A surfactant polymer wound dressing protects human keratinocytes from inducible necroptosis

Chronic wounds show necroptosis from which keratinocytes must be protected to enable appropriate wound re-epithelialization and closure. Poloxamers, a class of synthetic triblock copolymers, are known to be effective against plasma membrane damage (PMD). The purpose of this study is to evaluate the efficacy of a specific poloxamer, surfactant polymer dressing (SPD), which is currently used clinically as wound care dressing, against PMD in keratinocytes. Triton X-100 (TX100) at sub-lytic concentrations caused PMD as demonstrated by the efflux of calcein and by the influx of propidium iodide and FM1-43. TX100, an inducer of necroptosis, led to mitochondrial fragmentation, depletion of nuclear HMGB1, and activation of signaling complex associated with necroptosis (i.e., activation of RIP3 and phosphorylation of MLKL). All responses following exposure of human keratinocytes to TX100 were attenuated by pre- or co-treatment with SPD (100 mg/ml). The activation and translocation of phospho-MLKL to the plasma membrane, taken together with depletion of nuclear HMGB1, characterized the observed cell death as necroptosis. Thus, our findings show that TX100-induced plasma membrane damage and death by necroptosis were both attenuated by SPD, allowing keratinocyte survival. The significance of such protective effects of SPD on keratinocytes in wound re-epithelialization and closure warrant further studies.

The chronic wound microenvironment is harsh often characterized by un-resolved inflammation, elevated protease activity, low pH, high peroxide levels and large masses of dead and dying cells 1-4 . Surgical debridement and negative pressure wound therapy are commonly used to cleanse the wound microenvironment 5-7 . After appropriate wound bed preparation 8 , wound dressings are intended to maintain favorable changes in the wound microenvironment such that the body's healing responses may be effective in closing the wound [9][10][11] . In a wound defect on the skin, keratinocytes are the primary cells that achieve wound closure by re-epithelialization 12 . Thus, keratinocyte survival and migration are critically required to achieve this primary endpoint of wound healing. Wound dressings favoring keratinocyte survival in a hostile wound microenvironment are therefore of extraordinary significance.
At the open chronic wound-site, death of skin cells may be caused by a multitude of chemical mediators and mechanical injury. The chemical mediators in the form of pore-forming agents released by microbes directly compromise the integrity of the plasma membrane, potentially causing electrolyte imbalance and inflammasome activation [13][14][15][16][17][18][19][20][21][22] . On the other hand, chemical mediators in the form of reactive oxygen species, DAMPs (damage-associated molecular patterns), and cytokines emerging from the activity of pro-inflammatory cells breach plasma membrane integrity 17,[23][24][25][26][27][28][29] . Such an insult is known to cause necroptosis (a programmed form of necrosis) in the chronic wound 27,30 . Previously, necrosis was considered as uncontrolled or accidental cell death caused by physical or chemical trauma. However, current evidence demonstrates necroptosis is regulated by intrinsic cellular proteins such as receptor interacting protein 3 (RIP3) and mixed lineage kinase domain-like (MLKL). Necroptosis is characterized by swelling and coalescing of intracellular organelles, loss of cellular ATP content, rupture of the plasma membrane, and subsequent leakage of cell contents 31,32 .
ing the influx of PI and efflux of calcein as measured by flow cytometry and confocal microscopy. An increase in uptake of PI and efflux of calcein are markers of cell death. Exposure to 0.1, 0.2, and 0.3 mM of TX100 for 3 h led to ~4%, ~60%, and ~85%, respectively (Fig. 1A,B) as measured by flow cytometry. The confocal images (Fig. 1C,D) supports the flow cytometry data. For further experiments, 0.2 mM of TX100 was chosen since this dose was sublytic and below the critical micellar concentration (CMC) of TX100. TX100-induced cell death was evident at 3 h, as observed by flow cytometry (Fig. 1E,F). Time-dependent exposure (3, 6, and 9 h) of TX100 did not increase the cell death (Fig. 1E,F). Triton X-100 induced cell death was blocked by pre-and co-treatment with SPD. Exposure to SPD alone was not toxic to cells at concentrations of up to 100 mg/ml (Fig. S1). To assess the efficacy of SPD against TX100-induced cell death, HaCaT cells were pre-treated with SPD at different concentrations (20,50, and 100 mg/ml) for 24 h and then exposed cells to TX100 in the continued presence of SPD. Data from flow cytometry ( Fig. 2A,B) and confocal microscopy (Fig. 2C,D) demonstrate that SPD consistently reduced cell death in response to 3 h exposure to TX100 at 0.2 mM. Similar findings were observed in human epidermal keratinocytes (HEKa) cells, where pre-treatment with SPD at a concentration of 100 mg/ml was able to reduce TX100-induced cell death (Fig. S2). In addition, HaCaT cells were co-treated with TX100 and SPD simultaneously (without pretreatment with SPD), and cell death was analyzed by flow cytometry and confocal microscopy. Co-treatment with SPD was effective against TX100-induced cell death ( Fig. 2E-H). To test if the protective efficacy of SPD over TX100-induced cell death persisted long-term, HaCaT cells were co-treated with TX100 and SPD (100 mg/ ml) for 3 h, washed off, and then subjected to analysis of cell death after 24 h. Interestingly, TX100-induced cell death was significantly less when co-treated with SPD ( Fig. 3A-D).
