Revisiting aortic valve prosthesis choice in patients younger than 50 years: 10 years results of the AUTHEARTVISIT study.

OBJECTIVES: This population-based cohort study investigated mid-term outcome after surgical aortic valve replacement with a bioprosthetic or mechanical valve prosthesis in patients aged <50 years in a European social welfare state. METHODS: We analysed patient data from the main social insurance carriers in Austria (2010-2020). Subsequent patient-level record linkage with national health data provided patient characteristics and clinical outcome. Survival, reoperation, myocardial infarction, heart failure, embolic stroke or intracerebral haemorrhage, bleeding other than intracerebral haemorrhage and major adverse cardiac events were evaluated as outcomes. RESULTS: A total of 991 patients were analysed. Regarding demographics, no major differences between groups were observed. Multivariable Cox regression revealed no significant difference in overall survival (P = 0.352) with a median follow-up time of 6.2 years. Reoperation-free survival was decreased (hazard ratio = 1.560 [95% CI: 1.076-2.262], P = 0.019) and the risk for reoperation was increased (hazard ratio = 2.770 [95% CI: 1.402-5.472], P = 0.003) in patients who received bioprostheses. Estimated probability of death after reoperation was 0.23 (CL: 0.08-0.35) after 2 years and 0.34 (CL: 0.06-0.53) after 10 years over both groups. Regarding further outcomes, no significant differences between the two groups were observed. CONCLUSIONS: In patients below 50 years of age receiving aortic valve replacement, implantation of bioprostheses when compared to mechanical heart valve prostheses was associated with a significantly higher rate of reoperations and reduced reoperation-free survival. Nevertheless, we could not observe a difference in overall survival. However, long-term follow-up has to evaluate that a significantly lower rate of reoperations may translate in consistently improved long-term survival.

FAM3C/ILEI protein is elevated in psoriatic lesions and triggers psoriasiform hyperproliferation in mice.

FAM3C/ILEI is an important cytokine for tumor progression and metastasis. However, its involvement in inflammation remains elusive. Here, we show that ILEI protein is highly expressed in psoriatic lesions. Inducible keratinocyte-specific ILEI overexpression in mice (K5-ILEIind ) recapitulates many aspects of psoriasis following TPA challenge, primarily manifested by impaired epidermal differentiation and increased neutrophil recruitment. Mechanistically, ILEI triggers Erk and Akt signaling, which then activates STAT3 via Ser727 phosphorylation. Keratinocyte-specific ILEI deletion ameliorates TPA-induced skin inflammation. A transcriptomic ILEI signature obtained from the K5-ILEIind model shows enrichment in several signaling pathways also found in psoriasis and identifies urokinase as a targetable enzyme to counteract ILEI activity. Pharmacological inhibition of urokinase in TPA-induced K5-ILEIind mice results in significant improvement of psoriasiform symptoms by reducing ILEI secretion. The ILEI signature distinguishes psoriasis from healthy skin with uPA ranking among the top "separator" genes. Our study identifies ILEI as a key driver in psoriasis, indicates the relevance of ILEI-regulated genes for disease manifestation, and shows the clinical impact of ILEI and urokinase as novel potential therapeutic targets in psoriasis.

Lipids of Platelet-Rich Fibrin Reduce the Inflammatory Response in Mesenchymal Cells and Macrophages.

Platelet-rich fibrin (PRF) has a potent anti-inflammatory activity but the components mediating this effect remain unknown. Blood lipids have anti-inflammatory properties. The question arises whether this is also true for the lipid fraction of PRF. To answer this question, lipid fractions of solid and liquid PRF were tested for their potential to lower the inflammatory response of ST2 bone marrow stromal cells and primary bone marrow macrophages exposed to IL1ß and TNFα, and LPS, respectively. Cytokine production and the underlying signalling pathway were analysed by RT-PCR, immunoassays, and Western blotting. We report here that lipids from solid and liquid PRF substantially lowered cytokine-induced expression of IL6, CCL2 and CCL5 in ST2 cells. Moreover, the inflammatory response induced by Pam3CSK4, the agonist of Toll-like receptor (TLR) TLR2, was partially reduced by the lipid extracts in ST2 cells. The PRF lipids further reduced the LPS-induced expression of IL1ß, IL6 and CCL5 in macrophages at the transcriptional level. This was confirmed by showing the ability of PRF lipids to diminish IL6 at the protein level in ST2 cells and macrophages. Likewise, PRF lipid extracts reduced the phosphorylation of p38 and JNK and moderately decreased the phosphorylation of NFκB-p65 in ST2 cells. These findings suggest that the lipid fraction is at least partially responsible for the anti-inflammatory activity of PRF in vitro.

