Biased TCR gene usage in citrullinated Tenascin C specific T-cells in rheumatoid arthritis

We aimed to search for common features in the autoreactive T cell receptor (TCR) repertoire in patients with rheumatoid arthritis (RA), focusing on the newly identified candidate antigen citrullinated Tenascin C (cit-TNC). Mononuclear cells from peripheral blood or synovial fluid of eight RA-patients positive for the RA-associated HLA-DRB1*04:01 allele were in-vitro cultured with recently identified citrullinated peptides from Tenascin C. Antigen-specific T cells were isolated using peptide-HLA tetramer staining and subsequently single-cell sequenced for paired alpha/beta TCR analyses by bioinformatic tools. TCRs were re-expressed for further studies of antigen-specificity and T cell responses. Autoreactive T cell lines could be grown out from both peripheral blood and synovial fluid. We demonstrate the feasibility of retrieving true autoreactive TCR sequences by validating antigen-specificity in T cell lines with re-expressed TCRs. One of the Tenascin C peptides, cit-TNC22, gave the most robust T cell responses including biased TCR gene usage patterns. The shared TCR-beta chain signature among the cit-TNC22-specific TCRs was evident in blood and synovial fluid of different patients. The identification of common elements in the autoreactive TCR repertoire gives promise to the possibility of both immune monitoring of the autoimmune components in RA and of future antigen- or TCR-targeted specific intervention in subsets of patients.

Homing Peptide-Based Targeting of Tenascin-C and Fibronectin in Endometriosis

The current diagnostic and therapeutic strategies for endometriosis are limited. Although endometriosis is a benign condition, some of its traits, such as increased cell invasion, migration, tissue inflammation, and angiogenesis are similar to cancer. Here we explored the application of homing peptides for precision delivery of diagnostic and therapeutic compounds to endometriotic lesions. First, we audited a panel of peptide phages for the binding to the cultured immortalized endometriotic epithelial 12Z and eutopic stromal HESC cell lines. The bacteriophages displaying PL1 peptide that engages with angiogenic extracellular matrix overexpressed in solid tumors showed the strongest binding to both cell lines. The receptors of PL1 peptide, tenascin C domain C (TNC-C) and fibronectin Extra Domain-B (Fn-EDB), were expressed in both cells. Silver nanoparticles functionalized with synthetic PL1 peptide showed specific internalization in 12Z and HESC cells. Treatment with PL1-nanoparticles loaded with the potent antimitotic drug monomethyl auristatin E decreased the viability of endometriotic cells in 2D and 3D cultures. Finally, PL1-nanoparticless bound to the cryosections of clinical peritoneal endometriotic lesions in the areas positive for TNC-C and Fn-EDB immunoreactivities and not to sections of normal endometrium. Our findings suggest potential applications for PL1-guided nanoparticles in precision diagnosis and therapy of endometriosis.

Elevated Serum Tenascin-C Predicts Mortality in Critically Ill Patients With Multiple Organ Dysfunction

Background: Multiple organ dysfunction is a complex and lethal clinical feature with heterogeneous causes and is usually characterized by tissue injury of multiple organs. Tenascin-C (TNC) is a matricellular protein that is rarely expressed in most of the adult tissues, but re-induced following injury. This study aimed to evaluate serum TNC in predicting mortality in critically ill patients with multiple organ dysfunction.Methods: Adult critically ill patients with at least two organs dysfunction and an increase of Sequential Organ Failure Assess (SOFA) score ≥ 2 points within 7 days were prospectively enrolled into two independent cohorts. The emergency (derivation) cohort was a consecutive series and the patients were from Emergency Department. The inpatient (validation) cohort was a convenience series and the patients were from medical wards. Their serum samples at the first 24 h after enrollment were collected and subjected to TNC measurement using ELISA. The association between serum TNC level and 28-day all-cause mortality was investigated, and then the predictive value of serum TNC was analyzed.Results: A total of 110 patients with a median age of 64 years (53, 73) were enrolled in the emergency cohort. Compared to the survivors, serum TNC in the non-survivors was significantly higher (467.7 vs. 197.5 ng/ml, p < 0.001). Multivariate logistic regression analysis revealed that the association between serum TNC and 28-day mortality was independent of sepsis or critical illness scores such as SOFA, Acute Physiology and Chronic Health Evaluation (APACHE II), and Simplified Acute Physiology Score (SAPS II), respectively (p < 0.001 for each). The area under receiver operating characteristic curve of serum TNC for predicting mortality was 0.803 (0.717–0.888) (p < 0.001), similar with SOFA 0.808 (0.725–0.891), APACHE II 0.762 (0.667–0.857), and SAPS II 0.779 (0.685–0.872). The optimal cut-off value of serum TNC was 298.2 ng/ml. Kaplan–Meier analysis showed that the survival of patients with serum TNC ≥ 300 ng/ml was significantly worse than that of patients with serum TNC < 300 ng/ml. This result was validated in the inpatient cohort. The sensitivity and specificity of serum TNC ≥ 300 ng/ml for predicting mortality were 74.3 and 74.7% in the emergency cohort, and 63.0 and 70.1% in the inpatient cohort, respectively.Conclusion: Serum TNC was associated with mortality in critically ill patients with multiple organ dysfunction, and would be used as a prognostic tool for predicting mortality in this population.

