Identification of a novel role for TL1A/DR3 deficiency in acute respiratory distress syndrome that exacerbates alveolar epithelial disruption.

Alveolar epithelial barrier is a potential therapeutic target for acute respiratory distress syndrome (ARDS). However, an effective intervention against alveolar epithelial barrier has not been developed. Here, based on single-cell RNA and mRNA sequencing results, death receptor 3 (DR3) and its only known ligand tumor necrosis factor ligand-associated molecule 1A (TL1A) were significantly reduced in epithelium from an ARDS mice and cell models. The apparent reduction in the TL1A/DR3 axis in lungs from septic-ARDS patients was correlated with the severity of the disease. The examination of knockout (KO) and alveolar epithelium conditional KO (CKO) mice showed that TL1A deficiency exacerbated alveolar inflammation and permeability in lipopolysaccharide (LPS)-induced ARDS. Mechanistically, TL1A deficiency decreased glycocalyx syndecan-1 and tight junction-associated zonula occludens 3 by increasing cathepsin E level for strengthening cell-to-cell permeability. Additionally, DR3 deletion aggravated barrier dysfunction and pulmonary edema in LPS-induced ARDS through the above mechanisms based on the analyses of DR3 CKO mice and DR3 overexpression cells. Therefore, the TL1A/DR3 axis has a potential value as a key therapeutic signaling for the protection of alveolar epithelial barrier.

TL1A/DR3 Axis, A Key Target of TNF-a, Augments the Epithelial-Mesenchymal Transformation of Epithelial Cells in OVA-Induced Asthma.

Tumor necrosis factor (TNF)-like cytokine 1A (TL1A), a member of the TNF family, exists in the form of membrane-bound (mTL1A) and soluble protein (sTL1A). TL1A binding its only known functional receptor death domain receptor 3 (DR3) affects the transmission of various signals. This study first proposed that the TL1A/DR3 axis was significantly upregulated in patients and mice with both asthma and high TNF-a expression and in TNF-a-stimulated epithelial Beas-2B cells. Two independent approaches were used to demonstrate that the TL1A/DR3 axis of mice was strongly correlated with TNF-a in terms of exacerbating asthmatic epithelial-mesenchymal transformation (EMT). First, high expression levels of EMT proteins (e.g., collagen I, fibronectin, N-cadherin, and vimentin) and TL1A/DR3 axis were observed when mice airways were stimulated by recombinant mouse TNF-a protein. Moreover, EMT protein and TL1A/DR3 axis expression synchronously decreased after mice with OVA-induced asthma were treated with infliximab by neutralizing TNF-a activity. Furthermore, the OVA-induced EMT of asthmatic mice was remarkably improved upon the deletion of the TL1A/DR3 axis by knocking out the TL1A gene. TL1A siRNA remarkably intervened EMT formation induced by TNF-a in the Beas-2B cells. In addition, EMT was induced by the addition of high concentrations of recombinant human sTL1A with the cell medium. The TL1A overexpression via pc-mTL1A in vitro remarkably increased the EMT formation induced by TNF-a. Overall, these findings indicate that the TL1A/DR3 axis may have a therapeutic role for asthmatic with high TNF-a level.

Co-Expression and Functional Interactions of Death Receptor 3 and E-Selectin in Clear Cell Renal Cell Carcinoma.

Similar to the behavior of inflamed tubular epithelial cells, clear cell renal cell carcinoma (ccRCC) cells express death receptor 3 (DR3 or TNFSFR25) in situ, and expression increases with tumor grade. Surprisingly, E-selectin, which can be induced in endothelial cells by DR3 signaling, is also expressed by ccRCC cells and increases with tumor grade. In ccRCC organ cultures, addition of tumor necrosis factor-like 1A (TL1A or TNFSF15), the ligand for DR3, activates NF-κB and mitogen-activated protein kinases, induces both DR3 and E-selectin expression in an NF-κB-dependent manner, and promotes cell cycle entry. DR3 immunoprecipitated from ccRCC tissue contains sialyl Lewis X moieties (the ligand recognized by E-selectin), proximity ligation assays reveal DR3, and E-selectin interacts on ccRCC cells. Similar to that with the addition of TL1A, the addition of soluble E-selectin to ccRCC organ cultures activates NF-κB and mitogen-activated protein kinases in ccRCC cells and increases both DR3 and E-selectin expression and cell-cycle entry. In contrast, normal renal tubular epithelium, which poorly expresses DR3, is minimally responsive to either of these ligands. These data suggest a functional role for autocrine/paracrine DR3/E-selectin interactions in ccRCC and its progression, revealing a potential new target for therapeutic intervention.