SPD maintained the integrity of the plasma membrane following exposure to Triton X-100. Exposure to TX100 resulted in intense staining in the cytoplasm, indicating movement of the dye (FM1-43) and subsequent intercalation into membranes of the cytoplasmic organelles (Fig. 4A,B). The accumulation was significantly attenuated in the presence of SPD (Fig. 4A,B). SPD reduced nuclear depletion of HMGB1 and mitochondrial fragmentation. High mobility group protein B1 (HMGB1) is a nuclear protein vital for the maintenance of several nuclear functions, including replication, recombination, transcription, and DNA repair 44,45 . Nuclear depletion of HMGB1 is a marker of necroptosis 46 . We sought to assess the nuclear abundance of HMGB1 post-insult. Immunocytochemistry with an antibody specific to HMGB1 was performed to assess its cellular localization. Nuclear depletion of HMGB1, a classical marker of necroptosis, was observed in response to TX100 (Fig. 5A-E, S3). In agreement with protection against cell death, SPD at 100 mg/ml prevented the nuclear depletion of HMGB1 ( Fig. 5A-E,  S3). Yet another important role ascribed to HMGB1 is in mitochondrial quality control 47 . Thus, we examined if the mitochondria are influenced concomitant with HMGB1 depletion. For this purpose, cells were treated with SPD and TX-100 for 1 h, followed by staining with MitoTrackerRedCMXRos. Exposure to TX100 at 0.2 mM led to a fragmentation of the tubular mitochondria (Fig. 6A,B). The reduced length of mitochondria in response to TX100 was blocked by co-treatment with SPD (Fig. 6A,B). Triton X-100 induced upregulation of RIP3 kinase as well as activation of MLKL. Among the patterns of cell death in response to TX100, an involvement of apoptosis, including the canonical pathways, has been questioned [48][49][50] . Since there is a potential for the release of DAMP (such as HMGB1), we examined programmed necrosis, i.e., necroptosis, following treatment with TX100. Here, we focused on the signaling complex upstream of necroptosis. As a first step, we tested RIP3 expression by immunocytochemistry after TX100 treatment. Exposure to TX100 for 3 h led to an increase of RIP3, which was attenuated by the co-treatment with SPD ( Fig. 7A,B). To test the role of MLKL, we performed immunocytochemistry of phospho-MLKL. MLKL was found to be phosphorylated upon treatment with TX100, which was significantly attenuated by co-treatment with SPD (Fig. 8A

Discussion
SPD is employed as a water-soluble burn wound dressing and is FDA cleared 51,52 . SPD is not only well tolerated by patients, but anecdotal clinical evidence show beneficial effects in wound healing 53,54 . It is therefore of extraordinary significance to understand the mechanism of action of SPD. Having a hydrophilic polymer chain, SPD can form a reservoir for topical hydrophilic antimicrobials agents 55 . SPD can be removed from wound beds easily compared to standard silver sulfadiazine creams on the burn wounds. They can effectively disrupt bacterial biofilm infection 35      www.nature.com/scientificreports/ form of cell death is evident at the site of chronic wounds 56 . The present work characterizes TX100 as a potent inducer of necroptosis in human keratinocytes. At dosages achievable at the wound site, where SPD is delivered topically, SPD prevented necroptosis induced by TX100. Any sparing of keratinocyte death at the hostile wound microenvironment is likely to facilitate wound-re-epithelialization and therefore closure. As strategies to induce reproducible PMD, many approaches have been reported [57][58][59][60][61][62] . They include (a) laserinduced membrane damage, (b) exposure to pore-forming pathogenic cytotoxins, (c) mechanical injury by glass beads, shear stress, cellular contraction, cell scraping, and forcing cells through a needle, and (d) exposure to detergents 63,64 . Of these methods, the pore-forming toxins and detergents are suitable for induction of PMD simultaneously in a large number of cells 63 , and hence are highly suitable for high-throughput screening of potential therapeutic agents. However, in the case of bacterial cytotoxins, a significant disadvantage is several toxins would be required to produce membrane pores of varying sizes. With detergents, on the other hand, the extent of PMD can be tuned by varying their concentration and time of exposure. These variables can easily be controlled during the conduct of experiments. Controlled exposure to TX100, a non-ionic detergent, is widely employed in cell biological applications 50,65,66 . The interactions of TX100 with the plasma membrane in other cell types have been partly characterized. In particular, at sub-lytic doses, TX100 is known to permeabilize the plasma membrane and cause cell death by mechanisms not yet established [48][49][50][65][66][67] . The sub-lytic dose, however, corresponds to levels below CMC, which is ~ 0.24 mM 48 . Because of their polar head group, TX100 molecules disrupt hydrogen bonding present within the cell's lipid bilayer and lead to the destruction of the compactness and integrity of lipid membrane 65 . Cells undergo irreversible permeabilization of the membrane and structural collapse when the TX100 concentration reached the CMC 65 . The action of TX100 also depends on the composition of the lipid bilayer 68 . TX100 action starts by destabilization of the lipid component of the membranes (noncooperative binding of detergents to the membrane to the cooperative binding). This leads to the formation of membrane fragments of proteins and lipids with detergent shielded edges 69 . The changes in plasma membrane permeability and fluidity also plays a critical role in psoriasis 70 .