Biased TCR gene usage in citrullinated Tenascin C specific T-cells in rheumatoid arthritis

Objectives To search for common features in the autoreactive T cell receptor (TCR)-repertoire in patients with rheumatoid arthritis (RA), focusing on the newly identified candidate antigen citrullinated Tenascin C (cit-TNC). Methods Mononuclear cells from peripheral blood or synovial fluid of eight RA-patients positive for the RA-associated HLA-DRB1*04:01 allele were in-vitro cultured with recently identified citrullinated peptides from Tenascin C. Antigen-specific T cells were isolated using peptide-HLA tetramer staining and subsequently single-cell sequenced for paired alpha/beta TCR analyses by bioinformatic tools. TCRs were re-expressed for further studies of antigen-specificity and T cell responses. Results Autoreactive T cell lines could be grown out from both peripheral blood and synovial fluid. We demonstrate the feasibility of retrieving true autoreactive TCR sequences by validating antigen-specificity in T cell lines with re-expressed TCRs. One of the Tenascin C peptides, cit-TNC22, gave the most robust T cell responses including biased TCR gene usage patterns. The shared TCR-beta chain signature among the citTNC22-specific TCRs was evident in blood and synovial fluid of different patients. Conclusion The identification of common elements in the autoreactive TCR repertoire gives promise to the possibility of both immune monitoring of the autoimmune components in RA and of future antigen- or TCR-targeted specific intervention in subsets of patients.

Prostate tumor-induced stromal reprogramming generates Tenascin C that promotes prostate cancer metastasis through YAP/TAZ inhibition

Metastatic prostate cancer (PCa) in bone induces bone-forming lesions that enhance PCa progression. How tumor-induced bone formation enhances PCa progression is not known. We have previously shown that PCa-induced bone originates from endothelial cells (EC) that have undergone endothelial-to-osteoblast (EC-to-OSB) transition by tumor-secreted BMP4. Here, we show that EC-to-OSB transition leads to changes in the tumor microenvironment that increases the metastatic potential of PCa cells. We found that conditioned medium (CM) from EC-OSB hybrid cells increases the migration, invasion and survival of PC3-mm2 and C4-2B4 PCa cells. Quantitative mass spectrometry (iTRAQ) identified Tenascin C (TNC) as one of the major proteins secreted from EC-OSB hybrid cells. TNC expression in tumor-induced osteoblasts was confirmed by immunohistochemistry of MDA-PCa118b xenograft and human bone metastasis specimens. Mechanistically, BMP4 increases TNC expression in EC-OSB cells through the Smad1-Notch/Hey1 pathway. How TNC promotes PCa metastasis was next interrogated by in vitro and in vivo studies. In vitro studies showed that a TNC neutralizing antibody inhibits EC-OSB-CM-mediated PCa cell migration and survival. TNC knockdown decreased, while addition of recombinant TNC or TNC overexpression increased migration and anchorage-independent growth of PC3 or C4-2b cells. When injected orthotopically, PC3-mm2-shTNC clones decreased metastasis to bone, while C4-2b-TNC overexpressing cells increased metastasis to lymph nodes. TNC enhances PCa cell migration through α5β1 integrin-mediated YAP/TAZ inhibition. These studies elucidate that tumor-induced stromal reprogramming generates TNC that enhances PCa metastasis and suggest that TNC may be a target for PCa therapy.

Tenascin-C: Friend or Foe in Lung Aging?

