key: cord-0306817-9dq4vg3s authors: Direder, M.; Weiss, T.; Copic, D.; Vorstandlechner, V.; Laggner, M.; Mildner, C. S.; Klas, K.; Bormann, D.; Haslik, W.; Radtke, C.; Farlik, M.; Shaw, L.; Golabi, B.; Tschachler, E.; Hoetzenecker, K.; Ankersmit, H. J.; Mildner, M. title: Schwann cells contribute to keloid formation date: 2021-08-10 journal: nan DOI: 10.1101/2021.08.09.21261701 sha: 830e886b3011f2e9f07d4ae889bd143c12b11024 doc_id: 306817 cord_uid: 9dq4vg3s Keloids are disfiguring, hypertrophic scars with yet poorly understood pathomechanisms, which could lead to severe functional impairments. Here we analyzed the characteristics of keloidal cells by single cell sequencing and discovered the presence of an abundant population of Schwann cells that persisted in the hypertrophic scar tissue after wound healing. In contrast to normal skin, keloidal Schwann cells possess a repair-like phenotype and high cellular plasticity. Our data support the hypothesis that keloidal Schwann cells contribute to the formation of the extracellular matrix and are able to affect M2 polarization of macrophages. Indeed, we show that macrophages in keloids predominantly display a M2 polarization and produce factors that inhibit Schwann cell differentiation. Our data suggest a contribution of this cross-talk to the continuous expansion of keloids, and that targeting Schwann cells might represent an interesting novel treatment option for keloids. Keloids are fibroproliferative, protruding scar-like pathologies of the skin 1 characterized by a persisting, gradual growth beyond the margin of the wound into the surrounding healthy skin. In susceptible individuals even minor skin injuries, such as insect bites or vaccinations, can induce keloid formation 2, 3 . Although keloids show some tumor-like behavior, they do not metastasize. Nonetheless, keloids can cause severe pain, chronic pruritus, psychosocial impairment and movement restriction due to their scar-like character [3] [4] [5] [6] . Genetic predispositions as well as chronic inflammatory processes are being discussed in disease etiology 2,6-8 . As keloids are characterized by an increased proliferation of fibroblasts and extensive over-production of ECM components, keloid research has primarily focused on the involvement of fibroblasts in the development of these lesions 1 . Although these studies have identified numerous potentially pathology-related factors, the fundamental patho-mechanistic events driving keloid formation remain unclear 1 . The limited treatment options include steroid injections, -radiation, and surgery, but the majority of patients still suffer from high recurrence rates 4, 9, 10 , which underline the importance of identifying novel therapeutic approaches. Due to the pruritic and painful nature of keloids, a neuronal contribution to the pathogenesis is conceivable. The subepidermal nerve plexus of the skin is the largest sensory organ of the human body. There is growing evidence that cutaneous innervation plays an important role in mediating wound healing 15, 16 . It is, thus, surprising that a contribution of cutaneous nerves in keloid formation is poorly investigated [11] [12] [13] [14] . The major cellular constituents of peripheral nerves are Schwann cells. In the healthy skin, Schwann cells ensheath cutaneous axons and ensure the integrity and function of sensory neurons [17] [18] [19] . Recently, increasing attention was drawn to Schwann cells because of their ability to adopt a transient repair phenotype in response to peripheral nerve injury 20,21 . This process involves a de-differentiation step into a proliferative precursor or immature-like Schwann cell state and the acquisition of repair specific functions 22 . These dedicated repair Schwann cells express (neuro)trophic factors to support neuronal survival and form regeneration tracks (Bungner bands) to promote axonal outgrowth and guidance 23, 24 . Furthermore, repair Schwann cells release a plethora of chemokines and cytokines to attract macrophages, thereby contributing to clear the lesion from myelin debris and remodelling the ECM to facilitate nerve regeneration [25] [26] [27] [28] . Previous studies further support that the interaction between Schwann cells and macrophages affect their phenotype. While macrophages are known to regulate repair Schwann cell re-differentiation, Schwann