TEM1/endosialin/CD248 promotes pathologic scarring and TGF-ß activity through its receptor stability in dermal fibroblasts.

BACKGROUND: Pathologic scars, including keloids and hypertrophic scars, represent a common form of exaggerated cutaneous scarring that is difficult to prevent or treat effectively. Additionally, the pathobiology of pathologic scars remains poorly understood. We aim at investigating the impact of TEM1 (also known as endosialin or CD248), which is a glycosylated type I transmembrane protein, on development of pathologic scars. METHODS: To investigate the expression of TEM1, we utilized immunofluorescence staining, Western blotting, and single-cell RNA-sequencing (scRNA-seq) techniques. We conducted in vitro cell culture experiments and an in vivo stretch-induced scar mouse model to study the involvement of TEM1 in TGF-ß-mediated responses in pathologic scars. RESULTS: The levels of the protein TEM1 are elevated in both hypertrophic scars and keloids in comparison to normal skin. A re-analysis of scRNA-seq datasets reveals that a major profibrotic subpopulation of keloid and hypertrophic scar fibroblasts greatly expresses TEM1, with expression increasing during fibroblast activation. TEM1 promotes activation, proliferation, and ECM production in human dermal fibroblasts by enhancing TGF-ß1 signaling through binding with and stabilizing TGF-ß receptors. Global deletion of Tem1 markedly reduces the amount of ECM synthesis and inflammation in a scar in a mouse model of stretch-induced pathologic scarring. The intralesional administration of ontuxizumab, a humanized IgG monoclonal antibody targeting TEM1, significantly decreased both the size and collagen density of keloids. CONCLUSIONS: Our data indicate that TEM1 plays a role in pathologic scarring, with its synergistic effect on the TGF-ß signaling contributing to dermal fibroblast activation. Targeting TEM1 may represent a novel therapeutic approach in reducing the morbidity of pathologic scars.

Dominant dystrophic epidermolysis bullosa is associated with glycolytically active GATA3+ Th2 cells which may contribute to pruritus in lesional skin.

BACKGROUND: Dominant dystrophic epidermolysis bullosa (DDEB) is characterized by trauma-induced blisters and, in some individuals, intense pruritus. Precisely what causes itch in DDEB and optimal ways to reduce it have not been fully determined. OBJECTIVE: To characterize DDEB skin transcriptomes to identify therapeutic targets to reduce pruritus in patients. METHODS: We evaluated affected and unaffected skin biopsy samples from 6 DDEB subjects (all with the very itchy pruriginosa subtype), and 4 healthy individuals using bulk RNA-seq. Single-cell transcriptomes of affected (n=2) and unaffected (n=1) DDEB and healthy skin (n=2) were obtained. Dupilumab treatment was provided for three patients. RESULTS: The skin bulk transcriptome showed significant enrichment of Th1/2 and Th17 pathways in affected DDEB skin compared with non-lesional DDEB and healthy skin. Single-cell transcriptomics showed an association of glycolytically active GATA3+ Th2 cells in affected DDEB skin. Treatment with dupilumab in three people with DDEB led to significantly reduced VAS itch scores after 12 weeks (mean VAS=3.83) compared to pre-treatment (mean VAS=7.83). Bulk RNA-seq and qPCR showed that healthy skin and dupilumab-treated epidermolysis bullosa (EB) pruriginosa skin show very similar transcriptomic profiles, and reduced Th1/2 and Th17 pathway enrichment. CONCLUSIONS: Single-cell RNA-seq helps define an enhanced DDEB-associated Th2 profile and rationalizes drug repurposing of anti-Th2 drugs in treating DDEB pruritus.

Tofacitinib ameliorates skin inflammation in a case of severe autosomal recessive congenital ichthyosis.