Targeting TL1A/DR3 Signaling Offers a Therapeutic Advantage to Neutralizing IL13/IL4Rα in Muco-Secretory Fibrotic Disorders.

Mucus secretion is an important feature of asthma that highly correlates with morbidity. Current therapies, including administration of mucolytics and anti-inflammatory drugs, show limited effectiveness and durability, underscoring the need for novel effective and longer lasting therapeutic approaches. Here we show that mucus production in the lungs is regulated by the TNF superfamily member 15 (TL1A) acting through the mucus-inducing cytokine IL-13. TL1A induces IL13 expression by innate lymphoid cells leading to mucus production, in addition to promoting airway inflammation and fibrosis. Reciprocally, neutralization of IL13 signaling through its receptor (IL4Rα), completely reverses TL1A-induced mucus secretion, while maintaining airway inflammation and fibrosis. Importance of TL1A is further demonstrated using a preclinical asthma model induced by chronic house dust mite exposure where TL1A neutralization by genetic deletion or antagonistic blockade of its receptor DR3 protected against mucus production and fibrosis. Thus, TL1A presents a promising therapeutic target that out benefits IL13 in reversing mucus production, airway inflammation and fibrosis, cardinal features of severe asthma in humans.

Host versus cell-dependent effects of ß-arrestin 1 expression in prostate tumorigenesis.

Prostate cancer (PCa) constitutes a serious health challenge and remains one of the main causes of cancer-related death among men. The more aggressive form of the disease has been attributed to androgen independence, resulting in a lack of response to androgen deprivation therapy and sustained activation of other growth pathways. The scaffold proteins ß-arrestin 1 and 2 (ßarr1 and ßarr2), which are known to mediate G protein-coupled receptor desensitization and internalization, were also shown to modulate prostate tumorigenesis. ßarr1 is significantly overexpressed (>4-fold) in PCa cells relative to ßarr2. In this study, we investigated the effect of ßarr1 overexpression in PCa development and progression using the mouse and human PCa cell xenografts, and autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP) models deficient in ß-arrestin depletion of ßarr1 in TRAMP mice (TRAMP/ßarr1-/-) increased PCa growth and decreased overall survival relative to control TRAMP or TRAMP/ßarr2-/- animals. Prostate tissues from TRAMP/ßarr1-/- tumors displayed an increase in androgen receptor (AR) expression, whereas overexpression of ßarr1 in TRAMP-C1 (TRAMP-C1-ßarr1-GFP) which derived from TRAMP decreased AR expression, cell proliferation and tumor growth in nude mice xenografts, relative to control TRAMP-C1-GFP. Knockdown of ßarr1 expression in human MDA PCa 2b cells (MDA PCa 2b-ßarr1-/-) also decreased AR expression cell proliferation and tumor growth relative to control (MDA PCa 2b-Sham) cells. Interestingly, both TRAMP-C1-ßarr1-GFP and MDA PCa 2b-ßarr1-/- xenografts showed a decrease in AKT phosphorylation but an increase in MAPK activation. Altogether, the data indicate that the effect of ßarr1 in modulating AR signaling to regulate PCa aggressiveness is cell and host autonomous.

Aberrant Hypermethylation-Mediated Suppression of PYCARD Is Extremely Frequent in Prostate Cancer with Gleason Score ≥ 7.