cells promote the induction of M2 polarization of macrophages 29 . Macrophages play a crucial role in cutaneous wound healing by modulating the microenvironment during the different healing stages [30] [31] [32] . Especially M2-macrophages are associated with fibrosis and scarring and persist in keloids 31, 33, 34 . Moreover, a recent study in mice reported that the wound microenvironment 4 is a key determinant of Schwann cell behavior, influencing their proliferation status, reprogramming into mesenchymal-like cells, immune signaling and ECM production 35 . Indeed, Schwann cells have been shown to contribute not only to nerve regeneration but also to wound healing by regulating myofibroblast differentiation, epithelial proliferation and ECM formation 36 . Hence, Schwann cells are important players in cutaneous wound healing processes and might play an as yet underappreciated role in fibrotic processes. In the present study we performed single-cell RNA sequencing (scRNAseq) of keloids to analyze the entire cellular spectrum and the transcriptional landscape at a single cell resolution and to identify hitherto underestimated cellular and molecular players of keloid pathogenesis. Our analysis revealed a yet not described population of highly plastic keloidal Schwann cells. The vast majority of keloidal Schwann cells was not associated with axons, displayed a dedifferentiated, repair-like phenotype, and showed key features with a high potential for affecting ECM deposition and macrophage function. Our findings suggest that an abnormal reaction of Schwann cells to skin injuries contribute to keloid pathogenesis. 5 To investigate the cellular composition of keloids, we performed scRNAseq and compared our data with a recently published scRNAseq data set of normal human skin 37 . In total, transcriptomic data of 19598 cells from normal skin and 47478 cells from keloids were analyzed. After unbiased cluster generation (Fig. 1a) , cell clusters were identified with well- well-vascularized (smooth-muscle-actin positive) tissue of keloids compared to healthy skin ( Supplementary Fig. 2) . Strikingly, relative numbers of Schwann cells (S100B + /NGFR + ) were strongly increased in keloids (Fig. 1b) . To compare the SC populations between skin and keloid samples, IF staining for the Schwann cell marker S100B was performed. In healthy skin S100B stained epidermal Langerhans cells, melanocytes and dermal Schwann cells found in the subepidermal nerve plexus, nerve structures around sweat glands, and larger nerve bundles of the deep dermis (Fig. 1c) . By contrast, S100B staining of keloids showed a scattered and disorganized distribution of Schwann cells in the dermis (Fig. 1c) . Morphologically, Schwann cells in keloids showed an elongated, bipolar shape with a spindle-shaped body (Fig. 1d) . To further determine whether Schwann cells were associated with neurons, we stained 100 µm thick transversal dermal sections of normal skin and keloids with S100B and the axon marker PGP9.5. Throughout all dermal layers of healthy skin, Schwann cells were found to ensheath axons (Fig.2a) , while the vast majority of Schwann cells in keloids was not associated with axons up to 6 mm depth (Fig. 2b) (Supplementary movies [1] [2] [3] [4] [5] . Only in the deep dermis of keloids (6000-8500 µm), neuron-ensheathing Schwann cells were detected. To characterize the Schwann cell population in keloids in more detail, we performed subclustering and detected four Schwann cell subtypes exclusively present in keloids (SC-Repair, SC-EC, SC-FB and SC-Prolif). One major Schwann cell subtype, here referred to as SC-Skin was detected specifically in normal skin, and one Schwann cell subtype referred to 6 as SC-Promyel was present in both normal skin and keloids (SC-Promyel) ( Fig. 3a and Supplementary Fig. 3a) . A total of 370 genes were differentially expressed between skin-and all keloid-derived Schwann cells (144 up-and 226 down-regulated) (Fig. 3b) . The skin-specific Schwann cell cluster (SC-Skin) expressed several genes characteristic for myelinating Schwann cells [myelin basic protein (MBP), proteolipid protein (PLP1), peripheral myelin protein 22 (PMP22) and myelin protein zero (MPZ)] as well as genes commonly expressed in non-myelinating Schwann cells [neural cell adhesion molecule 1 (NCAM1), L1 cell adhesion molecule (L1CAM)] 