# Journal Pre-proof Anti-inflammatory macrophages are recruited to the keloids that obtain response to intra-lesional injection therapies Pre- and post-treatment biopsies from a doubleblinded, randomized, controlled trial Tuomas Komulainen, Matti Ylitörmä, Kristiina E. Hietanen, Juhani Järvinen, Ilkka Junttila, Ilkka S. Kaartinen, Tero A.H. Järvinen PII: S0022-202X(25)03519-5 DOI: Reference: JID 5059 To appear in: Journal of Investigative Dermatology Received Date: 6 June 2025 Revised Date: 29 September 2025 Accepted Date: 17 October 2025 Please cite this article as: Komulainen T, Ylitörmä M, Hietanen KE, Järvinen J, Junttila I, Kaartinen IS, Järvinen TAH, Anti-inflammatory macrophages are recruited to the keloids that obtain response to intra-lesional injection therapies Pre- and post-treatment biopsies from a double-blinded, randomized, controlled trial, *Journal of Investigative Dermatology* (2025), doi: [https://doi.org/10.1016/](https://doi.org/10.1016/j.jid.2025.10.597) [j.jid.2025.10.597](https://doi.org/10.1016/j.jid.2025.10.597). This is a PDF of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability. This version will undergo additional copyediting, typesetting and review before it is published in its final form. 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Published by Elsevier, Inc. on behalf of the Society for Investigative Dermatology. ![](https://assets.reachrx.ai/images/10.1016%E2%98%8Dj.jid.2025.10.597/_page_0_Picture_13.jpeg) ![](https://assets.reachrx.ai/images/10.1016%E2%98%8Dj.jid.2025.10.597/_page_1_Picture_0.jpeg) # Anti-inflammatory macrophages are recruited to the keloids that obtain response to intra-lesional injection therapies # Pre- and post-treatment biopsies from a double-blinded, randomized, controlled trial Tuomas Komulainen^{1,2^}, Matti Ylitörmä^{1,2^}, Kristiina E. Hietanen^1,3, Juhani Järvinen^1,2, Ilkka Junttila^1,4,5, Ilkka S. Kaartinen^{1,2^\*}, Tero A.H. Järvinen^1,2\* ¹Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland ²Department of Musculoskeletal Surgery and Diseases, Tampere University Hospital, Wellbeing Services County of Pirkanmaa, Tampere, Finland ³Central Finland Health Care District, Jyväskylä, Finland ⁴NordLab Oulu, Oulu University Hospital, Oulu, Finland ⁵Fimlab Laboratories, Tampere, Finland ^{^}These authors contributed equally This study was conducted in Tampere University, Pirkanmaa, Finland \* Correspondence: Prof. Tero Järvinen, M.D., Ph.D. https://orcid.org/0000-0002-4027-1759 Dr. Ilkka Kaartinen, M.D., Ph.D. https://orcid.org/0000-0001-9928- ## 6931 Faculty of Medicine and Health Technology Tampere University, Tampere, Finland Finland +358-44-285 4620 tero.jarvinen@tuni.fi Department of Plastic Surgery Tampere University Hospital, +358-50-305 4554 ilkka.kaartinen@pirha.fi Running title: Anti-inflammatory macrophages and keloid treatment #### Abbreviations: 5-FU = 5-fluorouracil - M1 = Inflammatory macrophage - M2 = Anti-inflammatory macrophage - TAC = Triamcinolone hexacetonide Key words: T-Lymphocytes, Myofibroblasts, Biomarkers, Injections, Corticosteroid Dear Editor, We explored the predictive value of immune cell populations to intra-lesional injections by using biopsies collected from the active border of keloids before and after injections in the doubleblinded randomized controlled trial comparing the efficacy of intralesional 5-fluorouracil (5-FU) and triamcinolone (TAC) injections in 48 human keloids (Hietanen et al. 2019). Responder (R) and non-responder (NR) keloids from both treatment arms were studied as separate and combined groups resulting in 25 keloids obtaining response (15 from the TAC- and 10 from the 5-FU-arm) and 23 non-responding keloids (10 from the TAC- and 13 from the 5-FU-arm). Each keloid was defined into three layers, superficial, middle and deep according to a histological classification system (Jiao et al. 2017). Immunohistochemistry using antibodies and quantitative virtual microscopy were used to detect and score immune cell populations in different keloid layers. A detailed description of the study is provided in the **Supplementary Text**. Different macrophage populations did not differ between responders and non-responders in any of the keloid layers before treatment, **Figure 1, Supplementary Figures S1 and S2**. M2/antiinflammatory macrophages (CD163+) increased in each dermal layer of the responder group during the treatment, being more common in the responders than in the non-responders in the entire keloid biopsies (NR 119 vs R 228 cells/mm², **P=0.0450**) and in the middle dermis (NR 37 vs. R 97, **P = 0.0168**) after the treatment. The M1/inflammatory macrophages (CD86+) showed small biological changes in responders and the "general" macrophages (CD68+) showed no changes during the treatment. M2s increased almost exclusively in the TAC-treated keloids. Their increase in the TAC-treated keloids was greater in the responders than in the non-responders in all keloid layers. The M2s increased significantly less in the 5-FU-treated than in the TAC-treated keloids. The TAC-arm responders had significantly more M2s than the 5-FU-treated responder and non-responder groups in entire keloid, in the superficial and in the middle dermis after the treatment. #### Supplementary Figure S3, Supplementary Tables S1-S4. Almost 70 % of myofibroblasts reside in the middle dermis in keloids (Komulainen et al. 2024). The inverse correlation between the M2s and myofibroblasts was statistically significant in the middle dermis (r = -0.51, P = 0.0008). Also, a positive correlation between the M2s and both blood vessels as well as with angiogenic blood vessels (VEGFR2+) was identified in the middle dermis. Supplementary Figure S4, Supplementary Tables S5-S10. None of the T cell populations (T cells (CD3+), helper T cells (CD4+), cytotoxic T cells (CD8+) and Tregs (FoxP3+)) showed difference between the responders and non-responders in any of the keloid layers before the treatment. T cells, and every T cell subtype were reduced in the superficial dermis during the intralesional injection treatments in the non-responders as well as in the responders. There were fewer Tregs in the responders than in the non-responders in the superficial dermis after the treatment, P = 0.0267. T cells or other T cell subtypes showed no differences in the middle, in the deep dermis or in the entire keloid biopsy. The Tregs decreased mainly in the responders of the TAC-arm. T cells were quantified in the overlying epidermis. There was no difference in the epidermal T cells between the non-responders and responders before or after the treatment, **Figure 2, Supplementary Figures S5-S6, Supplementary Tables S11-S18**. We also quantified Schwann cells, mature and langerin-expressing dendritic cells, high endothelial venules and neutrophils in the keloids. Taken together, Schwann cells were abundant #### Journal Pre-proof in keloids but showed no difference before or after treatment. Only random individual dendritic cells, neutrophils and high endothelial venules were detected in keloids before and after the intralesional injections. Some of the keloids were totally devoid of them. **Supplementary** #### Figures S7-S11, Supplementary Tables S19-S22. M2s and M1s determined from pre-treatment biopsies are not predictive of treatment response, but M2s are recruited to keloids that obtain clinical response to intralesional injection treatment. These changes are achieved mainly in the TAC-arm responders. M2s can promote resolution of tissue homeostasis without fibrosis (Zawadzki et al. 2025). Our results suggest that TAC affects keloids by influencing their "immune phenotype". Corticosteroids can induce M2-subpopulation that is angiogenic (Xu et al. 2025). We demonstrate that there is correlation between M2s and angiogenic blood vessels in the keloid middle dermis. M2 accumulation correlates inversely with the myofibroblasts in the middle dermis, where most myofibroblast reside (Komulainen et al. 2024). As it is still debated whether keloids represent a "myofibroblast disease", the potential association between the M2s and the myofibroblasts may have significance for other fibrotic diseases (Komulainen et al. 2024). We found that the T cells and Tregs decreased among the TAC-arm responders, particularly in the superficial dermis, during the intralesional injections. Tregs decreased more than other T cell populations in the responders. Tregs are key components of peripheral tolerance and play an immunosuppressive role (Chen et al. 2019). This implicates the potential importance of Tregs in alleviation of the keloid. It is not clear whether the abundance of Tregs is pathogenic, or merely present the general inflammatory state in keloids (Lee et al. 2023). Relatively low number of neutrophils and dendritic cells in keloids argues against active Th1- or Th17-type immune #### Journal Pre-proof responses. The accumulation of M2s is related to Th2 T cell response, which associates with profibrotic events, but also with tissue repair (Kokubo et al. 2022; Xu et al. 2025). One could argue that the analysis of the 5-FU-treated subgroup was unnecessary as its mechanism of action is unrelated to immune response. Yet these analyses offered a relevant internal reference to the TAC that functions by suppressing inflammation. Double-blinded RCT is an ideal way to assess the clinical response to the therapies. Pre- and post-treatment biopsies provide "window" to explore the biological state of the leading edge of keloid in response to the intra-lesional injection treatments. To the best of our knowledge no other material of same scientific rigor has been collected from the keloid RCTs. None of the immune cell populations is predictive of the response, but the M2 macrophages accumulate, while Tregs almost disappear, in the keloids that obtain response to intralesional injections, especially to corticosteroid. # **ETHICS STATEMENT** The double-blinded RCT was approved by the Ethics Committee of the Pirkanmaa Health Care District and recorded to online database of clinical research trials: ClinicalTrials.gov (#NCT02155439) (Hietanen et al. 2019). All participants were informed about the study protocol and provided written informed consent (Hietanen et al. 2019). ## DATA AVAILABILITY STATEMENT All relevant information is included in the manuscript. The original data is available from the corresponding authors (tero.jarvinen@tuni.fi and ilkka.kaartinen@pirha.fi). # **CONFLICT OF INTEREST STATEMENT** The authors state no conflict of interest. #### ACKNOWLEDGEMENTS We thank Sari Toivola and Marianne Karlsberg for their excellent laboratory work, and Giuseppe Rocca, Elisa Muñoz Pulido, Gina Alessandra López Toledo, Keylen Renee Nevárez Loya and Danielle Carvalho do Carmo for assistance in the investigation process. The guarantor of this original research article is Prof. Tero Järvinen, M.D., Ph.D. #### **Funding** This work was supported by the Academy of Finland (grant 287907 to TJ), the Competitive State Research Financing of the Expert Responsibility Area of Tampere University Hospital (grants 9R025, 9V010, 9X011 and 9V010 to IK and TJ), of Nordlab (grant KT0016 to IJ), and of Fimlab (grant X51409 to IJ), State funding for university-level health research, Tampere University Hospital, Wellbeing services county of Pirkanmaa (grants T62774 and T63764 to TJ and grants T62904 and T63914 to TK), the Finnish Medical Foundation (to TJ, and grant 6397 to TK), the Tampere University Hospital Support Foundation (to TJ), the Tampere Tuberculosis Foundation (to TJ and IJ), the Päivikki and Sakari Sohlberg Foundation (to TJ), the Orion Research Foundation, Foundation of Vappu Uuspää (to TJ), Instrumentarium Research Foundation (to TJ) and Foundation of Advancement of Laboratory Medicine (to IJ). The funding sources were not involved in conducting the research or in the preparation of the article. # **AUTHOR CONTRIBUTIONS STATEMENT** Conceptualization TAHJ, IK, TK, Formal analysis: TK, MY, Investigation KH, TK, MY, JJ Resources: TAHJ, IK, KH, Supervision; TAHJ, IK, KH, Visualization; TK, TAHJ, MY, Project administration; TAHJ, Funding acquisition; TAHJ, IK, TK, Writing - Original Draft TK, TAHJ, MY, Writing – Review & Editing; All