ORF1p Is a Potential Novel Diagnostic Marker for Differentiated Vulvar Intraepithelial Neoplasia.

Accurate diagnosis of differentiated vulvar intraepithelial neoplasia (dVIN) can be challenging as histomorphologic features may be subtle and overlap with nondysplastic lesions. In practice, aberrant p53 expression supports the diagnosis, but a substantial percentage retains wild-type p53. Recently, the retrotransposon long interspersed nuclear element 1 has been detected in distinct cancer types. We have now investigated the expression of the long interspersed nuclear element 1 encoded protein ORF1p in dysplastic and nondysplastic vulvar samples to assess its diagnostic value. Specimens of dVIN (n=29), high-grade squamous intraepithelial lesions (n=26), inflammatory vulvar lesions (n=20), lichen sclerosus (n=22), and normal vulvar epithelia (n=29) were included. ORF1p and p53 expression was determined using immunohistochemistry. The majority of dVIN [27/29 (93%)] and high-grade squamous intraepithelial lesions [20/26 (77%)] showed distinct (i.e. moderate or strong) ORF1p expression in the basal and suprabasal or all epithelial layers, respectively. Of note, ORF1p was present in all 4 cases of dVIN with wild-type p53 staining pattern. In contrast, ORF1p was negative or weakly expressed in most inflammatory lesions [14/20 (70%)] and lichen sclerosus [18/22 (82%), P <0.001]. Normal control epithelium exhibited negative staining in all cases. In conclusion, ORF1p might be a useful diagnostic marker for dVIN, especially in cases with retained wild-type p53.

EGR1 Is Implicated in Right Ventricular Cardiac Remodeling Associated with Pulmonary Hypertension.

BACKGROUND: Pulmonary hypertension (PH) is a vasoconstrictive disease characterized by elevated mean pulmonary arterial pressure (mPAP) at rest. Idiopathic pulmonary arterial hypertension (iPAH) and chronic thromboembolic pulmonary hypertension (CTEPH) represent two distinct subtypes of PH. Persisting PH leads to right ventricular (RV) hypertrophy, heart failure, and death. RV performance predicts survival and surgical interventions re-establishing physiological mPAP reverse cardiac remodeling. Nonetheless, a considerable number of PH patients are deemed inoperable. The underlying mechanism(s) governing cardiac regeneration, however, remain largely elusive. METHODS: In a longitudinal approach, we profiled the transcriptional landscapes of hypertrophic RVs and recovered hearts 3 months after surgery of iPAH and CTEPH patients. RESULTS: Genes associated with cellular responses to inflammatory stimuli and metal ions were downregulated, and cardiac muscle tissue development was induced in iPAH after recovery. In CTEPH patients, genes related to muscle cell development were decreased, and genes governing cardiac conduction were upregulated in RVs following regeneration. Intriguingly, early growth response 1 (EGR1), a profibrotic regulator, was identified as a major transcription factor of hypertrophic RVs in iPAH and CTEPH. A histological assessment confirmed our biocomputational results, and suggested a pivotal role for EGR1 in RV vasculopathy. CONCLUSION: Our findings improved our understanding of the molecular events driving reverse cardiac remodeling following surgery. EGR1 might represent a promising candidate for targeted therapy of PH patients not eligible for surgical treatment.

Clinical Relevance of Elevated Soluble ST2, HSP27 and 20S Proteasome at Hospital Admission in Patients with COVID-19.