Lung aging is characterized by lung function impairment, ECM remodeling and airspace enlargement. Tenascin-C (TNC) is a large extracellular matrix (ECM) protein with paracrine and autocrine regulatory functions on cell migration, proliferation and differentiation. This matricellular protein is highly expressed during organogenesis and morphogenetic events like injury repair, inflammation or cancer. We previously showed that TNC deficiency affected lung development and pulmonary function, but little is known about its role during pulmonary aging. In order to answer this question, we characterized lung structure and physiology in 18 months old TNC-deficient and wild-type (WT) mice. Mice were mechanically ventilated with a basal and high tidal volume (HTV) ventilation protocol for functional analyses. Additional animals were used for histological, stereological and molecular biological analyses. We observed that old TNC-deficient mice exhibited larger lung volume, parenchymal volume, total airspace volume and septal surface area than WT, but similar mean linear intercept. This was accompanied by an increase in proliferation, but not apoptosis or autophagy markers expression throughout the lung parenchyma. Senescent cells were observed in epithelial cells of the conducting airways and in alveolar macrophages, but equally in both genotypes. Total collagen content was doubled in TNC KO lungs. However, basal and HTV ventilation revealed similar respiratory physiological parameters in both genotypes. Smooth muscle actin (α-SMA) analysis showed a faint increase in α-SMA positive cells in TNC-deficient lungs, but a marked increase in non-proliferative α-SMA + desmin + cells. Major TNC-related molecular pathways were not up- or down-regulated in TNC-deficient lungs as compared to WT; only minor changes in TLR4 and TGFβR3 mRNA expression were observed. In conclusion, TNC-deficient lungs at 18 months of age showed exaggerated features of the normal structural lung aging described to occur in mice between 12 and 18 months of age. Correlated to the increased pulmonary function parameters previously observed in young adult TNC-deficient lungs and described to occur in normal lung aging between 3 and 6 months of age, TNC might be an advantage in lung aging.

The role of extracellular matrix biomolecules on endometrial epithelial cell attachment and cytokeratin 18 expression on gelatin hydrogels

The endometrium undergoes profound changes in tissue architecture and composition, both during the menstrual cycle as well as in the context of pregnancy. Dynamic remodeling processes of the endometrial extracellular matrix (ECM) are a major element of endometrial homeostasis, including changes across the menstrual cycle. A critical element of this tissue microenvironment is the endometrial basement membrane, a specialized layer of proteins that separates the endometrial epithelium from the underlying endometrial ECM. Bioengineering models of the endometrial microenvironment that present an appropriate endometrial ECM and basement membrane may provide an improved environment to study endometrial epithelial cell (EEC) function. Here, we exploit a tiered approach using two-dimensional high throughput microarrays and three-dimensional gelatin hydrogels to define patterns of EEC attachment and cytokeratin 18 (CK18) expression in response to combinations of endometrial basement membrane proteins. We identify combinations (collagen IV + tenascin C; collagen I + collagen III; hyaluronic acid + tenascin C; collagen V; collagen V + hyaluronic acid; collagen III; collagen I) that facilitate increased EEC attachment, increased CK18 intensity, or both. We also identify significant EEC mediated remodeling of the GelMA matrix environment via analysis of nascent protein deposition. Together, we report efforts to tailor the localization of basement membrane-associated proteins and proteoglycans in order to investigate tissue engineered models of the endometrial microenvironment.

Imaging the Alternatively Spliced D Domain of Tenascin C in Preclinical Models of Inflammatory Bowel Disease

Purpose To image colon-expressed alternatively spliced D domain of tenascin C in preclinical colitis models using near infrared (NIR) labeled targeted molecular imaging agents.Methods Human IgG and scFv fusion proteins specific to the alternatively spliced D domain of tenascin C were generated. Immunohistochemistry identified disease-specific expression of this extracellular matrix in mouse colitis models. Proteins were labeled with the NIR fluorophore IRDye 800CW via amine chemistry and intravenously dosed to evaluate targeting specificity in preclinical rodent and primate colitis models.Results The NIR labeled proteins successfully targeted colonic lesions in a murine model of colitis and appeared as distinct punctate spots. Co-administration of a blocking dose reduced the whole colon standardized uptake of the fluorescent dose >7-fold in mouse models. Estimates suggest local expression at >100 nM in diseased mouse colon. Macroscopic targeting specificity was not observed in diseased primate colon. Cellular level specificity was assessed via microscopy and immunohistochemistry.Conclusion Our imaging data suggest the alternatively spliced D domain of tenascin C is a promising target for delivery-based applications in inflammatory bowel diseases.