23 (Fig. 3c) . The Schwann cell cluster present in both tissues was enriched in the keloids, presumably representing pro-myelinating Schwann cells (SC-Promyel), characterized by reduced expression levels of MPZ and PLP1 and no MBP (Fig. 3c ). In line with our scRNAseq data, double staining of S100B with MBP confirmed that myelinating Schwann cells were present in healthy skin but absent from keloids (Fig. 5a ). The majority of Schwann cells present in keloids highly expressed nestin (NES), insulin-like growth factor-binding protein 3 (IGFBP3), insulin-like growth factor-binding protein 5 (IGFBP5), transforming growth factor beta-induced (TGFBI), TNF-alpha induced protein 6 (TNFAIP6) and cellular communication network factor 3 (CCN3) (Fig. 3a and 3d ) 21, [39] [40] [41] . To validate our transcriptomics data on the protein level, we performed double staining of S100B and nestin, visualizing that keloidal Schwann cells were highly positive for nestin (Fig. 4a) . Nestin is a known marker for neural precursor cells, involved in neuronal/glial development, which is also upregulated in human repair Schwann cells 42, 43 . To characterize the cellular state of keloidal Schwann cells in more detail, we conducted IF staining for SRY-box transcription factor 10 (SOX10) and nerve growth factor receptor (NGFR), which are both known to be upregulated in immature/de-differentiated Schwann cells, as well as the transcription factor JUN which was demonstrated to be a key factor determining the repair identity of Schwann cells 21 . NGFR was strongly expressed by keloidal Schwann cells but also weakly expressed by vascular cells (Fig. 4b ). SOX10 nuclear expression was exclusively found in keloidal Schwann cells (Fig.4c ). Of note, keloidal Schwann cell nuclei were positive for c-JUN (Fig. 4d ) but c-JUN expression was also found in other cells. Based on the characteristic elongated keloidal Schwann cell morphology together with the expression of markers associated with de-differentiation and repair, the major Schwann cell type in the keloids could be assigned to a repair-like cellular state (SC-Repair). In addition, we detected a SC cluster (SC-Prolif) with high expression of genes associated with cell division, such as marker of proliferation Ki-67 (MKI67) and DNA-Topoisomerase 2-alpha (TOP2A) exclusively in keloids but not in normal skin ( Fig. 3a and 3d) . IF staining of keloids with KI-67 in combination with NGFR, confirmed the presence of proliferating Schwann cells in situ, indicating that keloidal Schwann cells have the ability to re-enter the cell-cycle (Fig. 5c . IF stainings confirmed our single cell data and showed double positive cells for S100B and CD31 (SC-EC; Fig. 5d ) as well as S100B and CD90 in keloids (SC-FB; Fig. 5e ). In healthy skin, CD90-positivity was only observed in axons but not in Schwann cells (Fig. 5e) 44 . Compared to all other S100B-positive Schwann cells, SC-EC showed a different morphology, as they were oval shaped without extensions (Fig. 5d) In silico analysis of the Schwann cell differentiation behaviour in keloids Upon peripheral nerve injury, non-and myelinating Schwann cells start to de-differentiate, regain migratory and proliferative properties, and perform specific repair functions to support the regeneration of damaged nerves. 23,36 To investigate whether similar processes are obvious during keloid development, we performed pseudotime trajectory analysis. Pseudotime trajectory suggested that keloidal Schwann cells originate from differentiated myelinated Schwann cells (Fig. 6a) . Moreover, our calculation indicated that some of these repair Schwann cells acquired a proliferative state or de-differentiated into fibroblasts-like or endothelial-like Schwann cells (Fig. 6a) . We further investigated changes in Schwann cell gene expression along the pseudotime axis. While expression of genes associated with myelination (neuroblast differentiation-associated protein (AHNAK), caveolin 1 (CAV1), cluster of differentiation 9 (CD9), neuronal membrane glycoprotein M6-B (GPM6B), MBP, MPZ, PLP1 or PMP22) decreased over pseudotime (Fig. 6b) , the expression of genes associated with Schwann cell precursor cells, repair Schwann cells and nerve regeneration, such as CCN3, neurexin 1 (NRXN1), platelet derived growth factor alpha chain (PDGFA), pleiotrophin (PTN), protein tyrosine