Spatiotemporal regulation of acute wound healing by the NLRP3 inflammasome: dual roles in macrophage-fibroblast chemotaxis and phenotype during wound repair

Abstract

Background: The spatiotemporal regulation of inflammatory dynamics is critical for successful wound healing. However, the precise mechanistic role of the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome in orchestrating these processes remains incompletely characterized. This study aimed to delineate the specific mechanisms by which NLRP3 governs cellular and molecular events during wound healing. Methods: Multi-omics sequencing data were utilized to profile NLRP3 inflammasome activation dynamics in murine and human acute wound models. Nlrp3^−/− mice were generated using CRISPR-Cas9 technology. In vitro and in vivo functional assays were performed to assess NLRP3-dependent regulation of macrophage and fibroblast recruitment, polarization, and phenotype modulation. Results: NLRP3ispredominantlyexpressedinmacrophagesandneutrophilsduringtheinflammatoryphaseofwoundhealing. Global deletion of Nlrp3 reduces IL-1 β, the main downstream effector, attenuates CCL/CXCL chemokine signaling, decreases both inflammatory and pro-reparative cell infiltration, and disrupts the phenotypic switching of macrophages and fibroblasts, collectively delaying wound closure. However, the resulting low-inflammatory microenvironment in Nlrp3^−/− mice may upregulate Wnt and Notch signaling early in the repair phase, curbing fibrosis and promoting appendage regeneration. Partial IL-1 β blockade in WT mice recapitulates the NLRP3-null phenotype, whereas IL-1 β reconstitution in knockout mice accelerates healing but increases fibrosis. Moreover, the NLRP3 protein also modulates fibroblast phenotype independently of inflammasome activation via a ROS-dependent mechanism. Conclusion: NLRP3 exerts dual-phase regulatory roles in wound healing: (i) during inflammation, it drives chemokine- mediated macrophage/fibroblast recruitment and M1 polarization while suppressing fibroblast-mediated repair via IL-1 β signaling; (ii) later, NLRP3 deficiency enhances Wnt/Notch signaling, promoting structural restoration despite transiently delayed healing. Moreover, fibroblasts with high NLRP3 expression engage an inflammasome-independent NLRP3/ROS axis that augments activation of TGF- β/Smad signaling. These findings position NLRP3 as a potential therapeutic target for modulating phase-specific inflammatory and regenerative responses.

Keywords

NLRP3 inflammasome / Wound healing / Spatiotemporal regulation / Chemotaxis / Macrophage polarization / Fibroblast

References

[1] Peña OA, Martin P. Cellular and molecular mechanisms of skin wound healing. Nat Rev Mol Cell Biol 2024; 25: 599-616. https://doi.org/10.1038/s41580-024-00715-1.

[2] Cañedo-Dorantes L, Cañedo-Ayala M. Skin acute wound healing: a comprehensive review. Int J Inflamm 2019; 2019: 1-15. https://doi.org/10.1155/2019/3706315.

[3] Martin P, Pardo-Pastor C, Jenkins RG, Rosenblatt J. Imperfect wound healing sets the stage for chronic diseases. Science 2024; 386: eadp2974. https://doi.org/10.1126/science.adp2974.

[4] Monaghan MG, Borah R, Thomsen C, Browne S. Thou shall not heal: overcoming the non-healing behaviour of diabetic foot ulcers by engineering the inflammatory microenvironment. Adv Drug Deliv Rev 2023; 203: 115120. https://doi.org/10.1016/j.addr.2023.115120.

[5] Artlett CM. Inflammasomes in wound healing and fibrosis. J Pathol 2013; 229: 157-67. https://doi.org/10.1002/path.4116.

[6] Akbal A, Dernst A, Lovotti M, Mangan MSJ, McManus RM, Latz E. How location and cellular signaling combine to activate the NLRP3 inflammasome. Cell Mol Immunol 2022; 19: 1201-14. https://doi.org/10.1038/s41423-022-00922-w.

[7] Swanson KV, Deng M, Ting JP-Y. The NLRP3 inflammasome: molecular activation and regulation to therapeutics. Nat Rev Immunol 2019; 19: 477-89. https://doi.org/10.1038/s41577-019-0165-0.

[8] Vinaik R, Abdullahi A, Barayan D, Jeschke MG. NLRP3 inflammasome activity is required for wound healing after burns. Transl Res 2020; 217: 47-60. https://doi.org/10.1016/j.trsl.2019.11.002.