The non-ionic detergent, TX100, is known to elicit plasma membrane damage (demonstrated usually by transiently increased membrane permeability) and subsequent cell death [48][49][50]66,67,71,72 . In some cells, TX100 induces apoptotic cell death [48][49][50]66,67 . Lipid homeostasis of the skin is unique such that lipid signaling is a key contributor to the turn-over of skin cells and the lipid composition of the skin contributes to its barrier function 73 . It is therefore not surprising that keratinocytes respond differently to detergents. For example, NP-40 is a non-ionic detergent like TX100 yet it induces necrosis in HaCaT keratinocytes 74 . Our choice to make use of TX100 to induce experimental PMD turned out to be a useful strategy to produce damage in a large number of cells simultaneously. At sub-lytic concentrations, TX100 produced transient membrane permeabilization without causing immediate cell death. Such membrane permeabilization is known to be a characteristic feature of necroptosis 29 . SPD, by itself, showed no cytotoxicity. When co-treated to cells and subsequently removed, SPD retained its cytoprotective properties indicating of successful cellular uptake by a magnitude that was functional relevant. Cytoprotective effects of SPD co-treated with TX100 demonstrated that SPD is not only safe on its own but that it did not chemically interact with TX100 in a harmful manner. FM1-43 is a fluorescent membrane probe 75 . It is not fluorescent in an aqueous medium. However, the dye becomes intensely fluorescent when it intercalates into the outer leaflet of the plasma membrane 75 . The fluorescence of FM1-43 is only at the membrane. An increase in fluorescence is an indicator of plasma membrane damage 76 . In keratinocytes, TX100 dependent increase in FM1-43 fluorescence establishes the ability of the detergent to cause PMD. PMD is known to occur during necroptosis 29 . This work is the first to recognize the value of FM1-43 as a productive approach to detect early necroptosis events. Understanding of mitochondrial changes during the course of necroptosis has led to the observation that the RIP1-RIP3 complex initiates mitochondrial fission 77 . Co-ordinated with DNA replication, mitochondrial fission divides the organelle. In a healthy cell, mitochondria exist as a dynamic network of fission and fusion 78 . During necroptosis, the balance favors fission and manifests as reduced mitochondrial length 79 . MitoTrackerRedCMXRos is a cationic dye, which accumulates in the mitochondrial membrane of living cells 80 . It is intrinsically fluorescent, binds irreversibly to the polarized mitochondrial membrane, and does not require reduction or oxidation for the emission of fluorescence 80 . Thus, it is well suited to study mitochondrial fission during necroptosis. TX100 dependent reduction in mitochondrial length, indicative of fission, was completely spared in the presence of SPD.

Figure 2.
