MLKL-dependent necrosis is commonly associated with adverse pathologies; however, our study reveals that MLKL is essential for normal skin wound healing. In vivo experiments demonstrate that MLKL deficiency results in delayed wound repair. While MLKL is traditionally thought to affect wound healing through modulation of necrosis-induced inflammation, our findings suggest that its role extends beyond this mechanism. Firstly, MLKL overexpression persists throughout the entire wound healing process, from incision to closure, rather than being restricted to the inflammation phase. Secondly, MLKL deficiency negatively impacts both the wound healing rate and the expression of growth-related factors. Thirdly, the regulatory effect of macrophages on myofibroblast activity is diminished in the absence of MLKL. Conversely, while myofibroblasts can activate macrophages, this activation is partially inhibited by MLKL−/− myofibroblasts. Our study also establishes that PGE2 functions as an intermediary in the interaction between macrophages and myofibroblasts. In summary, MLKL plays a crucial role in skin wound healing by mediating a complex network of cellular interactions and cytokine expressions, thereby contributing to the establishment of a tissue repair microenvironment at the wound site.
The initial damage stimulus induces a conformational change in cells at the wound site, leading to plasma membrane translocation and the lethal permeation of the lipid bilayer. This process results in the release of cellular contents and the subsequent activation of an inflammatory response 17. Pro-inflammatory chemokines and cytokines released by activated macrophages, primarily M1 macrophages, initiate the recruitment and activation of additional immune cells, including neutrophils and bone marrow-derived monocytes, to the injury site. These immune cells further amplify inflammation to eliminate damage signals and necrotic cells 18. Necroptosis is mediated by RIPK3 19. RIPK3 deficiency significantly delays wound closure and impairs wound healing quality, as evidenced by delayed re-epithelialization, angiogenesis, granulation tissue formation, and collagen deposition 14. MLKL, a functional substrate for RIPK3, acts as an adaptor protein in necrosis signal transduction 20. Both RIPK3 and MLKL overexpression are observed at early stages of skin wound healing. Additionally, three other targets (Sycp2, HK3 and TNF-α) are overexpressed at the wound site. Sycp2, a synaptonemal complex protein, is associated with meiosis 21. HK3 is essential for initiating glycolysis 22. Both Sycp2 and HK3 may be involved in cellular differentiation related to inflammatory and immune responses 22. TNF-α is crucial in both apoptosis and necroptosis, and its interaction with MLKL in necroptosis is well-documented 23. Therefore, the co-overexpression of TNF-α and MLKL could be observed at wound sites as expected. In MLKL−/−mice, delayed skin wound closure and impaired morphological characteristics throughout the healing process are observed. Additionally, the concentration of inflammatory cytokines, including PGE2, TNF-α, and IL-6, is lower in the wound tissue of MLKL−/− mice compared to C57BL/6J mice three days post-injury. Serum levels of TNF-α and IL-6 are also reduced in MLKL−/− mice, while PGE2 levels are elevated in their serum after injury. The wound site data are likely more indicative of the local inflammatory microenvironment than serum data, which reflects a systemic rather than a localized response 24. MLKL may reduce the inflammatory response at the wound site through two potential mechanisms: it could directly regulate the synthesis of inflammatory cytokines, leading to decreased intracellular cytokine production in MLKL−/− mice. Alternatively, reduced cell death, as evidenced by altered expression of P53, Bcl-2, and Caspase-3, could result in less cellular content release, potentially reducing inflammatory cytokine release in MLKL−/− wound sites. It is possible that both mechanisms contribute to the reduced inflammatory cytokine release observed in MLKL−/− skin wounds. Overall, these results suggest that MLKL deficiency impairs skin wound healing, likely due to diminished inflammatory responses at the wound site, particularly during the early stages of healing.
As wound healing progresses from the pro-inflammatory to the pro-healing phase, it transitions into the remodeling stage. This stage is marked by a shift in macrophage phenotype from pro-inflammatory (M1) to anti-inflammatory (M2) 25. The debate continues on whether M2 macrophages at injury sites arise from blood monocytes or result from the phenotypic conversion of M1 macrophages 4. These anti-inflammatory macrophages are crucial for inflammation resolution and the progression of remodeling. They contribute to the healing process through their regenerative properties by secreting angiogenic and growth factors, cytokines, and chemokines, such as metalloproteinases (MMPs), VEGF, IL-8, TGF-β, IL-10, and arginase, which are essential for recruiting and activating other cells. Depletion of macrophages during the regenerative phase can directly or indirectly impact wound revascularization, matrix production, and re-epithelialization 16. Our study reveals that MLKL and RIPK3 are sustained in the wound area during mid- and late-stages of healing, indicating that RIPK3-MLKL signaling may be important not only in the early inflammatory phase but also in the later stages of wound repair. We found that MLKL deficiency impairs the expression of growth-related factors, such as EGF, VEGF, MMP-9, and ERα, during the mid- and late-stages of wound healing. This suggests that MLKL plays a role in regulating growth factor expression at the wound site during the later stages of healing. The delayed wound regeneration observed in MLKL−/− mice, as evidenced by H&E staining, aligns with these findings. Specifically, collagen fibers and mature granulation tissue were significantly more abundant in wounds of C57BL/6J mice compared to MLKL−/− mice at later stages of healing.