Epigenetic gene silencing by aberrant DNA methylation leads to loss of key cellular pathways in tumorigenesis. In order to analyze the effects of DNA methylation on prostate cancer, we established LNCaP-derived human prostate cancer cells that can pharmacologically induce global reactivation of hypermethylated genes by the methyl-CpG targeted transcriptional activation (MeTA) method. The MeTA suppressed the growth of LNCaP-derived cells and induced apoptosis. Microarray analysis indicated that PYCARD (PYD and CARD domain containing) encoding an apoptosis-inducing factor was upregulated by 65-fold or more after treatment with MeTA. We analyzed DNA methylation statuses using 50 microdissected primary prostate cancer tissues and found an extremely high frequency of tumor-specific promoter hypermethylation of PYCARD (90%, 45/50). Moreover, DNA methylation status was significantly associated with Gleason score (P = 0.0063); the frequency of tumor-specific hypermethylation was 96% (44/46) in tumors with Gleason score ≥ 7, whereas that in tumors with Gleason score 6 was 25% (1/4). Immunohistochemical analyses using these 50 cases indicated that only 8% (4/50) of cancerous tissues expressed PYCARD, whereas 80% (40/50) of corresponding normal prostate epithelial and/or basal cells expressed PYCARD. In addition, there was no relationship between PYCARD immunostaining and the Gleason score in cancerous tissue and surrounding normal tissue. Inducible expression of PYCARD inhibited cell proliferation by induction of apoptosis. These results suggest that aberrant methylation of PYCARD is a distinctive feature of prostate cancers with Gleason score ≥ 7 and may play an important role in escaping from apoptosis in prostatic tumorigenesis.

Syndecan 4, galectin 2, and death receptor 3 (DR3) as novel proteins in pathophysiology of preeclampsia.

INTRODUCTION: Preeclampsia has the highest rate of obstetric morbidity and mortality. METHODS: We recruited 21 women with preeclampsia and 27 women with uncomplicated pregnancies. We used a quantitative protein macroarray that allowed for analysis of 40 proteins. RESULTS: We found a statistically significant increase in the concentration of DR3, LIF and a significant decrease of VEGF, PlGF, syndecan-4 and galectin-2, in the plasma of women with preeclampsia. CONCLUSIONS: There are no previous studies assessing syndecan 4, galectin 2, and DR3 concentrations in women with preeclampsia; Our results indicate these proteins are new factors that play important roles in the immunological pathomechanism of preeclampsia.

Direct signaling of TL1A-DR3 on fibroblasts induces intestinal fibrosis in vivo.

Tumor necrosis factor-like cytokine 1A (TL1A, TNFSF15) is implicated in inflammatory bowel disease, modulating the location and severity of inflammation and fibrosis. TL1A expression is increased in inflamed mucosa and associated with fibrostenosing Crohn's disease. Tl1a-overexpression in mice causes spontaneous ileitis, and exacerbates induced proximal colitis and fibrosis. Intestinal fibroblasts express Death-receptor 3 (DR3; the only know receptor for TL1A) and stimulation with TL1A induces activation in vitro. However, the contribution of direct TL1A-DR3 activation on fibroblasts to fibrosis in vivo remains unknown. TL1A overexpressing naïve T cells were transferred into Rag-/- , Rag-/- mice lacking DR3 in all cell types (Rag-/-Dr3-/-), or Rag-/- mice lacking DR3 only on fibroblasts (Rag-/-Dr3∆Col1a2) to induce colitis and fibrosis, assessed by clinical disease activity index, intestinal inflammation, and collagen deposition. Rag-/- mice developed overt colitis with intestinal fibrostenosis. In contrast, Rag-/-Dr3-/- demonstrated decreased inflammation and fibrosis. Despite similar clinical disease and inflammation as Rag-/-, Rag-/-Dr3∆Col1a2 exhibited reduced intestinal fibrosis and attenuated fibroblast activation and migration. RNA-Sequencing of TL1A-stimulated fibroblasts identified Rho signal transduction as a major pathway activated by TL1A and inhibition of this pathway modulated TL1A-mediated fibroblast functions. Thus, direct TL1A signaling on fibroblasts promotes intestinal fibrosis in vivo. These results provide novel insight into profibrotic pathways mediated by TL1A paralleling its pro-inflammatory effects.