[9] Ding Y, Ding X, Zhang H, Li S, Yang P, Tan Q. Relevance of NLRP3 Inflammasome-related pathways in the pathology of diabetic wound healing and possible therapeutic targets. Oxidative Med Cell Longev 2022; 2022: 1-15. https://doi.org/10.1155/2022/9687925.

[10] Huang C, Ogawa R. Role of Inflammasomes in keloids and hypertrophic scars-lessons learned from chronic diabetic wounds and skin fibrosis. IJMS 2022; 23: 6820. https://doi.org/10.3390/ijms23126820.

[11] Pyrillou K, Burzynski LC, Clarke MCH. Alternative pathways of IL-1 activation, and its role in health and disease. Front Immunol 2020; 11: 613170. https://doi.org/10.3389/fimmu.2020.613170.

[12] Tsukui T, Wolters PJ, Sheppard D. Alveolar fibroblast lineage orchestrates lung inflammation and fibrosis. Nature 2024; 631: 627-34. https://doi.org/10.1038/s41586-024-07660-1.

[13] Bünemann E, Hoff N-P, Buhren BA, Wiesner U, Meller S, Bölke E, et al. Chemokine ligand-receptor interactions critically regulate cutaneous wound healing. Eur J Med Res 2018; 23: 4. https://doi.org/10.1186/s40001-017-0299-0.

[14] Ridiandries A, Tan JTM, Bursill CA. The role of chemokines in wound healing. IJMS 2018; 19: 3217. https://doi.org/10.3390/ijms19103217.

[15] Amuso VM, Haas MR, Cooper PO, Chatterjee R, Hafiz S, Salameh S, et al. Fibroblast-mediated macrophage recruitment supports acute wound healing. J Invest Dermatol 2024; 145: 1781-1797.e8. https://doi.org/10.1016/j.jid.2024.10.609.

[16] Jin L, Zhou S, Zhao S, Long J, Huang Z, Zhou J, et al. Early short-term hypoxia promotes epidermal cell migration by activating the CCL2-ERK1/2 pathway and epithelial-mesenchymal transition during wound healing. Burns Trauma 2024; 12: tkae017. https://doi.org/10.1093/burnst/tkae017.

[17] Duan X, Yuan X, Yao B, Song W, Li Z, Enhejirigala, et al. The role of CTHRC1 in promotion of cutaneous wound healing. Signal Transduct Target Ther 2022; 7: 183. https://doi.org/10.1038/s41392-022-01008-9.

[18] Chen L, Arbieva ZH, Guo S, Marucha PT, Mustoe TA, DiPietro LA. Positional differences in the wound transcriptome of skin and oral mucosa. BMC Genomics 2010; 11: 471. https://doi.org/10.1186/1471-2164-11-471.

[19] Sinha S, Sparks HD, Labit E, Robbins HN, Gowing K, Jaffer A, et al. Fibroblast inflammatory priming determines regenerative versus fibrotic skin repair in reindeer. Cell 2022; 185: 4717-4736.e25. https://doi.org/10.1016/j.cell.2022.11.004.

[20] Hu KH, Kuhn NF, Courau T, Tsui J, Samad B, Ha P, et al. Transcriptional space-time mapping identifies concerted immune and stromal cell patterns and gene programs in wound healing and cancer. Cell Stem Cell 2023; 30: 885-903.e10. https://doi.org/10.1016/j.stem.2023.05.001.

[21] Mascharak S, Talbott HE, Januszyk M, Griffin M, Chen K, Davitt MF, et al. Multi-omic analysis reveals divergent molecular events in scarring and regenerative wound healing. Cell Stem Cell 2022; 29: 315-327.e6. https://doi.org/10.1016/j.stem.2021.12.011.

[22] Liu Z, Bian X, Luo L, Björklund ÅK, Li L, Zhang L, et al. Spatiotemporal single-cell roadmap of human skin wound healing. Cell Stem Cell 2024; 32: 479-498.e8. https://doi.org/10.1016/j.stem.2024.11.013.

[23] Cai Y, Xiong M, Xin Z, Liu C, Ren J, Yang X, et al. Decoding aging-dependent regenerative decline across tissues at single-cell resolution. Cell Stem Cell 2023; 30: 1674-1691.e8. https://doi.org/10.1016/j.stem.2023.09.014.

[24] 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. https://doi.org/10.3389/fphys.2018.00419.

[25] Z J, L X, W C, L Y, W Y, M C, et al. NLRP3 inflammasome mediates M1 macrophage polarization and IL-1β production in inflammatory root resorption. J Clin Periodontol 2020; 47: 451-60. https://doi.org/10.1111/jcpe.13258.