Autosomal recessive congenital ichthyosis (ARCI) is a genetically heterogeneous disorder manifesting aberrant skin scaling and increased transepidermal water loss (TEWL). Current treatments for ARCI are limited and sub-optimal. We studied a 27-year-old man with ARCI resulting from a homozygous missense variant in TGM1 (transglutaminase 1). RNA-sequencing of lesional skin revealed aberrant JAK-STAT signalling, providing a rationale for innovative treatment with a Janus kinase inhibitor. We prescribed oral tofacitinib (11 mg daily) for 26 weeks. Rapid improvements in erythema and fissuring manifested within the first month. Sustained reductions in 5-D itch scale and Dermatology Life Quality Index (DLQI) scores were also observed. TEWL decreased for the first 10 weeks but increased thereafter. Tofacitinib down-regulated inflammatory genes and pathways, while enhancing skin barrier markers. Moreover, TGM1 distribution was normalized although enzymatic activity remained deficient. This study suggests that oral tofacitinib may be a useful therapy to consider in patients with ARCI.

A Significant Association between Type 1 Diabetes and Helicobacter pylori Infection: A Meta-Analysis Study.

Background and Objectives: Type 1 diabetes mellitus (T1DM) is a chronic and serious condition that is characterized by inadequate pancreatic-ß-cells' insulin production. The connection between T1DM and Helicobacter pylori infection remains uncertain. This study aimed to conduct a systematic meta-analysis to examine the association between H. pylori infection, hemoglobin A1c levels, and the development of T1DM. Materials and Methods: The initial search identified 451 articles on the association between H. pylori infection and T1DM. Among them, 12 articles had 2797 participants who met the inclusion criteria for an advanced meta-analysis. Results: A significant association was observed between H. pylori infection and T1DM (OR 1.77, 95% CI 1.47-2.12, p < 0.0001), with heterogeneity: Tau2 = 0.47; Chi2 = 57.07, df = 11 (p < 0.0001); I2 = 81%. Subgroup analysis showed that H. pylori infection was significantly associated with a longer duration of T1DM and higher hemoglobin A1c levels (p < 0.001 for both) but not with age at T1DM diagnosis (p = 0.306). Conclusions: These findings contribute to the understanding of the association between H. pylori infection and T1DM and highlight the potential role of H. pylori in influencing the duration and glycemic control of diabetes. Therefore, pediatric patients who have longstanding T1DM and poor glycemic control should be screened for H. pylori infection.

Transcriptomic responses of peripheral blood mononuclear cells to cyclosporin and etanercept in a female infant with juvenile generalized pustular psoriasis.

Generalized pustular psoriasis (GPP) is a rare but severe form of psoriasis. An early onset of the diseases is correlated with mutations among IL36RN, CARD14, AP1S3, MPO and SERPINA3 genes. Systemic biological agents including anti-TNF-α, anti-IL-17, anti-IL-12/IL-23, anti-IL1R, anti-IL1ß and anti-IL-36R act as novel treatment methods for GPP. Herein we report a female infant clinically diagnosed with GPP since she was 10-month-old. Results of whole-exome sequencing (WES) and Sanger sequencing revealed a reported heterozygous IL36RN (c.115+6T>C) and another reported heterozygous SERPINA3 frame-shifting variant (c.1247_1248del). Initial cyclosporin treatment for the patient led to a partial remission of the symptoms. However, the patient reached nearly total remission of pustules and erythema after anti-TNF-α inhibitor etanercept treatment. Results of further RNA sequencing (RNA-seq) done on peripheral blood mononuclear cells correlated with the clinical responses, showing that cyclosporin suppressed a portion of the neutrophil-related genes, while most genes associated with neutrophil activation, neutrophil-mediated immunity and degranulation were downregulated by the subsequent etanercept treatment. We report this case to demonstrate WES and RNA-seq in combination could come in handy in reaching a precise diagnosis and in evaluating or even predicting the molecular alterations underlying clinical treatment effectiveness.

Aberrant expression of interleukin-17A in mast cells contributes to the pathogenesis of hidradenitis suppurativa.

