of Tlr4 was confirmed by examining MACS splenic monocyte/macrophages from Tlr4^f/fLyz2^Cre+ mice and littermate controls (Tlr4^f/fLyz2^Cre−) (Fig. 5A). Wounds were generated in the Tlr4^f/fLyz2^Cre+ mice and their littermate controls, and wound closure was analyzed daily. Wound closure was markedly delayed at early time points in the Tlr4^f/fLyz2^Cre+ mice (Fig. 5B). Additionally, to determine if TLR4⁺ monocytes/macrophages can restore healing, we performed an congenic adoptive transfer. Monocyte/macrophages (CD3⁻, CD19⁻, Ly6G⁻, NK1.1⁻, CD11c⁻, and CD11b⁺) were isolated from Tlr4^+/+ mice expressing CD45.1 and injected into the peripheral blood of wounded (day 1) Tlr4^−/− mice expression CD45.2. To confirm the transferred monocytes appropriately tracked to the cutaneous wound, on day 4 postinjury (day 3 posttransfer), wound myeloid cells (CD3⁻CD19⁻Ly6G⁻CD11b⁺) were analyzed for CD45.1 and CD45.2 expression. A marked percentage of wound myeloid cells was found to express CD45.1 (Fig. 5C), indicating transferred myeloid cells appropriately tracked to the cutaneous wound in agreement with our previous publications (16, 37, 38). To confirm these cells were functional, we also analyzed cytokine expression and found Tlr4^+/+→Tlr4^−/− wound myeloid cells displayed increased cytokine expression in comparison with Tlr4^−/−→Tlr4^−/− (Fig. 5D). Importantly, wound healing was significantly improved in Tlr4^+/+→Tlr4^−/− in comparison with Tlr4^+/+→Tlr4^−/− (Fig. 5E). These results suggest that myeloid-specific TLR4 was sufficient to partially rescue wound healing in these mice, likely by restoring the initial inflammatory phase necessary for proper wound healing (Fig. 6).

In this study, we identify that TLR4 expression is significantly elevated in tissue myeloid cells following cutaneous tissue injury and remains elevated during the wound healing course. The increased TLR4 expression is, in part, due to increased expression of the histone methyltransferase, MLL1, and its resulting methylation at the activating H3K4 site on the TLR4 promoter. Additionally, our results show that global TLR4 and MyD88 deficiency, as well as myeloid-specific TL4 deficiency, results in impaired wound healing exhibiting decreased expression of well-established proinflammatory mediators that are critical for normal tissue repair. Furthermore, when myeloid-specific TLR4 was replenished via adoptive transfer, wound healing was restored, suggesting that myeloid-specific TLR4 was both necessary and sufficient to partially rescue wound healing (Fig. 6).

The role of TLR4 in wound healing has previously been investigated in other models, but these have failed to identify regulators of the TLR4 expression following tissue injury and lack the importance of cell specificity with downstream TLR4 signaling pathways. Within the context of thermal burn injury, TLR4 has been shown to provide an important role in leukocyte adhesion and cytokine release (39). Similarly within diabetes, TLR4 expression and signaling are significantly increased in diabetic patients and db/db mice (40). Knockdown of TLR4 in diabetic mice resulted in attenuated inflammation as measured by circulating chemokines and improved wound healing, suggesting that sustained TLR4 activation may be detrimental in diabetic wounds (41, 42). In contrast to the detrimental effects of TLR4 in diabetic wound healing, in nondiabetic wound healing, TLR4 signaling may be instrumental in the early phase, as demonstrated in our current study, with TLR4-deficient mice displaying markedly prolonged wound healing associated with decreased inflammatory gene expression. Previous work has demonstrated that wounds in Tlr2^−/−, Tlr4^−/−, and double-knockout Tlr2^−/−/Tlr4^−/− mice exhibited attenuated healing and decreased global wound Tgfβ and Ccl5 expression relative to wild-type animals (43). However, the dynamic epigenetic regulation of TLR4 expression, as well as cell specificity, has not previously been examined. Additionally, other studies have not interrogated the specific contributions of downstream adapter proteins, such as MyD88-dependent signaling, and their contribution to inflammation and wound healing. MyD88 is the most common adaptor molecule involved in most TLR signaling, with the exception of TLR3, which uses TRIF/TRAM. However, TLR4 has also been shown to signal through noncanonical intracellular pathways, which may partially explain why the MyD88-deficient mice had a less robust delay in wound closure in comparison with the TLR4 knockout. Indeed, there is growing evidence of “noncanonical” TLR4 signaling effectors (e.g., phosphatase and tensin homolog and integrins and the epidermal growth factor receptor) as important downstream participants (44).