phosphatase receptor type Z1 (PTPRZ1), proliferation-inducing protein 33 (SPARCL1) and zinc finger e-box binding homeobox 2 (ZEB2) increased (Fig. 6c) [45] [46] [47] [48] [49] [50] [51] [52] . Furthermore, factors associated with cell migration, including calreticulin (CALR), TGFBI and tenascin c (TNC) [53] [54] [55] , increased along the pseudotime trajectory. A more complete set of genes regulated along the pseudotime trajectory is provided in supplementary Fig. 6 . These findings 8 support the hypothesis that keloidal Schwann cells originate from differentiated myelinated Schwann cells by decreasing the expression of myelin genes while upregulating genes involved in repair mechanisms and migration. Keloidal Schwann cells contribute to the formation of the extracellular matrix Pseudotime trajectory analysis indicated functional changes of Schwann cells in keloids. To investigate the transcriptional differences and their possible functional consequences, we utilized GO-Term enrichment analyses. Genes highly expressed in normal skin Schwann cells were strongly associated with myelination and neuron development ( Supplementary Fig. 7a ), as well as membrane assembly, macrophage chemotaxis and dendritic cell differentiation ( Supplementary Fig. 7b ). GO-terms of the keloid-specific Schwann cell clusters differed significantly from those of Schwann cells present in normal skin. The most prominent GOterms found in SC-Prolif were associated with cell proliferation processes ( Supplementary Fig. 8a ). Genes specifically expressed in SC-EC were mainly associated with the regulation of inflammatory response, and response to bacteria but also with vasculature development and regulation of epithelial cell differentiation ( Supplementary Fig. 8b ). The gene set enriched in SC-FB showed strong association with processes regulating the production and assembly of the ECM (Supplementary Fig. 8c ). Interestingly, genes strongly expressed in SC-Repair were associated with organization of the ECM, response to wound healing and connective tissue development (Fig. 6d) . Overall, the expression of many genes associated with ECM production and assembly 56 was significantly up-regulated in keloids (Supplementary Fig. 9 -11) and various matrix-associated genes were enriched specifically in keloidal Schwann cells (Fig. 6e ). Since inflammatory processes have been reported to be involved in the pathogenesis of keloids, we further investigated the expression of factors related to skin inflammation. However, the expression of inflammatory mediators was even down-regulated in keloids compared to healthy skin ( Supplementary Fig. 12 ) and not regulated in Schwann cells (Fig. 6f ). Together these data indicate a contribution of Schwann cells to the organization of the ECM but not the inflammatory milieu in keloids. Since the majority of Schwann cells in keloids were not associated with axons and displayed a repair-related phenotype, we next explored whether similar Schwann cell populations are found in cutaneous neurofibroma type 1 (NF1), a benign skin-tumor originating from Schwann cells 57 . Therefore, we compared our data set with previously published scRNAseq data of NF1 58 . The re-calculated UMAP (Fig. 7a ) and cluster markers (Fig. 7b ) differed significantly is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 10, 2021. 9 between SC-NF1, SC-Skin and most keloidal Schwann cells. Only the SC-EC cluster showed high transcriptional resemblance with SC-NF1 (Fig. 7a , left and middle panel; red oval) resulting in a new shared cluster after combined calculation (Fig. 7a , right panel and Fig. 7c ). Cells of this combined cluster displayed a transcriptional profile associated with inflammatory processes (Supplementary Fig. 13a) . Importantly, no repair-related Schwann cells were found in NF1. In addition, pseudotime trajectory of the combined data set showed that SC-Repair and SC-NF1 represent two separate branches, both originating from myelinating skin Schwann cells (Fig. 7d) . Expression of genes associated with matrix formation was decreased in NF1, suggesting that NF1-derived Schwann cells are not involved in tissue remodelling processes ( Fig. 7e) . However, expression of several inflammatory mediators, such as interleukin 6 (IL6), interleukin 8 (CXCL8), nuclear receptor 4A1 (NR4A1) and nuclear receptor 4A2 (NR4A2) was