Miscommunication between macrophages and fibroblasts is recognized as a critical factor that can shift the balance from physiological repair to pathological fibrosis 8. It is well-documented that macrophage depletion during wound healing leads to a reduction in fibroblast numbers and that macrophages play a role in scavenging TGF-β, a key regulator of fibroblast/myofibroblast activation 18. Supernatants from C57BL/6J M1 macrophages were found to enhance the expression of VEGF and MMP-9 in fibroblasts. This effect was partially inhibited when supernatants from MLKL−/− M1 macrophages were used instead, indicating that MLKL in macrophages may regulate myofibroblast activity. Additionally, PGE2 synthesis, which is known to increase at both early and late stages of wound healing 26. We hypothesized that PGE2 is a potential mediator in the cross-talk between M1 macrophages and myofibroblasts, as we found that COX-2 (the upstream enzyme of PGE2 synthesis) expression and PGE2 levels in MLKL−/− wound tissue were lower than in C57BL/6J wound tissue (Figure. 7). To investigate this further, we replenished PGE2 in a co-culture system of MLKL−/− M1 macrophages and myofibroblasts. This restoration of PGE2 led to a recovery in the expression patterns of VEGF and MMP-9 in myofibroblasts. Conversely, MLKL in myofibroblasts was found to regulate the synthesis of IL-6, NO, and TNF-α in M1 macrophages through PGE2. These findings suggest that PGE2 may act as an intermediary in the MLKL-mediated interaction between M1 macrophages and myofibroblasts. Regarding the mechanism behind the reduced PGE2 concentration in supernatants from MLKL−/− macrophages (both M1 and M2) and myofibroblasts, two potential explanations exist: MLKL deficiency might directly reduce PGE2 synthesis in these cells, or it might delay membranolysis, trapping PGE2 within the cells. Our data, particularly the reduced COX-2 expression in MLKL−/− cells, support the first possibility. Taken together, MLKL may play a crucial role in maintaining the early-stage inflammatory microenvironment at the wound site by modulating the interaction between M1 macrophages and fibroblasts.
M2 macrophages produce anti-inflammatory cytokines and growth factors that mitigate inflammation, activate fibroblasts, and promote angiogenesis and wound contraction 18. It has been demonstrated that ablation of macrophages reduces myofibroblast numbers in the wound bed, impairs myofibroblast function, and hinders wound healing 6,7. Prolonged pathological stimulation leading to unresolved crosstalk between M2 macrophages and fibroblasts often results in pathological fibrosis or chronic wounds 27. Thus, understanding the interaction between M2 macrophages and fibroblasts is essential. We observed that the expression of growth factors EGF, VEGF, and MMP-9 peaks at the late stage of wound healing; however, their levels were lower in MLKL−/− wound sites compared to C57BL/6J sites. MLKL in M2 macrophages appears to regulate VEGF and MMP-9 expression in myofibroblasts, suggesting that MLKL deficiency might lead to reduced growth factor expression at the wound site. Additionally, MLKL in fibroblasts can regulate the expression of arginase, Ym1, and IL-10 in M2 macrophages, indicating that MLKL in fibroblasts may also influence M2 macrophage activity (Figure. 6). Ym1 and arginase, which are predominantly expressed in macrophages at injury sites, play crucial roles in wound healing and resolution 16. Arginase contributes to wound healing through the production of L-ornithine, with its absence impairing cutaneous wound healing and its overproduction contributing to fibrosis 28. IL-10 promotes the differentiation of macrophages towards pro-regenerative phenotypes 29. PGE2 released from myofibroblasts, along with its receptor agonist, enhances arginase activity in M2 macrophages, further supporting the role of PGE2 in regulating M2 macrophage activity 30. We also found reduced COX-2 and PGE2 synthesis in MLKL−/− M2 macrophages. Similar to its role in the interaction between M1 macrophages and myofibroblasts, PGE2 is involved in the interaction between M2 macrophages and myofibroblasts. These findings highlight the importance of MLKL and PGE2 in mediating the complex interactions between macrophages and fibroblasts during wound healing (Figure. 10).
In conclusion, this study provides direct evidence that MLKL plays a crucial role in skin wound healing by modulating the wound site's microenvironment. We have also elucidated the involvement of MLKL in the interaction between macrophages and fibroblasts, identifying PGE2 as a potential intermediary in the MLKL-mediated communication between both M1 and M2 macrophages and fibroblasts.