[26] SIRT1 Promotes M2 Microglia Polarization via Reducing ROS-Mediated NLRP3 Inflammasome Signaling After Subarachnoid Hemorrhage- PubMed https://pubmed.ncbi.nlm.nih.gov/34899720/ (accessed 6 October 2024).

[27] Wang Y, Wan L, Zhang Z, Li J, Qu M, Zhou Q. Topical calcitriol application promotes diabetic corneal wound healing and reinnervation through inhibiting NLRP3 inflammasome activation. Exp Eye Res 2021; 209: 108668. https://doi.org/10.1016/j.exer.2021.108668.

[28] Shapouri-Moghaddam A, Mohammadian S, Vazini H, Taghadosi M, Esmaeili S-A, Mardani F, et al. Macrophage plasticity, polarization, and function in health and disease. J Cell Physiol 2018; 233: 6425-40. https://doi.org/10.1002/jcp.26429.

[29] Jiang D, Guo R, Machens H-G, Rinkevich Y. Diversity of fibroblasts and their roles in wound healing. Cold Spring Harb Perspect Biol 2023; 15: a041222. https://doi.org/10.1101/cshperspect.a041222.

[30] CD201+ fascia progenitors choreograph injury repair- PubMed https://pubmed.ncbi.nlm.nih.gov/37968392/ (24 accessed April 2025).

[31] Anders H-J, Suarez-Alvarez B, Grigorescu M, Foresto-Neto O, Steiger S, Desai J, et al. The macrophage phenotype and inflammasome component NLRP3 contributes to nephrocalcinosis-related chronic kidney disease independent from IL-1-mediated tissue injury. Kidney Int 2018; 93: 656-69. https://doi.org/10.1016/j.kint.2017.09.022.

[32] Mulay SR. Multifactorial functions of the inflammasome component NLRP3 in pathogenesis of chronic kidney diseases. Kidney Int 2019; 96: 58-66. https://doi.org/10.1016/j.kint.2019.01.014.

[33] Bracey NA, Gershkovich B, Chun J, Vilaysane A, Meijndert HC, Wright JR, et al. Mitochondrial NLRP3 protein induces reactive oxygen species to promote Smad protein signaling and fibrosis independent from the inflammasome. J Biol Chem 2014; 289: 19571-84. https://doi.org/10.1074/jbc.M114.550624.

[34] Alexanian M, Padmanabhan A, Nishino T, Travers JG, Ye L, Pelonero A, et al. Chromatin remodelling drives immune cell-fibroblast communication in heart failure. Nature 2024; 635: 434-43. https://doi.org/10.1038/s41586-024-08085-6.

[35] Xie X, Wang P, Jin M, Wang Y, Qi L, Wu C, et al. IL-1β-induced epithelial cell and fibroblast transdifferentiation promotes neutrophil recruitment in chronic rhinosinusitis with nasal polyps. Nat Commun 2024; 15: 9101. https://doi.org/10.1038/s41467-024-53307-0.

[36] Mangan MSJ, Olhava EJ, Roush WR, Seidel HM, Glick GD, Latz E. Targeting the NLRP3 inflammasome in inflammatory diseases. Nat Rev Drug Discov 2018; 17: 688. https://doi.org/10.1038/nrd.2018.149.

[37] Li H, Wang Z, Zhou F, Zhang G, Feng X, Xiong Y, et al. Sustained activation of NLRP3 inflammasome contributes to delayed wound healing in aged mice. Int Immunopharmacol 2023; 116: 109828. https://doi.org/10.1016/j.intimp.2023.109828.

[38] Nagar A, Bharadwaj R, Mof S, Roy A. What are NLRP3-ASC specks? An experimental progress of 22 years of inflammasome research. Front Immunol 2023; 14: 1188864. https://doi.org/10.3389/fimmu.2023.1188864.

[39] Higashikuni Y, Liu W, Numata G, Tanaka K, Fukuda D, Tanaka Y, et al. NLRP3 Inflammasome activation through heart-brain interaction initiates cardiac inflammation and hypertrophy during pressure overload. Circulation 2023; 147: 338-55. https://doi.org/10.1161/CIRCULATIONAHA.122.060860.

[40] Cliff CL, Squires PE, Hills CE. Tonabersat suppresses priming/activation of the NOD-like receptor protein-3 (NLRP3) inflammasome and decreases renal tubular epithelial-to-macrophage crosstalk in a model of diabetic kidney disease. Cell Commun Signal 2024; 22: 351. https://doi.org/10.1186/s12964-024-01728-1.