strongly upregulated in the NF1-derived Schwann cells (Fig. 7f) , indicating that they contribute to tissue inflammation. Hence, our comparison shows that Schwann cells in keloids and Schwann cells in NF1 differ drastically in their ability to affect tissue remodeling and inflammation. Since denervated Schwann cells are known to interact with macrophages 26 , we next We then focused our analysis on Schwann cell-derived factors that are known to influence macrophage function. Expression data of keloidal Schwann cells revealed several genes coding for secreted proteins, which are involved in the regulation of macrophage function. One of the strongest upregulated genes in SC-Repair was tumor necrosis factor alpha-induced protein 6 (TNFAIP-6) (Fig. 8c) , which has been shown to inhibit inflammation 59-61 and promote is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 10, 2021. ; https://doi.org/10.1101/2021.08.09.21261701 doi: medRxiv preprint M2 polarization of macrophages 62, 63 . In addition, we found an upregulation of CCN3 in keloidal Schwann cells (Fig. 8c) , a growth factor known to promote macrophage recruitment and differentiation into a M2 phenotype 64 . Interestingly, CCN3 was also upregulated in melanocytes in keloids. In accordance with a M2-promoting environment, CC-chemokine ligand 2 (CCL2), a chemokine important for the recruitment of inflammatory M1 macrophages in wounds 65, 66 , was almost completely absent in keloids (Fig. 8c) . Furthermore, CCL3 expression was strongly downregulated in keloids (Fig. 8c) . Since CCL2 and CCL3 in combination with TNF-alpha (TNF) are known to enhance the production and release of the ECM-degrading enzyme matrix metallopeptidase 9 (MMP9) in monocytes 67 , we next investigated MMP9 levels in our data set. While MMP9 was expressed by macrophages and dendritic cells in healthy skin, we detected no MMP9 expression in keloids (Fig. 8c) , and the total release of MMP9 protein was strongly reduced in keloids (Fig. 8d) . Macrophages have been reported to regulate Schwann cell dynamics during nerve regeneration by supporting Schwann cell re-myelinisation and maturation through growth arrest-specific 6 (GAS6) 68 . Indeed, GAS6 expression was significantly decreased in MAC-M2 and MAC-M1/M2 in keloids (Fig. 8c ). Among the strongest upregulated genes in SC-Repair was IGFBP5, a known pro-fibrotic factor supporting macrophage migration and conversion of monocytes into mesenchymal cells (Fig. 8c) [69] [70] [71] . IGFBP3, another member of the IGFBP protein family with known anti-inflammatory activity 72 was also significantly upregulated in keloidal Schwann cells (Fig. 8c) . To validate our scRNAseq data, we quantified several of the identified factors in skin and keloid biopsy lysates. We detected elevated CCN3 levels, while CCL2, MMP9, and GAS6 were significantly decreased in keloids compared to healthy skin (Fig. 8d ). In addition, immunofluorescence staining confirmed increased IGFBP5 in the dermis of keloids compared to normal skin (Fig. 8e ). Together, these data indicate a crosstalk of keloidal Schwann cells and macrophages promoting increased matrix production by Schwann cells whilst inhibiting matrix degradation by macrophages. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 10, 2021. To date, the pathophysiological processes underlying keloid development are still poorly understood. Thus, we here performed a comprehensive scRNAseq approach of keloids supported by confocal microscopy imaging. Our findings introduce Schwann cells as novel players involved in keloid pathology, and support their plasticity as promising therapeutic target. However, further studies are necessary to address this question. Tumor innervation and the contribution of the nervous system to keloid pathology have been hardly explored so far, and previous publications are contradictory [13] [14] [15] [16] . As the major constituents of nerves are axons and Schwann cells, it was striking that the majority of keloidal Schwann cells were not associated with an axon. These axon-free Schwann cells had a spindle shaped morphology, comparable to that recently described for repair Schwann cells 20,24 . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 10, 2021. ; 12 wound closure 36 . Of note, a connection between Schwann cell-density in the skin and impaired wound healing has also been demonstrated in humans 73 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 10, 2021. ; Our study revealed an important cross-talk between Schwann cells and macrophages ( Supplementary Fig. 16 ). Macrophages are known to significantly contribute to the stromal milieu by affecting repair processes, tissue inflammation and matrix remodeling in a macrophage subtype-specific manner 30-32 . Whereas M1-macrophages are pro-inflammatory and matrix-degrading, M2-macrophages contribute to the composition of the ECM 31 . In line with previous publications 33,34 , we mainly detected M2-macrophages in our keloids. In addition, we identified one macrophage population with intermediate M1/M2 gene expression pattern and one subpopulation sharing gene sets specific for M2-macrophages and fibroblasts. The ability of macrophages to convert into mesenchymal cells is well documented and IGFBP5, one of the strongest expressed factors in keloidal Schwann cells, has been shown to support this process 71, 76 . Although several genes associated with epithelial to mesenchymal transition, such as SNAI1, SNAI2, ZEB1, ZEB2, TWIST1, were not detected in this cell population (data not shown) 77 , our data indicate that, similar to Schwann cells, macrophages also show a high plasticity in keloids. Our data further revealed that repair Schwann cells present in keloids produced several factors affecting macrophage function. For example, we detected high levels of TNFAIP6, IGFBP5 and CCN3, all known to regulate migration, activation and polarization of macrophages [62] [63] [64] 71 . Interestingly, CCL2 was strongly down-regulated in keloidal Schwann cells, contributing to significantly reduced overall CCL2 protein levels of keloids. As CCL2 is one of the most important factors provoking the accumulation of M1-type macrophages in the wound area 65, 66 , reduced CCL2 levels might represent a crucial initial step in the development of keloids. CCL3 and TNF- were also down-regulated in keloids. All three factors together are known to be important for the production of macrophage-derived MMP9 67 . Indeed, MMP9 protein production was almost completely abolished in keloids. Interestingly, lack of MMP9 does not only contribute to less degradation of the ECM, but also affects Schwann cell function, as MMP9 has been shown to inhibit Schwann cell de-differentiation and proliferation 78 . In summary, our study indicates that the interaction of repair-like Schwann cells and macrophages in keloids leads to increased matrix deposition, which could be responsible for their infinite growth. It is tempting to speculate that intervention at any point of this cycle might represent a promising treatment option. Our data suggest that especially the cellular plasticity of keloidal Schwann cells presents a promising therapeutic target. Unfortunately, little effort has been put in the development of proper model systems to study keloid formation and progression, and the few available models lack important cell types, including Schwann cells. Therefore, possible therapeutic options still need to be tested in costly ex vivo cultures or directly in clinical studies. This highlights the urgent need for standardized, high quality preclinical model systems to develop efficient treatment procedures for keloids. Together, our study opens a new perspective on the pathogenesis of keloids, which could significantly improve the treatment of this skin disease in the future. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 10, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 10, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 10, 2021. ; https://doi.org/10.1101/2021.08.09.21261701 doi: medRxiv preprint For enrichment analysis, Metascape was used 87 . A p-value cutoff of 0.05 and a minimum enrichment score of 2 was set. For cryopreservation, tissues were washed with PBS and fixed in 4.5% formaldehyde solution, neutral buffered (SAV Liquid Production GmbH, Flintsbach am Inn, Germany) for 24 hours at 4°C. Specimens were washed with PBS for 24 hours and dehydrated by sequential incubation with 10%, 25%, and 42% succrose for 24 hours each. Tissues were snap-frozen in optimal cutting temperature compound (OCT compound, TissueTek, Sakura, Alphen aan den Rijn, The Netherlands) and stored at -80°C. Ten µm sections were cut using a cryotome (Leica, Wetzlar, Germany) and dried for 30 min at room temperature. Cryosections were immersed in PBS followed by blocking and permeabilization with 1% BSA, 5% goat serum (DAKO, Glostrup, Denmark), and 0.3% Triton-X (Sigma Aldrich) in PBS for 15 min. For paraffin embedding, tissues were washed with PBS and 6 mm punches were obtained. Biopsies were cut in a half and each part was fixed in 4.5% formaldehyde solution overnight and embedded in paraffin. After de-paraffinization and hydration, sections were boiled in Target Retrieval Solution (DAKO) using a 2100 Antigen Retriever (DAKO) followed by three washes with PBS for 5 minutes. Antibody details, dilutions, and incubation times are listed in Supplementary Table S3 . A washing step consisted of three washes with PBS for 5 minutes and was performed after each antibody incubation step. If stained for S100 (DAKO), primary antibodies were diluted in the ready to use S100 antibody Confocal images are depicted as maximum projection of total z-stacks. All stainings were performed on cryo-as well as paraffin-preserved tissue samples. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 10, 2021. For statistical evaluation, GraphPad Prism 8 software (GraphPad Software Inc., La Jolla, CA, USA) was used. Normal distribution within a group was tested by Shapiro-Wilk test. Comparison between two groups with normal distribution was performed with paired t-test. Independent groups without normal distribution were compared by Mann-Whitney-U-Test. Asterisks were used to mark p-values: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ScRNASeq data are available in NCBI´s Gene Expression Omnibus (GEO) and accessible through GEP series accession number GSE181316. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 10, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 10, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 10, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 10, 2021. Nerve-autonomous Schwann cell distribution throughout the whole keloidal dermis Bird´s-eye view of 100 µm-thick sections of (a) healthy skin and (b) keloids dermal sheets. S100B indicates Schwann cells and PGP 9.5 nerve fibers. Vertical reference bar illustrates depth of the depicted sheet region below the skin surface. Scale bar: 100 µm. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 10, 2021. Immunofluorescence staining confirmed predicted Schwann cell subtypes in keloid tissue Immunostainings of keloidal Schwann cells for (a) S100B and Nestin, (b) S100B and nerve growth factor receptor (NGFR), (c) S100B and SRY-box transcription factor 10 (SOX10) and is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 10, 2021. ; https://doi.org/10.1101/2021.08.09.21261701 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 10, 2021. ; https://doi.org/10.1101/2021.08.09.21261701 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 10, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 10, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 10, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 10, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 10, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 10, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 10, 2021. ; https://doi.org/10.1101/2021.08.09.21261701 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 10, 2021. ; https://doi.org/10.1101/2021.08.09.21261701 doi: medRxiv preprint S100B DAPI NGFR is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 10, 2021. ; https://doi.org/10.1101/2021.08.09.21261701 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint TNFRSF12A TNFAIP6 TNC TMSB10 TGFBI TBCA SPARCL1 S100A6 S100A4 S100A16 S100A13 S100A11 S100A10 PTPRZ1 PTN PPP1R14B PPFIBP1 PMEPA1 PLS3 PHLDA2 PDGFA NID1 NES MYL6 LSM7 ITGB1 IGFBP5 IGFBP3 GAS7 FABP5 ENC1 EMP3 ELN CSRP2 COL8A1 COL7A1 COL4A2 COL4A1 COL18A1 COL12A1 CDH6 CDH2 CCND1 CCN3 ATP5F1E ABI2 GO annotated Gene is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 10, 2021. ; https://doi.org/10.1101/2021.08.09.21261701 doi: medRxiv preprint LGALS1 LGALS3 MMP15 CCN3 SPARC SPARCL1 TGFBI TIMP1 TIMP2 TIMP3 TNC is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 10, 2021. ; https://doi.org/10.1101/2021.08.09.21261701 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 10, 2021. ; https://doi.org/10.1101/2021.08.09.21261701 doi: medRxiv preprint