BACKGROUND: Hidradenitis suppurativa (HS) significantly diminishes the quality of life for patients. Delayed diagnosis represents a significant challenge in effectively managing HS. OBJECTIVES: To identify and characterize the key mediator in HS. METHODS: Bioinformatic transcriptomic analysis was applied to identify potential candidates contributing to the disease process of HS. Skin samples from 40 patients with HS, four with psoriasis and 29 with normal skin were included. The expression of interleukin (IL)-17A was evaluated and compared among samples of normal skin, psoriatic skin and skin from different stages of HS by immunohistochemistry or dual-colour immunofluorescence. In vitro experiments and RNA sequencing analysis were also conducted to validate the expression of IL-17A and its pathogenic effect in HS. RESULTS: Transcriptomic database analyses identified IL-17 signalling as a potential contributor to HS. In HS, the predominant IL-17A+ cell population was identified as mast cells. IL-17A+ mast-cell density was significantly elevated in HS, especially in samples with advanced Hurley stages, compared with normal skin and psoriasis samples. The close contact between IL-17A+ mast cells and IL-17 receptor A (IL-17RA)-expressing keratinocytes was demonstrated, along with the significant effects of IL-17A on keratinocyte cell proliferation and HS pathogenic gene expression. Treatment with biologics (brodalumab or adalimumab) reduced the severity of the disease and the number of IL-17A+ mast cells in affected tissues. CONCLUSIONS: The presence of high-density IL-17A+ mast cells may serve as a valuable pathological marker for diagnosing HS. Moreover, developing therapeutic drugs targeting IL-17A+ mast cells may provide a new approach to treating HS.

ASC-J9 Blocks Cell Proliferation and Extracellular Matrix Production of Keloid Fibroblasts through Inhibiting STAT3 Signaling.

Keloids are a fibrotic skin disorder caused by abnormal wound healing and featuring the activation and expansion of fibroblasts beyond the original wound margin. Signal transducer and activator of transcription 3 (STAT3) has been found to mediate the biological functions of keloid fibroblasts (KFs). Therefore, we aimed to demonstrate whether ASC-J9, an inhibitor of STAT3 phosphorylation, can suppress the activation of KFs. Western blotting results showed that ASC-J9 inhibited the levels of COL1A1 and FN1 proteins, which were upregulated in KFs, by decreasing the expression of pSTAT3 and STAT3. RNA sequencing and in vitro studies further demonstrated that ASC-J9 treatment of KFs reduced cell division, inflammation, and ROS generation, as well as extracellular matrix (ECM) synthesis. ELISA assays verified that ASC-J9 treatment significantly mitigated IL-6 protein secretion in KFs. Transmission electron microscopy images revealed that ASC-J9 induced the formation of multilamellar bodies in KFs, which is associated with autophagy-related signaling. These results suggested that inhibiting a vicious cycle of the ROS/STAT3/IL-6 axis by ASC-J9 may represent a potential therapeutic approach to suppress cell proliferation and ECM production in KFs.

CD248 Regulates Inflammation and Encapsulation in Silicone-Related Capsule Formation.

BACKGROUND: Capsular contracture is the most common reason for having a secondary breast implant operation. The failure of the implanted device and discomfort are related to foreign body response, which involves a pathologic encapsulation. An up-regulated expression of CD248 was previously demonstrated to modulate inflammation and fibrosis. The authors hypothesized that CD248 contributes to foreign body reaction and contracture during silicone-stimulated capsule formation. METHODS: A murine capsular contracture model was established to correlate CD248 with capsular contracture. The timing and site of CD248 expression were characterized by protein analysis and histologic examination. The capsules between wild-type mice and CD248 knockout mice were compared in this model to verify the possible role of CD248 in silicone-related capsule formation. RESULTS: CD248 was expressed in the peri-silicone implant capsule by stromal fibroblast and perivascular fibroblast. CD248 was overexpressed on day 4 and down to a constant level, but it was still up-regulated through day 21 to day 56 after silicone implantation. The CD248 knockout mice showed a prolonged inflammation period, whereas the wild-type mice developed a thinner but more collagenous capsule. CONCLUSIONS: In conclusion, an effective murine capsular contracture model was established to study the relationship between CD248 and capsular contracture. CD248 may play a role in inflammation and encapsulation during silicone implantation. CD248 deletion in mice contributed to a loose and irregular collagen bundle in a capsule area, implying a decrease in contracture. Therefore, CD248 could be a potential therapeutic target in capsular contracture. CLINICAL RELEVANCE STATEMENT: CD248 may play a role in inflammation and encapsulation during silicone implantation. It could be a potential therapeutic target in clinical capsular contracture.