Although, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) represents one of the biggest challenges in the world today, the exact immunopathogenic mechanism that leads to severe or critical Coronavirus Disease 2019 (COVID-19) has remained incompletely understood. Several studies have indicated that high systemic plasma levels of inflammatory cytokines result in the so-called "cytokine storm", with subsequent development of microthrombosis, disseminated intravascular coagulation, and multiorgan-failure. Therefore, we reasoned those elevated inflammatory molecules might act as prognostic factors. Here, we analyzed 245 serum samples of patients with COVID-19, collected at hospital admission. We assessed the levels of heat shock protein 27 (HSP27), soluble suppressor of tumorigenicity-2 (sST2) and 20S proteasome at hospital admission and explored their associations with overall-, 30-, 60-, 90-day- and in-hospital mortality. Moreover, we investigated their association with the risk of ventilation. We demonstrated that increased serum sST2 was uni- and multivariably associated with all endpoints. Furthermore, we also identified 20S proteasome as independent prognostic factor for in-hospital mortality (sST2, AUC = 0.73; HSP27, AUC = 0.59; 20S proteasome = 0.67). Elevated sST2, HSP27, and 20S proteasome levels at hospital admission were univariably associated with higher risk of invasive ventilation (OR = 1.8; p < 0.001; OR = 1.1; p = 0.04; OR = 1.03, p = 0.03, respectively). These findings could help to identify high-risk patients early in the course of COVID-19.

Transcriptional Differences in Lipid-Metabolizing Enzymes in Murine Sebocytes Derived from Sebaceous Glands of the Skin and Preputial Glands.

Sebaceous glands are adnexal structures, which critically contribute to skin homeostasis and the establishment of a functional epidermal barrier. Sebocytes, the main cell population found within the sebaceous glands, are highly specialized lipid-producing cells. Sebaceous gland-resembling tissue structures are also found in male rodents in the form of preputial glands. Similar to sebaceous glands, they are composed of lipid-specialized sebocytes. Due to a lack of adequate organ culture models for skin sebaceous glands and the fact that preputial glands are much larger and easier to handle, previous studies used preputial glands as a model for skin sebaceous glands. Here, we compared both types of sebocytes, using a single-cell RNA sequencing approach, to unravel potential similarities and differences between the two sebocyte populations. In spite of common gene expression patterns due to general lipid-producing properties, we found significant differences in the expression levels of genes encoding enzymes involved in the biogenesis of specialized lipid classes. Specifically, genes critically involved in the mevalonate pathway, including squalene synthase, as well as the sphingolipid salvage pathway, such as ceramide synthase, (acid) sphingomyelinase or acid and alkaline ceramidases, were significantly less expressed by preputial gland sebocytes. Together, our data revealed tissue-specific sebocyte populations, indicating major developmental, functional as well as biosynthetic differences between both glands. The use of preputial glands as a surrogate model to study skin sebaceous glands is therefore limited, and major differences between both glands need to be carefully considered before planning an experiment.

The serine proteases dipeptidyl-peptidase 4 and urokinase are key molecules in human and mouse scar formation.

Despite recent advances in understanding skin scarring, mechanisms triggering hypertrophic scar formation are still poorly understood. In the present study, we investigate mature human hypertrophic scars and developing scars in mice at single cell resolution. Compared to normal skin, we find significant differences in gene expression in most cell types present in scar tissue. Fibroblasts show the most prominent alterations in gene expression, displaying a distinct fibrotic signature. By comparing genes upregulated in murine fibroblasts during scar development with genes highly expressed in mature human hypertrophic scars, we identify a group of serine proteases, tentatively involved in scar formation. Two of them, dipeptidyl-peptidase 4 (DPP4) and urokinase (PLAU), are further analyzed in functional assays, revealing a role in TGFß1-mediated myofibroblast differentiation and over-production of components of the extracellular matrix in vitro. Topical treatment with inhibitors of DPP4 and PLAU during scar formation in vivo shows anti-fibrotic activity and improvement of scar quality, most prominently after application of the PLAU inhibitor BC-11. In this study, we delineate the genetic landscape of hypertrophic scars and present insights into mechanisms involved in hypertrophic scar formation. Our data suggest the use of serine protease inhibitors for the treatment of skin fibrosis.

DNA hypomethylation leads to cGAS-induced autoinflammation in the epidermis.

DNA methylation is a fundamental epigenetic modification, important across biological processes. The maintenance methyltransferase DNMT1 is essential for lineage differentiation during development, but its functions in tissue homeostasis are incompletely understood. We show that epidermis-specific DNMT1 deletion severely disrupts epidermal structure and homeostasis, initiating a massive innate immune response and infiltration of immune cells. Mechanistically, DNA hypomethylation in keratinocytes triggered transposon derepression, mitotic defects, and formation of micronuclei. DNA release into the cytosol of DNMT1-deficient keratinocytes activated signaling through cGAS and STING, thus triggering inflammation. Our findings show that disruption of a key epigenetic mark directly impacts immune and tissue homeostasis, and potentially impacts our understanding of autoinflammatory diseases and cancer immunotherapy.