[41] Walraven M, Talhout W, Beelen RHJ, van Egmond M, Ulrich MMW. Healthy human second-trimester fetal skin is deficient in leukocytes and associated homing chemokines. Wound Repair Regen 2016; 24: 533-41. https://doi.org/10.1111/wrr.12421.

[42] Hu MS, Borrelli MR, Hong WX, Malhotra S, Cheung ATM, Ransom RC, et al. Embryonic skin development and repair. Organogenesis 2018; 14: 46-63. https://doi.org/10.1080/15476278.2017.1421882.

[43] Kirby CS, Islam N, Wier E, Alphonse MP, Sweren E, Wang G, et al. RNase L represses hair follicle regeneration through altered innate immune signaling. J Clin Invest 2025; 135: e172595. https://doi.org/10.1172/JCI172595.

[44] Accogli T, Hibos C, Milian L, Geindreau M, Richard C, Humblin E, et al. The intrinsic expression of NLRP3 in Th17 cells promotes their protumor activity and conversion into Tregs. Cell Mol Immunol 2025; 22: 541-56. https://doi.org/10.1038/s41423-025-01281-y.

[45] Ali N, Zirak B, Rodriguez RS, Pauli ML, Truong H-A, Lai K, et al. Regulatory T cells in skin facilitate epithelial stem cell differentiation. Cell 2017; 169: 1119-1129.e11. https://doi.org/10.1016/j.cell.2017.05.002.

[46] Liu Z, Hu X, Liang Y, Yu J, Li H, Shokhirev MN, et al. Glucocorticoid signaling and regulatory T cells cooperate to maintain the hair-follicle stem-cell niche. Nat Immunol 2022; 23: 1086-97. https://doi.org/10.1038/s41590-022-01244-9.

[47] Liu X, Chen J, Yue S, Zhang C, Song J, Liang H, et al. NLRP3-mediated IL-1β in regulating the imbalance between Th17 and Treg in experimental autoimmune prostatitis. Sci Rep 2024; 14: 18829. https://doi.org/10.1038/s41598-024-69512-2.

[48] Sun J, Jia W, Qi H, Huo J, Liao X, Xu Y, et al. An Antioxidative and active shrinkage hydrogel Integratedly promotes Re-epithelization and skin constriction for enhancing wound closure. Adv Mater 2024; 36: e2312440. https://doi.org/10.1002/adma.202312440.

[49] Mao J, Xia W, Wu Y, Li M, Zhao Y, Zhai P, et al. Biosynthesis of Lysosomally escaped apoptotic bodies inhibits Inflammasome synthesis in macrophages. Research (Wash DC) 2025; 8: 0581. https://doi.org/10.34133/research.0581.

[50] Yang Y, Chen N, Fan J, Fan L, Cai Y, Xue L, et al. Spatiotemporal immunomodulation of macrophages via NLRP3/IL-1β pathway by Core-Shell microneedles to promote healing of biofilm-infected diabetic ulcers. Small 2025; 21: e2505179. https://doi.org/10.1002/smll.202505179.

[51] Lv D, Cao X, Zhong L, Dong Y, Xu Z, Rong Y, et al. Targeting phenylpyruvate restrains excessive NLRP3 inflammasome activation and pathological inflammation in diabetic wound healing. Cell Reports Medicine 2023; 4: 101129. https://doi.org/10.1016/j.xcrm.2023.101129.

[52] Mu X, Wu X, He W, Liu Y, Wu F, Nie X. Pyroptosis and inflammasomes in diabetic wound healing. Front Endocrinol 2022; 13: 950798. https://doi.org/10.3389/fendo.2022.950798.

[53] Wolf SJ, Audu CO, Moon JY, Joshi AD, Melvin WJ, Barrett EC, et al. Diabetic wound keratinocytes induce macrophage JMJD3-mediated Nlrp3 expression via IL-1R signaling. Diabetes 2024; 73: 1462-72. https://doi.org/10.2337/db23-0968.

[54] Liu C, Zhang Q, Zhou H, Jin L, Liu C, Yang M, et al. GLP-1R activation attenuates the progression of pulmonary fibrosis via disrupting NLRP3 inflammasome/PFKFB3-driven glycolysis interaction and histone lactylation. J Transl Med 2024; 22: 954. https://doi.org/10.1186/s12967-024-05753-z.