The Role of the TL1A/DR3 Axis in the Activation of Group 2 Innate Lymphoid Cells in Subjects with Eosinophilic Asthma.

Rationale: Group 2 innate lymphoid cells (ILC2s) are critical for type 2 inflammation. In murine models of asthma, some ILC2s remain activated in the absence of epithelial cell-derived cytokine signaling, implicating alternate stimulatory pathways. DR3 (death receptor 3), a member of the tumor necrosis factor receptor superfamily, is expressed on ILC2s. Genome-wide association studies report an association between DR3 ligand, TL1A (tumor necrosis factor-like protein 1A), and chronic inflammatory conditions.Objectives: We investigated the TL1A/DR3 axis in airway ILC2 biology in eosinophilic asthma.Methods: Stable subjects with mild asthma were subject to allergen inhalation challenge, and DR3 expression on sputum cells was assessed. We investigated cytokine regulation of DR3 expression on ILC2s and steroid sensitivity. Airway TL1A was assessed in sputum from subjects with mild asthma and subjects with prednisone-dependent severe eosinophilic asthma.Measurements and Main Results: There was a significant increase in sputum DR3+ ILC2s 24 hours after allergen challenge, and DR3 expression on ILC2s was upregulated by IL-2, IL-33, or TSLP in vitro. Stimulation with TL1A significantly increased IL-5 expression by ILC2s and was attenuated by dexamethasone, an effect that was negated in the presence of TSLP. Airway TL1A levels were increased 24 hours after allergen challenge in subjects with mild asthma but were significantly greater in those with severe eosinophilic asthma. The highest levels were detected in subjects with severe asthma with airway autoimmune responses. C1q+ immune complexes from the sputa of subjects with severe asthma with high autoantibody levels stimulated TL1A production by monocytes.Conclusions: The TL1A/DR3 axis is a costimulator of ILC2s in asthma, particularly in the airways of patients with a predisposition to autoimmune responses.

Pathologic up-regulation of TNFSF15-TNFRSF25 axis sustains endothelial dysfunction in unprovoked venous thromboembolism.

AIMS: The pathogenetic mechanisms underlying unprovoked venous thromboembolism (uVTE) are largely unknown. In this study, we investigated the molecular mechanisms involved in uVTE pathogenesis by using ex vivo expanded endothelial colony-forming cells (ECFCs), which represent a valuable non-invasive tool for the assessment of endothelial function. METHODS AND RESULTS: We isolated and expanded ECFCs from the peripheral blood of uVTE patients and observed that these cells underwent earlier senescence and showed lower growth rate compared with ECFCs obtained from healthy donors. Through microarray expression profiling, we demonstrated that 2905 genes were differentially expressed between patients and controls. Among them, the anti-angiogenic cytokine TNF superfamily member 15 (TNFSF15) and its death-receptor TNFRSF25 were up-regulated in uVTE ECFCs, and this finding was validated by RT-qPCR. TNFSF15 up-regulation was confirmed at the protein level in ECFC supernatants, and the in vivo relevance of these findings was further corroborated by demonstrating that also the plasmatic levels of TNFSF15 are increased in uVTE patients. After proving that exogenous TNFSF15 exerts pro-apoptotic and anti-proliferative activity on control ECFCs, we demonstrated through blocking experiments that TNFSF15 up-regulation contributes to impaired survival and proliferation of uVTE ECFCs. CONCLUSION: By providing evidence that TNFSF15 impairs ECFC functions crucial to endothelial repair, and that uVTE patients have increased TNFSF15 levels both ex vivo and in vivo, the results of this study suggest that pathologic up-regulation of TNFSF15-TNFRSF25 axis may contribute to uVTE pathogenesis, and may represent the target for novel therapeutic strategies aimed at preventing recurrences in uVTE patients.

P21 and P27 promote tumorigenesis and progression via cell cycle acceleration in seminal vesicles of TRAMP mice.