Another murine model that has been recently used is the C3H/HeJ mice that have a genetic deficiency of TLR4. This model demonstrated delayed wound healing accompanied by elevated global levels of Tlr2, Tgfβ, and fibrosis in the wounded skin (22). Although the findings in this study corroborate our own findings regarding the importance of TLR4 in wound healing, the Ch3H/HeJ represents an imprecise system to analyze the sole effect of TLR4 (45, 46). The C3H/HeJ mice have a spontaneous mutation resulting in loss of the TLR4 gene but also have chromosomal inversion of chromosome 6, which could lead to unknown abnormalities other than TLR4. Despite the lack of exogenous mouse mammary tumor virus, virgin and breeding females may still develop some mammary tumors later in life. Thus, C3H/HeJ mice have several immunological abnormalities that may affect the function of TLR4.

The precise timing of both the initiation and resolution of inflammation is essential for restoring tissue integrity following injury. The first phase of wound healing is an inflammatory response that is characterized by tissue destruction and clearance of invading pathogens. In contrast, the second phase involves resolution of inflammation and tissue repair (3, 4). Healing of cutaneous wounds is a complex biological event that results from the interplay of a large number of resident cells (fibroblasts and keratinocytes) as well as infiltrating cell types, including leukocytes, monocytes/macrophages, and dendritic cells. Given the influential role of TLRs in wound healing, studies have attempted to identify the specific cell type expressing TLR4 vital for wound repair. However, this has been complicated as there is a lack of consensus in distinguishing monocytes, dendritic cells, and macrophages within the skin during periods of wound healing using surface markers (47, 48). Previous studies have investigated the role of TLR4 in wound healing but have focused on fibroblasts and CD19 B lymphocytes cells (22, 49). Within the current study, through a novel myeloid-specific, TLR4-deficient murine model and adoptive transfer, we demonstrated that myeloid-specific TLR4 and its downstream MyD88 signaling is necessary for sterile wound healing. The importance of myeloid-specific TLR4 is likely driven by the notion that the TLR4 pathway is critical for the initial inflammatory phase of wound healing, as myeloid cells are predominately responsible for initial cytokine release during the first phase of wound healing and, in turn, play a key role in the orchestration of subsequent phases (7, 31).

Accumulating evidence suggests that epigenetic regulation of gene expression influences immune cell phenotypes within both disease states, such as diabetes, as well as the normal response to injury (8, 9, 16). At present, a paucity of data exists on epigenetic-based mechanisms that regulate wound macrophage plasticity. Within the current study, we demonstrate that wound myeloid cells display increased expression of Mll1 and increased methylation at H3K4 of the TLR4 promoter, resulting in dynamic TLR4 expression during the wound healing course. The dependence of TLR4 on MLL1 was further supported when analyzing mice deficient in MLL1 in the myeloid-specific lineage (Mll1^f/fLyz2^Cre+), in which wounds from these mice showed significantly decreased Tlr4 expression. H3K4me3 of gene promoter regions is associated with active gene expression (12), and we have previously shown that H3K4me3 is involved in the regulation of inflammatory cytokine production in diabetic wound macrophages through the actions of the epigenetic methyltransferase MLL1 (16). The dynamic epigenetic regulation of TLR4 is important, as previous studies have shown that immune cell phenotypes are continuing evolving during the course of wound healing, and aberrances in this process can lead to delayed tissue repair (37). The role of epigenetic modifications on TLRs has previously been investigated in the regulation of TLR2 within diabetic wound healing, demonstrating that altered CpG methylation status on the TLR2 promoter may correlate with diabetic foot ulcer severity (50). However, to date, no studies have investigated the role of epigenetic modification of the TLR4 promoter pathway in nondiabetic wound healing.

Although this study produces insight into the mechanism behind TLR4 and myeloid inflammation in cutaneous wound healing, some limitations must be addressed. Myeloid cells play an important role in tissue repair following injury; however, there is evidence that TLR4 is also expressed in keratinocytes, fibroblasts, and B cells (49, 51). This may partially explain why the adoptive transfer of myeloid-specific TLR4 cells partially restores wound healing, as TLR4 may also regulate other cell types, including epithelial cells. Further, there are multiple ligands that stimulate a TLR4-dependent response, and at this time, we are unable to determine which specific ligand is involved during cutaneous injury, but it likely represents a combination of ligands, including hyaluron and skin microbiota. Last, although H3K4me3 suggests a potential mechanism for increased TLR4 expression in wound macrophage, we acknowledge that other epigenetic modifications may play a role in TLR4 production. Indeed, other epigenetic enzymes have been shown to play a role in aberrant myeloid cell function in pathological states (50, 52, 53). Thus, further studies assessing the role of other specific epigenetic enzymes in the regulation of TLR4 signaling would be useful.

In summary, we showed myeloid-specific TLR4 is important for normal inflammation during tissue repair and is epigenetically driven by MLL1. Activating the TLR4- and MyD88-specific signaling, at least early in the wound healing course, may be a reasonable therapeutic strategy for regulating the initial inflammatory response in pathologic conditions that impair wound healing.

We thank Robin Kunkel for her assistance with the graphical illustrations.

The authors have no financial conflicts of interest.