Pre-and co-treatment of human keratinocytes with SPD protected against TX100-induced cell death. (A-D) Human keratinocytes (HaCaT) were pretreated with SPD (100 mg/ml) for 24 h and then exposed to TX100 (0.2 mM) for 3 h in the continued presence of SPD (100 mg/ml). (A,B) Cells were stained with PI for flow cytometry. Flow cytometry histograms and quantification of %PI-positive cells. Data represent mean ± SEM (n = 4). *p < 0.05 compared to control (TX100-untreated). † p < 0.05 compared to TX100-treated group. (C,D) Cells were stained with Calcein and PI for confocal microscopy. Representative images and quantification of %PI-positive cells are shown. Data represent mean ± SEM (n = 3-4). *p < 0.05 compared to control (TX100untreated). † p < 0.05 compared to TX100-treated group. (E-H) Human keratinocytes (HaCaT) were co-treated with SPD (100 mg/ml) and TX100 (0.2 mM) for 3 h. (E,F) Cells were stained with PI for flow cytometry. Flow cytometry histograms and quantification of %PI-positive cells. Data represent mean ± SEM (n = 4). *p < 0.05 compared to control (TX100-untreated). † p < 0.05 compared to TX100-treated group. (G, H) Cells were stained with Calcein and PI for confocal microscopy. Representative images and quantification of %PI-positive cells are shown. Scale, 10 µm. Data represent mean ± SEM (n = 3-4). *p < 0.05 compared to control (TX100-untreated). † p < 0.05 compared to TX100-treated group. www.nature.com/scientificreports/ HMGB1 is a ubiquitous nuclear protein, the release of which is a characteristic feature of necroptosis 46 . Nuclear HMGB1 is not only released as a DAMP but is also associated with mitochondrial quality control 47 . Loss of nuclear HMGB1 is known to cause mitochondrial fission 81,82 . In the nucleus, the cell-survival functions of HMGB1 are attributed to its ability to facilitate assembly of DNA binding proteins on chromatin 83 . Once released from the nucleus, HMGB1 interacts with specific TLRs and incites local inflammation [84][85][86] . Thus, the release of nuclear HMGB1 is counterproductive to the wound healing cascade where timely resolution of inflammation is critical 2 . TX100 caused rapid and overt depletion of nuclear HMGB1. When expressed as the ratio of nuclear:extranuclear HMGB1, TX100 caused marked depletion which was significantly rescued under conditions of SPD co-treatment.
The canonical pathway for the induction of necroptosis entails activation of RIP1K-RIP3K, followed by activation of phospho-MLKL. Activation of RIP1K, a serine-threonine kinase causes oligomerization and autophosphorylation of RIP3K at Ser227 87-90 . RIP3K, thus activated, drives phosphorylation of MLKL 28,87,91,92 . The phosphorylated MLKL is translocated to the plasma membrane leading to pore formation reminiscent of gasdermins in pyroptosis 28,87,91,92 . As a pseudokinase, MLKL does not target any protein for phosphorylation. Instead, upon activation, MLKL is trafficked to the plasma membrane 28,31,87,93 . Upon interaction with inositide lipids associated with the plasma membrane, MLKL induces necroptosis causing cell death 91 . In particular, MLKL at the plasma membrane form pores that are directly implicated in cell death. In this work, TX100 activated RIP3 and generated phospho-MLKL. Moreover, in agreement with the spared depletion of nuclear HMGB1, the abundance of both RIP3 and phospho-MLKL were blunted in the presence of SPD. Inhibition of keratinocyte necroptosis is known to be a protective effect against psoriatic inflammation 94 . The clinical relevance of SPD may thus be broader than the realm of chronic wound management.
Taken together, this work shows that SPD, clinically used as wound care dressing, is likely to have functions beyond its reported effects on biofilm management. Necroptotic death, known to be abundant in chronic wounds, can be managed by SPD. The effects of this poloxamer are evident in multiple signaling events leading www.nature.com/scientificreports/ to necroptosis. Sparing of keratinocyte nuclear HMGB1 is likely not only to spare these cells enabling re-epithelialization, but also to manage chronic inflammation caused by release of this DAMP molecule. Additional studies testing these multifaceted effects of SPD in a chronic wound setting are thus warranted.

Materials and methods
Surfactant polymer dressing. PluroGel (Medline Industries, Inc. Northfield, IL), which contains Poloxamer 188, was employed as SPD in all the experiments. SPD was reconstituted to 1 g/ml in Dulbecco's phosphate-buffered saline (DPBS, Gibco, Carlsbad, CA # 14190250) to make the stock solution which was then filtered using 0.2 µm filter. Further, it was diluted in media and used to treat the cells. Characterization of cell death by flow cytometry. Cells were grown in twelve-well plates at a seeding density of 0.1million/ml. After reaching confluence, typically after 24 h, cells were treated with TX100 with and without SPD. Cell viability was measured using propidium iodide staining as previously described [97][98][99] . After  Statistical analyses. All the data are reported as mean ± SEM of three to nine experiments, as indicated in respective figure legends. One-way ANOVA was used to determine significant differences. A p value < 0.05 was considered statistically significant.

Data availability
All data generated or analyzed during this study are included in this published article (and its Supplementary Information files).  . SPD blunted TX100-induced phosphorylation of MLKL. Human keratinocytes (HaCaT) were exposed to TX100 (0.2 mM) for 3 h in presence of SPD (100 mg/ml). Cells were then fixed and immunostained with phospho-MLKL antibody. (A,B) Representative microscopic images and quantification of phospho-MLKL was assessed by confocal microscopy. Scale, 10 µm. Data represent mean ± SEM (n = 4). *p < 0.05 compared to control (TX100-untreated). † p < 0.05 compared to TX100-treated group.