Comparing the efficacy of γ- and electron-irradiation of PBMCs to promote secretion of paracrine, regenerative factors.

Cell-free secretomes represent a promising new therapeutic avenue in regenerative medicine, and γ-irradiation of human peripheral blood mononuclear cells (PBMCs) has been shown to promote the release of paracrine factors with high regenerative potential. Recently, the use of alternative irradiation sources, such as artificially generated ß- or electron-irradiation, is encouraged by authorities. Since the effect of the less hazardous electron-radiation on the production and functions of paracrine factors has not been tested so far, we compared the effects of γ- and electron-irradiation on PBMCs and determined the efficacy of both radiation sources for producing regenerative secretomes. Exposure to 60 Gy γ-rays from a radioactive nuclide and 60 Gy electron-irradiation provided by a linear accelerator comparably induced cell death and DNA damage. The transcriptional landscapes of PBMCs exposed to either radiation source shared a high degree of similarity. Secretion patterns of proteins, lipids, and extracellular vesicles displayed similar profiles after γ- and electron-irradiation. Lastly, we detected comparable biological activities in functional assays reflecting the regenerative potential of the secretomes. Taken together, we were able to demonstrate that electron-irradiation is an effective, alternative radiation source for producing therapeutic, cell-free secretomes. Our study paves the way for future clinical trials employing secretomes generated with electron-irradiation in tissue-regenerative medicine.

The secretome of stressed peripheral blood mononuclear cells increases tissue survival in a rodent epigastric flap model.

Reconstructive surgery transfers viable tissue to cover defects and to restore aesthetic and functional properties. Failure rates after free flap surgery range from 3 to 7%. Co-morbidities such as diabetes mellitus or peripheral vascular disease increase the risk of flap failure up to 4.5-fold. Experimental therapeutic concepts commonly use a monocausal approach by applying single growth factors. The secretome of γ-irradiated, stressed peripheral blood mononuclear cells (PBMCsec) resembles the physiological environment necessary for tissue regeneration. Its application led to improved wound healing rates and a two-fold increase in blood vessel counts in previous animal models. We hypothesized that PBMCsec has beneficial effects on the survival of compromised flap tissue by reducing the necrosis rate and increasing angiogenesis. Surgery was performed on 39 male Sprague-Dawley rats (control, N = 13; fibrin sealant, N = 14; PBMCsec, N = 12). PBMCsec was produced according to good manufacturing practices (GMP) guidelines and 2 ml were administered intraoperatively at a concentration of 2.5 × 107 cells/ml using fibrin sealant as carrier substance. Flap perfusion and necrosis (as percentage of the total flap area) were analyzed using Laser Doppler Imaging and digital image planimetry on postoperative days 3 and 7. Immunohistochemical stainings for von Willebrand factor (vWF) and Vascular Endothelial Growth Factor-receptor-3 (Flt-4) were performed on postoperative day 7 to evaluate formation of blood vessels and lymphatic vessels. Seroma formation was quantified using a syringe and flap adhesion and tissue edema were evaluated clinically through a cranial incision by a blinded observer according to previously described criteria on postoperative day 7. We found a significantly reduced tissue necrosis rate (control: 27.8% ± 8.6; fibrin: 22.0% ± 6.2; 20.9% reduction, p = .053 vs. control; PBMCsec: 19.1% ± 7.2; 31.1% reduction, p = .012 vs. control; 12.9% reduction, 0.293 vs. fibrin) together with increased vWF+ vessel counts (control: 70.3 ± 16.3 vessels/4 fields at 200× magnification; fibrin: 67.8 ± 12.1; 3.6% reduction, p = .651, vs. control; PBMCsec: 85.9 ± 20.4; 22.2% increase, p = .045 vs. control; 26.7% increase, p = .010 vs. fibrin) on postoperative day 7 after treatment with PBMCsec. Seroma formation was decreased after treatment with fibrin sealant with or without the addition of PBMCsec. (control: 11.9 ± 9.7 ml; fibrin: 1.7 ± 5.3, 86.0% reduction, 0.004 vs. control; PBMCsec: 0.6 ± 2.0; 94.8% reduction, p = .001 vs. control; 62.8% reduction, p = .523 vs. fibrin). We describe the beneficial effects of a secretome derived from γ-irradiated PBMCs on tissue survival, angiogenesis, and clinical parameters after flap surgery in a rodent epigastric flap model.