authors. ![](https://assets.reachrx.ai/images/10.1016%E2%98%8Dj.jid.2025.10.597/_page_9_Picture_3.jpeg) # **REFERENCES** Chen Y, Jin Q, Fu X, Qiao J, Niu F. Connection between T regulatory cell enrichment and collagen deposition in keloid. Exp. Cell Res. 2019;383(2):111549 Hietanen KE, Järvinen TA, Huhtala H, Tolonen TT, Kuokkanen HO, Kaartinen IS. Treatment of keloid scars with intralesional triamcinolone and 5-fluorouracil injections - a randomized controlled trial. J. Plast. Reconstr. Aesthetic Surg. 2019;72(1):4–11 Jiao H, Zhang T, Fan J, Xiao R. The Superficial Dermis May Initiate Keloid Formation: Histological Analysis of the Keloid Dermis at Different Depths. Front. Physiol. 2017;8:885 Kokubo K, Onodera A, Kiuchi M, Tsuji K, Hirahara K, Nakayama T. Conventional and pathogenic Th2 cells in inflammation, tissue repair, and fibrosis. Front. Immunol.; 2022;13 Komulainen T, Daymond P, Hietanen KE, Kaartinen IS, Järvinen TAH. Myofibroblasts reside in the middle dermis of the keloids but do not predict the response to injection therapies: a doubleblinded, randomized, controlled trial. Front. Med. 2024;11:1293028 Lee C-C, Tsai C-H, Chen C-H, Yeh Y-C, Chung W-H, Chen C-B. An updated review of the immunological mechanisms of keloid scars. Front. Immunol. 2023;14:1117630 Xu J, Ding L, Mei J, Hu Y, Kong X, Dai S, et al. Dual roles and therapeutic targeting of tumorassociated macrophages in tumor microenvironments. Sig Transduct. Target. Ther. 2025;10(1):268 Zawadzki A, Leeming DJ, Sanyal AJ, Anstee QM, Schattenberg JM, Friedman SL, et al. Hot and Cold Fibrosis: The Role of Serum Biomarkers to assess the Immune Mechanisms and ECM-Cell Interactions in Human Fibrosis. J. Hepatol. 2025;S0168-8278(25)00148-5 #### **Legends for Figures** Figure 1. M2 and M1 macrophages increase in the keloids responding to intralesional injections. We collected biopsies from the active border of 48 keloids before and after intralesional injections in RCT studying the efficacy of TAC- and 5-FU injections. The keloids were classified as a non-responders (NR) or responders (R) after the treatment. M2 (CD163+) and M1 (CD86+) macrophages were detected with antibodies. M2s showed major increase in the dermis of the responders (a). M2s (b-m) in keloids superficial, middle, and deep dermis before (pre) and after (post) treatment. Bar 50 $\mu$ m b-m. Orange dot = each separate data point, Middle line = geom. mean, Whiskers = 95 % CI of geom. mean. \* = P < 0.05, \*\* = P < 0.01, \*\*\* = P < 0.001, \*\*\*\* = P < 0.001. Used statistical tests reported in Supplementary table 25. Images were taken with Olympus VS200 slide scanner, Olympus VS-264C, Olympus PlanXApo 10x/0.40, WD 3.1 mm (Air). Brightness of the images was adjusted in post-production to highlight the immunohistochemical staining. **Figure 2.** Tregs disappear from the keloids' superficial dermis during intralesional injection treatments. Different T cell populations, T helper cells (CD4+), cytotoxic T cells (CD8+) and regulatory T cells (Tregs, FOXP3+), were quantified in the keloids' dermal layers and the results are presented in panel a. Tregs were reduced in the responders' (R) superficial dermis. Presentative images of each T cell subtype in the responders' superficial dermis before (pre) and after (post) the treatment showing T helper cells, cytotoxic T cells and Tregs. Black arrow heads pointing to Tregs. NR = non-responders. Orange dot = each separate data point, Middle line = geom. mean, Whiskers = 95 % CI of geom. mean. \* = P < 0.05. Bars 20 µm. Used statistical tests reported in Supplementary table 26. Images were taken with Olympus VS200 slide scanner, Olympus VS-264C, Olympus PlanXApo 10x/0.40, WD 3.1 mm (Air). Brightness of the images was adjusted in post-production to highlight the immunohistochemical staining. Journal Pre-proof ![](https://assets.reachrx.ai/images/10.1016%E2%98%8Dj.jid.2025.10.597/_page_13_Figure_0.jpeg) ![](https://assets.reachrx.ai/images/10.1016%E2%98%8Dj.jid.2025.10.597/_page_14_Figure_0.jpeg) ![](https://assets.reachrx.ai/images/10.1016%E2%98%8Dj.jid.2025.10.597/_page_14_Figure_1.jpeg)