Histone Trimethylations and HDAC5 Regulate Spheroid Subpopulation and Differentiation Signaling of Human Adipose-Derived Stem Cells.

Human adipose-derived stem cells (ASCs) have shown immense potential for regenerative medicine. Our previous work demonstrated that chitosan nano-deposited surfaces induce spheroid formation and differentiation of ASCs for treating sciatic nerve injuries. However, the underlying cell fate and differentiation mechanisms of ASC-derived spheroids remain unknown. Here, we investigate the epigenetic regulation and signaling coordination of these therapeutic spheroids. During spheroid formation, we observed significant increases in histone 3 trimethylation at lysine 4 (H3K4me3), lysine 9 (H3K9me3), and lysine 27 (H3K27me3), accompanied by increased histone deacetylase (HDAC) activities and decreased histone acetyltransferase activities. Additionally, HDAC5 translocated from the cytoplasm to the nucleus, along with increased nuclear HDAC5 activities. Utilizing single-cell RNA sequencing (scRNA-seq), we analyzed the chitosan-induced ASC spheroids and discovered distinct cluster subpopulations, cell fate trajectories, differentiation traits, and signaling networks using the 10x Genomics platform, R studio/language, and the Ingenuity Pathway Analysis (IPA) tool. Specific subpopulations were identified within the spheroids that corresponded to a transient reprogramming state (Cluster 6) and the endpoint cell state (Cluster 3). H3K4me3 and H3K9me3 were discovered as key epigenetic regulators by IPA to initiate stem cell differentiation in Cluster 6 cells, and confirmed by qPCR and their respective histone methyltransferase inhibitors: SNDX-5613 (a KMT2A inhibitor for H3K4me3) and SUVi (an SUV39H1 inhibitor for H3K9me3). Moreover, H3K9me3 and HDAC5 were involved in regulating downstream signaling and neuronal markers during differentiation in Cluster 3 cells. These findings emphasize the critical role of epigenetic regulation, particularly H3K4me3, H3K9me3, and HDAC5, in shaping stem cell fate and directing lineage-specific differentiation.

Profibrotic Subsets of SPP1+ Macrophages and POSTN+ Fibroblasts Contribute to Fibrotic Scarring in Acne Keloidalis.

Acne keloidalis is a primary scarring alopecia characterized by longstanding inflammation in the scalp causing keloid-like scar formation and hair loss. Histologically, acne keloidalis is characterized by mixed leukocytic infiltrates in the acute stage followed by a granulomatous reaction and extensive fibrosis in the later stages. To further explore its pathogenesis, bulk RNA sequencing, single-cell RNA sequencing, and spatial transcriptomics were applied to occipital scalp biopsy specimens of lesional and adjacent no-lesional skin in patients with clinically active disease. Unbiased clustering revealed 19 distinct cell populations, including 2 notable populations: POSTN+ fibroblasts with enriched extracellular matrix signatures and SPP1+ myeloid cells with an M2 macrophage phenotype. Cell communication analyses indicated that fibroblasts and myeloid cells communicated by SPP1 signaling networks in lesional skin. A reverse transcriptomics in silico approach identified corticosteroids as possessing the capability to reverse the gene expression signatures of SPP1+ myeloid cells and POSTN+ fibroblasts. Intralesional corticosteroid injection greatly reduced SPP1 and POSTN gene expression as well as acne keloidalis disease activity. Spatial transcriptomics and immunofluorescence staining verified microanatomic specificity of SPP1+ myeloid cells and POSTN+ fibroblasts with disease activity. In summary, the communication between POSTN+ fibroblasts and SPP1+ myeloid cells by SPP1 axis may contribute to the pathogenesis of acne keloidalis.

Inflammation in Wound Healing and Pathological Scarring.