Persistence of mature dendritic cells, TH2A, and Tc2 cells characterize clinically resolved atopic dermatitis under IL-4Rα blockade.

Therapeutic options for autoimmune diseases typically consist of broad and targeted immunosuppressive agents. However, sustained clinical benefit is rarely achieved, as the disease phenotype usually returns after cessation of treatment. To better understand tissue-resident immune memory in human disease, we investigated patients with atopic dermatitis (AD) who underwent short-term or long-term treatment with the IL-4Rα blocker dupilumab. Using multi-omics profiling with single-cell RNA sequencing and multiplex proteomics, we found significant decreases in overall skin immune cell counts and normalization of transcriptomic dysregulation in keratinocytes consistent with clearance of disease. However, we identified specific immune cell populations that persisted for up to a year after clinical remission while being absent from healthy controls. These populations included LAMP3 + CCL22+ mature dendritic cells, CRTH2 + CD161 + T helper ("TH2A") cells, and CRTAM + cytotoxic T cells, which expressed high levels of CCL17 (dendritic cells) and IL13 (T cells). TH2A cells showed a characteristic cytokine receptor constellation with IL17RB, IL1RL1 (ST2), and CRLF2 expression, suggesting that these cells are key responders to the AD-typical epidermal alarmins IL-25, IL-33, and TSLP, respectively. We thus identified disease-linked immune cell populations in resolved AD indicative of a persisting disease memory, facilitating a rapid response system of epidermal-dermal cross-talk between keratinocytes, dendritic cells, and T cells. This observation may help to explain the disease recurrence upon termination of immunosuppressive treatments in AD, and it identifies potential disease memory-linked cell types that may be targeted to achieve a more sustained therapeutic response.

Epilipidomics of Senescent Dermal Fibroblasts Identify Lysophosphatidylcholines as Pleiotropic Senescence-Associated Secretory Phenotype (SASP) Factors.

During aging, skin accumulates senescent cells. The transient presence of senescent cells, followed by their clearance by the immune system, is important in tissue repair and homeostasis. The persistence of senescent cells that evade clearance contributes to the age-related deterioration of the skin. The senescence-associated secretory phenotype of these cells contains immunomodulatory molecules that facilitate clearance but also promote chronic damage. Here, we investigated the epilipidome-the oxidative modifications of phospholipids-of senescent dermal fibroblasts, because these molecules are among the bioactive lipids that were recently identified as senescence-associated secretory phenotype factors. Using replicative- and stress- induced senescence protocols, we identified lysophosphatidylcholines as universally elevated in senescent fibroblasts, whereas other oxidized lipids displayed a pattern that was characteristic for the used senescence protocol. When we tested the lysophosphatidylcholines for senescence-associated secretory phenotype activity, we found that they elicit chemokine release in nonsenescent fibroblasts but also interfere with toll-like receptor 2 and 6/CD36 signaling and phagocytic capacity in macrophages. Using matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry imaging, we localized two lysophosphatidylcholine species in aged skin. This suggests that lysophospholipids may facilitate immune evasion and low-grade chronic inflammation in skin aging.

Dysregulation of tissue and serum microRNAs in organ transplant recipients with cutaneous squamous cell carcinomas.

Micro RNAs (miRNAs) are considered as promising biomarkers for skin cancer. By next generation sequencing (NGS), we measured and compared miRNA expression profiles of tumor, peri-lesional, and control tissue of immunosuppressed organ transplant recipients (OTR), suffering from localized cutaneous squamous cell carcinoma (cSCC). Out of 490 miRNAs detected in cSCC tissue, 23 significantly regulated miRNAs were selected for quantitative targeted analysis in serum samples of our patients and control sera from OTR without cSCC. Two onco-miRNAs (mir-1290, mir-1246), previously identified as crucial drivers for tumor initiation and cancer progression, were significantly and exclusively upregulated in both tumor tissue and serum. This finding suggests that miRNA dysregulation in cSCC tissue is reflected in serum even in patients without advanced disease and highly differentiated cSCCs.

TGF-ß in the Secretome of Irradiated Peripheral Blood Mononuclear Cells Supports In Vitro Osteoclastogenesis.