[55] Yang S, Feng Y, Chen L, Wang Z, Chen J, Ni Q, et al. Disulfiram accelerates diabetic foot ulcer healing by blocking NET formation via suppressing the NLRP3/Caspase-1/GSDMD pathway. Transl Res 2023; 254: 115-27. https://doi.org/10.1016/j.trsl.2022.10.008.

[56] Zhu M, Xu R, Yuan J, Wang J, Ren X, Cong T, et al. Tracking-seq reveals the heterogeneity of off-target effects in CRISPR-Cas9-mediated genome editing. Nat Biotechnol 2025; 43: 799-810. https://doi.org/10.1038/s41587-024-02307-y.

[57] Tsai SQ, Nguyen NT, Malagon-Lopez J, Topkar VV, Aryee MJ, Joung JK. CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets. Nat Methods 2017; 14: 607- 14. https://doi.org/10.1038/nmeth.4278.

[58] Wienert B, Wyman SK, Richardson CD, Yeh CD, Akcakaya P, Porritt MJ, et al. Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq. Science 2019; 364: 286-9. https://doi.org/10.1126/science.aav9023.

[59] Tsai SQ, Zheng Z, Nguyen NT, Liebers M, Topkar VV, Thapar V, et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol 2015; 33: 187-97. https://doi.org/10.1038/nbt.3117.

[60] Du W, Zhao L, Diao K, Zheng Y, Yang Q, Zhu Z, et al. A versatile CRISPR/Cas9 system off-target prediction tool using language model. Commun Biol 2025; 8: 882. https://doi.org/10.1038/s42003-025-08275-6.

[61] Yampolsky M, Bachelet I, Fuchs Y. Reproducible strategy for excisional skin-wound-healing studies in mice. Nat Protoc 2024; 19: 184-206. https://doi.org/10.1038/s41596-023-00899-4.

[62] Wang X, Ge J, Tredget EE, Wu Y. The mouse excisional wound splinting model, including applications for stem cell transplantation. Nat Protoc 2013; 8: 302-9. https://doi.org/10.1038/nprot.2013.002.

[63] Dekoninck S, Hannezo E, Sifrim A, Miroshnikova YA, Aragona M, Malfait M, et al. Defining the design principles of skin epidermis postnatal growth. Cell 2020; 181: 604-620.e22. https://doi.org/10.1016/j.cell.2020.03.015.

[64] Fu J, Schroder K, Wu H. Mechanistic insights from inflammasome structures. Nat Rev Immunol 2024; 24: 518-35. https://doi.org/10.1038/s41577-024-00995-w.

[65] Huang X, Li T, Zhang G, Chen J, Li T, Yang S, et al. AIM2 activation mediated by RIPK1/3-dependent mitochondrial DNA release drives A β1-40-induced retinal pigment epithelium injury. Cell Commun Signal 2025; 23: 301. https://doi.org/10.1186/s12964-025-02294-w.

[66] Zhang Y, Zhang G, Dong B, Pandeya A, Cui J, dos S VS, et al. Pyroptosis of pulmonary fibroblasts and macrophages through NLRC4 inflammasome leads to acute respiratory failure. Cell Rep 2025; 44: 115479. https://doi.org/10.1016/j.celrep.2025.115479.

[67] Denes A, Coutts G, Lénárt N, Cruickshank SM, Pelegrin P, Skinner J, et al. AIM2 and NLRC4 inflammasomes contribute with ASC to acute brain injury independently of NLRP3. Proc Natl Acad Sci USA 2015; 112: 4050-5. https://doi.org/10.1073/pnas.1419090112.

[68] Abbasi S, Sinha S, Labit E, Rosin NL, Yoon G, Rahmani W, et al. Distinct regulatory programs control the latent regenerative potential of dermal fibroblasts during wound healing. Cell Stem Cell 2020; 27: 396-412.e6. https://doi.org/10.1016/j.stem.2020.07.008.

[69] Lim CH, Sun Q, Ratti K, Lee S-H, Zheng Y, Takeo M, et al. Hedgehog stimulates hair follicle neogenesis by creating inductive dermis during murine skin wound healing. Nat Commun 2018; 9: 4903. https://doi.org/10.1038/s41467-018-07142-9.

[70] Yang Y, Chu C, Liu L, Wang C, Hu C, Rung S, et al. Tracing immune cells around biomaterials with spatial anchors during large-scale wound regeneration. Nat Commun 2023; 14: 5995. https://doi.org/10.1038/s41467-023-41608-9.