Significance: The aberrant inflammation during wound healing results in pathological scarring, such as hypertrophic scars and keloids. This adversely affects the quality of life of patients due to the disfiguring appearance as well as the symptoms of itch and pain. This review summarizes the up-to-date knowledge of the immunopathogenesis and treatment options for pathological scars. Recent Advances: With the advent of new technologies, combined with in vitro and in vivo wound models, several inflammatory cells have been shown to have both direct and indirect effects on both wound healing and pathological scarring. Critical Issues: Expansion of pro-fibrotic immune cells such as M2 macrophages, dendritic cells, mast cells, and Th2 cells leads to fibroblast transition to myofibroblasts via transforming growth factor-ß1 signaling pathway. Appropriate management of such inflammatory responses during wound healing remains a critical issue during clinical practice. Future Directions: Regulating inflammation response during wound healing may be a potential therapeutic option for avoiding or reducing pathological scars.

Loss of RhoE Function in Dermatofibroma Promotes Disorganized Dermal Fibroblast Extracellular Matrix and Increased Integrin Activation.

Dermatofibromas (DFs) are common, benign fibrous skin tumors that can occur at any skin site. In most cases, DFs are solitary and sporadic, but a few are multiple and familial, and the mechanisms leading to these lesions are currently unclear. Using exome sequencing, we have identified a heterozygous variant in a pedigree with autosomal dominant multiple familial DF within RND3 (c.692C>T,p.T231M) that encodes for the small GTPase RhoE, a regulator of the actin cytoskeleton. Expression of T231M-RhoE or RhoE depletion using CRISPR in human dermal fibroblasts increased proliferation and adhesion to extracellular matrix through enhanced ß1 integrin activation and more disorganized matrix. The enzyme PLOD2 was identified as a binding partner for RhoE, and the formation of this complex was disrupted by T231M-RhoE. PLOD2 promotes collagen cross-linking and activation of ß1 integrins, and depleting PLOD2 in T231M-RhoE-expressing cells reduced T231M-RhoE-mediated ß1 integrin activation and led to increased matrix alignment. Immunohistochemical analysis revealed reduced expression of RhoE but increased expression of PLOD2 in the dermis of DF skin samples compared with that of the controls. Our data show that loss of RhoE function leads to increased PLOD2 activation, enhancing integrin activation and leading to a disorganized extracellular matrix, contributing to DF.

Adipose transcriptome in the scalp of androgenetic alopecia.

Previous studies have shown how adipocytes can modulate the activity of hair follicle stem cells. However, the role of adipocytes in the pathogenesis of androgenetic alopecia (AGA) remains unknown. We aimed to determine signaling pathways related to the adipose tissue changes in the human scalp with AGA through RNA-seq analysis. RNA was isolated from the adipose tissues derived from the bald (frontal) and normal (occipital) scalps of male patients with AGA (n = 4). The pooled RNA extracts from these samples were subjected to RNA sequencing, followed by differential gene expression and pathway analysis. Our gene expression analysis identified 1,060 differentially expressed genes, including 522 upregulated and 538 downregulated genes in the bald AGA scalp. Biological pathways pertaining to either adipose tissue metabolism or the hair cycle were generated in our pathway analysis. Downregulation of the peroxisome proliferator-activated receptor (PPAR) signaling pathway was noted to be significant in the bald scalp. Expression of adipogenic markers (e.g., PPARG, FABP4, PLN1, and ADIPOQ) was also decreased in the bald site. These findings imply that adipogenesis becomes downregulated in AGA, specifically within the bald scalp adipose. Our results lead to the hypothesis that PPARγ-mediated adipogenesis in the scalp adipose, via crosstalk with signaling pathways involved in hair cycling, might play a role in AGA.

Genetic Diagnosis of Rubinstein-Taybi Syndrome With Multiplex Ligation-Dependent Probe Amplification (MLPA) and Whole-Exome Sequencing (WES): Case Series With a Novel CREBBP Variant.