Osteoclastogenesis required for bone remodeling is also a key pathologic mechanism of inflammatory osteolysis being controlled by paracrine factors released from dying cells. The secretome of irradiated, dying peripheral blood mononuclear cells (PBMCs) has a major impact on the differentiation of myeloid cells into dendritic cells, and macrophage polarization. The impact on osteoclastogenesis, however, has not been reported. For this aim, we used murine bone marrow macrophages exposed to RANKL and M-CSF to initiate osteoclastogenesis, with and without the secretome obtained from γ-irradiated PBMCs. We reported that the secretome significantly enhanced in vitro osteoclastogenesis as determined by means of histochemical staining of the tartrate-resistant acid phosphatase (TRAP), as well as the expression of the respective target genes, including TRAP and cathepsin K. Considering that TGF-ß enhanced osteoclastogenesis, we confirmed the TGF-ß activity in the secretome with a bioassay that was based on the increased expression of IL11 in fibroblasts. Neutralizing TGF-ß by an antibody decreased the ability of the secretome to support osteoclastogenesis. These findings suggested that TGF-ß released by irradiated PBMCs could enhance the process of osteoclastogenesis in vitro.

Single-cell transcriptomics combined with interstitial fluid proteomics defines cell type-specific immune regulation in atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is the most common chronic inflammatory skin disease, but its complex pathogenesis is only insufficiently understood, resulting in still limited treatment options. OBJECTIVE: We sought to characterize AD on both transcriptomic and proteomic levels in humans. METHODS: We used skin suction blistering, a painless and nonscarring procedure that can simultaneously sample skin cells and interstitial fluid. We then compared results with conventional biopsies. RESULTS: Suction blistering captured epidermal and most immune cells equally well as biopsies, except for mast cells and nonmigratory CD163+ macrophages that were only present in biopsy isolates. Using single-cell RNA sequencing, we found comparable transcriptional profiles of key inflammatory pathways between blister and biopsy AD, but suction blistering was superior in cell-specific resolution for high-abundance transcripts (KRT1/KRT10, KRT16/KRT6A, S100A8/S100A9), which showed some background signals in biopsy isolates. Compared with healthy controls, we found characteristic upregulation of AD-typical cytokines such as IL13 and IL22 in Th2 and Th22 cells, respectively, but we also discovered these mediators in proliferating T cells and natural killer T cells, that also expressed the antimicrobial cytokine IL26. Overall, not T cells, but myeloid cells were most strongly enriched in AD, and we found dendritic cell (CLEC7A, amphiregulin/AREG, EREG) and macrophage products (CCL13) among the top upregulated proteins in AD blister fluid proteomic analyses. CONCLUSION: These data show that by using cutting-edge technology, suction blistering offers several advantages over conventional biopsies, including better transcriptomic resolution of skin cells, combined with proteomic information from interstitial fluid, unraveling novel inflammatory players that shape the cellular and proteomic microenvironment of AD.

PIWIL-2 and piRNAs are regularly expressed in epithelia of the skin and their expression is related to differentiation.

PIWI proteins play multiple roles in germline stem cell maintenance and self-renewal. PIWI-interacting RNAs (piRNAs) associate with PIWI proteins, form effector complexes and maintain genome integrity and function in the regulation of gene expression by epigenetic modifications. Both are involved in cancer development. In this study, we investigated the expression of PIWIL-2 and piRNAs in normal human skin and epithelial tumors and its regulation during keratinocyte (KC) differentiation. Immunohistochemistry showed that PIWIL-2 was regularly expressed in the epidermis and adnexal tissue with strongest expression in sebaceous glands. Cell culture studies revealed an association of PIWIL-2 expression with the state of differentiated KC. In contrast, the PIWIL-2 expression pattern did not correlate with stem cell compartments or malignancy. piRNAs were consistently detected in KC in vitro by next-generation sequencing and the expression levels of numerous piRNAs were regulated during KC differentiation. Epidermal piRNAs were predominantly derived from processed snoRNAs (C/D-box snoRNAs), tRNAs and protein coding genes. Our data indicate that components of the PIWIL-2-piRNA pathway are present in epithelial cells of the skin and are regulated in the context of KC differentiation, suggesting a role of somatic gene regulation. However, putative roles in the maintenance of stem cell compartments or the development of malignancy in the skin were not supported by this study.

Butyrate Decreases ICAM-1 Expression in Human Oral Squamous Cell Carcinoma Cells.