The Keloid Disorder: Heterogeneity, Histopathology, Mechanisms and Models Keloids and hypertrophic scars Keloids and hypertrophic scars New insights on keloids, hypertrophic scars, and striae DLQI scores in patients with keloids and hypertrophic scars: a prospective case control study Keloids in various races. A review of 175 cases Study of 1,000 patients with keloids in South India Epidemiology of keloids in normally pigmented Africans and African people with albinism: population-based cross-sectional survey International clinical recommendations on scar management Novel signals controlling embryonic Schwann cell development, myelination and dedifferentiation Sox10 is required for Schwann cell identity and progression beyond the immature Schwann cell stage The transcription factor Sox10 is a key regulator of peripheral glial development Proteomics and transcriptomics of peripheral nerve tissue and cells unravel new aspects of the human Schwann cell repair phenotype The adhesion GPCR Gpr56 regulates oligodendrocyte development via interactions with Gα12/13 and RhoA Developmentally regulated expression of Thy-1 in structures of the mouse sensory-motor system Zeb2 is essential for Schwann cell differentiation, myelination and nerve repair The giant protein AHNAK involved in morphogenesis and laminin substrate adhesion of myelinating Schwann cells Schwann cell caveolin-1 expression increases during myelination and decreases after axotomy Schwann cell plasticity regulates neuroblastic tumor cell differentiation via epidermal growth factor-like protein 8 Glial M6B stabilizes the axonal membrane at peripheral nodes of Ranvier Schwann cell CD9 expression is regulated by axons Secretome analysis of nerve repair mediating Schwann cells reveals Smaddependent trophism Regulation of tyrosine phosphorylation and protein tyrosine phosphatases during oligodendrocyte differentiation Mechanism of miR-148b inhibiting cell proliferation and migration of Schwann cells by regulating CALR Fibroblast-derived tenascin-C promotes Schwann cell migration through β1-integrin dependent pathway during peripheral nerve regeneration Enhanced Expression of TGFBI Promotes the Proliferation and Migration of Glioma Cells The extracellular matrix: Tools and insights for the "omics" era Modeling tumors of the peripheral nervous system associated with Neurofibromatosis type 1: Reprogramming plexiform neurofibroma cells Human cutaneous neurofibroma matrisome revealed by single-cell RNA sequencing The link module from human TSG-6 inhibits neutrophil migration in a hyaluronan-and inter-alpha -inhibitor-independent manner Mesenchymal stem cells transplantation suppresses inflammatory responses in global cerebral ischemia: contribution of TNF-α-induced protein 6 TSG-6 protein, a negative regulator of inflammatory arthritis, forms a ternary complex with murine mast cell tryptases and heparin TSG-6 Inhibits Oxidative Stress and Induces M2 Polarization of Hepatic Macrophages in Mice With Alcoholic Hepatitis via Suppression of STAT3 Activation TNFα-stimulated gene-6 (TSG6) activates macrophage phenotype transition to prevent inflammatory lung injury Prostate cancer-derived CCN3 induces M2 macrophage infiltration and contributes to angiogenesis in prostate cancer microenvironment CCR2 promotes hepatic fibrosis in mice Hepatic recruitment of the inflammatory Gr1+ monocyte subset upon liver injury promotes hepatic fibrosis Chemokine stimulation of monocyte matrix metalloproteinase-9 requires endogenous TNF-alpha Macrophages Regulate Schwann Cell Maturation after Nerve Injury Insulin-like growth factor binding protein 5 induces skin fibrosis: A novel murine model for dermal fibrosis Insulin-like growth factor-binding protein-5 induces pulmonary fibrosis and triggers mononuclear cellular infiltration The pro-fibrotic factor IGFBP-5 induces lung fibroblast and mononuclear cell migration Suppression of IGF binding protein-3 by palmitate promotes hepatic inflammatory responses Rarefaction of the peripheral nerve network in diabetic patients is associated with a pronounced reduction of terminal Schwann cells An inflammatory gene signature distinguishes neurofibroma Schwann cells and macrophages from cells in the normal peripheral nervous system How does the Schwann cell lineage form tumors in NF1? 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