Rubinstein-Taybi Syndrome (RSTS) is a rare congenital disease with distinctive facial features, broadening of the thumbs and halluces, and developmental delay. RSTS is caused by de novo genetic alterations in CREBBP and the homologous EP300 genes. In this study, we established a genetic diagnostic protocol by integrating multiplex ligation-dependent probe amplification (MLPA) and whole-exome sequencing (WES). Five patients clinically diagnosed with RSTS were enrolled for genetic testing. Germline DNA was extracted from the peripheral blood of the patients and their families. One patient (case 1) was identified as harboring a large heterozygous deletion in the 16p13.3 region, spanning the CREBBP gene. Three patients (Cases 2-4) harbored different CREBBP variants (c.2608C>T:p.Gln870Ter,c.4404_4405del:p.Thr1468fs,c.3649C>T:p.Gln1217Ter). No causative variants were identified for the fifth RSTS patient (case 5). Here, we propose a molecular diagnostic protocol that identified causative genetic alterations in 4/5 of the patients, yielding a molecular diagnostic rate of 80%. Given the rarity of RSTS, more research is needed to explore its pathogenesis and mechanism.

Tumor endothelial marker 1 is upregulated in heart after cardiac injury and participates in cardiac remodeling.

Tumor endothelial marker 1 (TEM1) is a transmembrane glycoprotein that appears on mesenchymal lineage-derived cells during embryogenesis, but its expression greatly reduces after birth. Re-upregulation of TEM1 is found in tumor angiogenesis, organ fibrosis and wound healing indicating its potential role in tissue remodeling and repair. The expression level and function of TEM1 in adult heart are unknown. In explanted hearts from heart failure (HF) patients received cardiac transplantation, immunofluorescence staining showed TEM1 was expressed in cardiomyocytes (CMs) and cardiac fibroblasts. Bioinformatics analysis showed TEM1 upregulation in mouse heart after coronary ligation. Cardiac TEM1 expression was reconfirmed in mouse HF induced by coronary ligation or doxorubicin injection. TEM1 expression increased in cultured CMs stimulated with mechanical stretch, doxorubicin and hypoxia. Further studies showed recombinant TEM1 (rTEM1) was a functional protein that influenced cell behaviors of CMs. It directly activated Erk and Akt through interaction with PDGF receptor. TEM1lacZ/lacZ mice had less collagen deposition and worse cardiac function than wild type mice. These results indicate that TEM1 expression increases in the heart after cardiac injury and works as a functional protein that participates in cardiac remodeling.

Role of tumor endothelial marker 1 (Endosialin/CD248) lectin-like domain in lipopolysaccharide-induced macrophage activation and sepsis in mice.

Deleterious hyper-inflammation resulting from macrophage activation may aggravate sepsis and lead to lethality. Tumor endothelial marker 1 (TEM1), a type I transmembrane glycoprotein containing six functional domains, has been implicated in cancer and chronic sterile inflammatory disorders. However, the role of TEM1 in acute sepsis remains to be determined. Herein we explored the functional significance of the TEM1 lectin-like domain (TEM1D1) in monocyte/macrophage activation and sepsis using TEM1D1-deleted (TEM1LeD/LeD) transgenic mice and recombinant TEM1D1 (rTEM1D1) protein. Under stimulation with lipopolysaccharides (LPS) or several other toll-like receptor agonists, TEM1LeD/LeD macrophages produced lower levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 than wild-type TEM1wt/wt macrophages. Compared with TEM1wt/wt macrophages, LPS-macrophage binding and intracellular mitogen-activated protein kinase (MAPK)/nuclear factor (NF)-κB activation were suppressed in TEM1LeD/LeD macrophages. In vivo, TEM1D1 deletion improved survival in LPS-challenged mice with reduction of circulating TNF-α and IL-6 and alleviation of lung injury and pulmonary leukocyte accumulation. In contrast, rTEM1D1 could bind to LPS and markedly suppress LPS-macrophage binding, MAPK/NF-κB signaling in macrophages and proinflammatory cytokine production. Treatment with rTEM1D1 improved survival and attenuated circulating TNF-α and IL-6, lung injury and pulmonary accumulation of leukocytes in LPS-challenged mice. These findings demonstrated differential roles for the TEM1 lectin-like domain in macrophages and soluble TEM1 lectin-like domain in sepsis. TEM1 in macrophages mediates LPS-induced inflammation via its lectin-like domain, whereas rTEM1D1 interferes with LPS-induced macrophage activation and sepsis.