Short-chain fatty acids (SCFA) are bacterial metabolites that can be found in periodontal pockets. The expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) within the epithelium pocket is considered to be a key event for the selective transmigration of leucocytes towards the gingival sulcus. However, the impact of SCFA on ICAM-1 expression by oral epithelial cells remains unclear. We therefore exposed the oral squamous carcinoma cell line HSC-2, primary oral epithelial cells and human gingival fibroblasts to SCFA, namely acetate, propionate and butyrate, and stimulated with known inducers of ICAM-1 such as interleukin-1-beta (IL1ß) and tumor necrosis factor-alfa (TNFα). We report here that butyrate but not acetate or propionate significantly suppressed the cytokine-induced ICAM-1 expression in HSC-2 epithelial cells and primary epithelial cells. The G-protein coupled receptor-43 (GPR43/ FFAR2) agonist but not the histone deacetylase inhibitor, trichostatin A, mimicked the butyrate effects. Butyrate also attenuated the nuclear translocation of p65 into the nucleus on HSC-2 cells. The decrease of ICAM-1 was independent of Nrf2/HO-1 signaling and phosphorylation of JNK and p38. Nevertheless, butyrate could not reverse an ongoing cytokine-induced ICAM-1 expression in HSC-2 cells. Overall, these observations suggest that butyrate can attenuate cytokine-induced ICAM-1 expression in cells with epithelial origin.

Deciphering the functional heterogeneity of skin fibroblasts using single-cell RNA sequencing.

Though skin fibroblasts (FB) are the main cell population within the dermis, the different skin FB subsets are not well characterized and the traditional classification into reticular and papillary FBs has little functional relevance. To fill the gap of knowledge on FB diversity in human skin, we performed single-cell RNA sequencing. Investigation of marker genes for the different skin cell subtypes revealed a heterogeneous picture of FBs. When mapping reticular and papillary FB markers, we could not detect cluster specificity, suggesting that these two populations show a higher transcriptional heterogeneity than expected. This finding was further confirmed by in situ hybridization, showing that DPP4 was expressed in both dermal layers. Our analysis identified six FB clusters with distinct transcriptional signatures. Importantly, we could demonstrate that in human skin DPP4+ FBs are the main producers of factors involved in extracellular matrix (ECM) assembly. In conclusion, we provide evidence that hitherto considered FB markers are not ideal to characterize skin FB subpopulations in single-cell sequencing analyses. The identification of DPP4+ FBs as the main ECM-producing cells in human skin will foster the development of anti-fibrotic treatments for the skin and other organs.

Tissue-regenerative potential of the secretome of γ-irradiated peripheral blood mononuclear cells is mediated via TNFRSF1B-induced necroptosis.

Peripheral blood mononuclear cells (PBMCs) have been shown to produce and release a plethora of pro-angiogenetic factors in response to γ-irradiation, partially accounting for their tissue-regenerative capacity. Here, we investigated whether a certain cell subtype of PBMCs is responsible for this effect, and whether the type of cell death affects the pro-angiogenic potential of bioactive molecules released by γ-irradiated PBMCs. PBMCs and PBMC subpopulations, including CD4+ and CD8+ T cells, B cells, monocytes, and natural killer cells, were isolated and subjected to high-dose γ-irradiation. Transcriptome analysis revealed subpopulation-specific responses to γ-irradiation with distinct activation of pro-angiogenic pathways, cytokine production, and death receptor signalling. Analysis of the proteins released showed that interactions of the subsets are important for the generation of a pro-angiogenic secretome. This result was confirmed at the functional level by the finding that the secretome of γ-irradiated PBMCs displayed higher pro-angiogenic activity in an aortic ring assay. Scanning electron microscopy and image stream analysis of γ-irradiated PBMCs revealed distinct morphological changes, indicative for apoptotic and necroptotic cell death. While inhibition of apoptosis had no effect on the pro-angiogenic activity of the secretome, inhibiting necroptosis in stressed PBMCs abolished blood vessel sprouting. Mechanistically, we identified tumor necrosis factor (TNF) receptor superfamily member 1B as the main driver of necroptosis in response to γ-irradiation in PBMCs, which was most likely mediated via membrane-bound TNF-α. In conclusion, our study demonstrates that the pro-angiogenic activity of the secretome of γ-irradiated PBMCs requires interplay of different PBMC subpopulations. Furthermore, we show that TNF-dependent necroptosis is an indispensable molecular process for conferring tissue-regenerative activity and for the pro-angiogenic potential of the PBMC secretome. These findings contribute to a better understanding of secretome-based therapies in regenerative medicine.