Transgenic adenocarcinoma mouse prostate (TRAMP) model is established to mimic human prostate cancer progression, where seminal vesicle lesions often occur and has been described as phyllodes-like epithelial-stromal tumors. However, the molecular mechanism regulating tumorigenesis and progression in seminal vesicles of TRAMP mice remains largely unknown. In this study, C57BL/6 TRAMP mice were found to have a significantly shorter lifespan than wild-type (WT) mice and all of the seminal vesicles were markedly increased in size and weight with age from 24 weeks exhibiting a clearly papillary-phyllode pattern, though no obvious difference was observed in multiple organs including heart, liver, spleen, lungs, kidneys, testicles and bone between TRAMP and WT mice, and less than 10% of TRAMP mice developed prostate tumors. Western blotting showed Cyclin (CCN) B1 and CCND1 were remarkably overexpressed in seminal vesicle tumors of TRAMP mice at 24 weeks of age and increased with age till the end of trial, which was confirmed by Immunohistochemistry (IHC). P21 and P27 were also significantly augmented, whereas P53 and phosphorylated P53 (p-P53) were constantly expressed in normal controls and P53 did not appear to be mutated. Not only cyclin-dependent kinase (CDK) 1 and phosphorylated forkhead box protein (FOX) O1 but also CDK4, CDK6 and phosphorylated retinoblastoma-associated protein (RB) had similar increase trends, so did epidermal growth factor receptor (EGFR), AKT serine/threonine kinase (AKT), and their respective phosphorylation levels. Signal transducer and activator of transcription (STAT) 3, p-STAT3, enhancer of zeste homolog 2 (EZH2) and EZH2 mediated trimethylation of histone H3 lysine 27 (H3K27me3) were considerably elevated, too. Taken together, this finding suggests P21 and P27 promote carcinogenesis and development in seminal vesicles of TRAMP mice via accelerating cell cycle progression, in which oncogenic transformation of P21 and P27 might be through regulation of EGFR-AKT signaling.

Activation of DR3 signaling causes loss of ILC3s and exacerbates intestinal inflammation.

TNF-like ligand 1 A (TL1A) and death receptor 3 (DR3) are a ligand-receptor pair involved in the pathogenesis of inflammatory bowel disease. Group 3 innate lymphoid cells (ILC3s) regulate intestinal immunity and highly express DR3. Here, we report that activation of DR3 signaling by an agonistic anti-DR3 antibody increases GM-CSF production from ILC3s through the p38 MAPK pathway. GM-CSF causes accumulation of eosinophils, neutrophils and CD11b+CD11c+ myeloid cells, resulting in loss of ILC3s from the intestine in an IL-23-dependent manner and exacerbating colitis. Blockade of GM-CSF or IL-23 reverses anti-DR3 antibody-driven ILC3 loss, whereas overexpression of IL-23 induces loss of ILC3s in the absence of GM-CSF. Neutralization of TL1A by soluble DR3 ameliorates both DSS and anti-CD40 antibody-induced colitis. Moreover, ILC3s are required for the deleterious effect of anti-DR3 antibodies on innate colitis. These findings clarify the process and consequences of DR3 signaling-induced intestinal inflammation through regulation of ILC3s.

Epigenetic modulators hydralazine and sodium valproate act synergistically in VEGI-mediated anti-angiogenesis and VEGF interference in human osteosarcoma and vascular endothelial cells.

Vascular endothelial growth inhibitor (VEGI; also referred to as TNFSF15 or TL1A) is involved in the modulation of vascular homeostasis. VEGI is known to operate via two receptors: Death receptor­3 (DR3) and decoy receptor­3 (DcR3). DR3, which is thus far the only known functional receptor for VEGI, contains a death domain and induces cell apoptosis. DcR3 is secreted as a soluble protein and antagonizes VEGI/DR3 interaction. Overexpression of DcR3 and downregulation of VEGI have been detected in a number of cancers. The aim of the present study was to investigate the effects of sodium valproate (VPA), a histone deacetylase inhibitor, in combination with hydralazine hydrochloride (Hy), a DNA methylation inhibitor, on the expression of VEGI and its related receptors in human osteosarcoma (OS) cell lines and human microvascular endothelial (HMVE) cells. Combination treatment with Hy and VPA synergistically induced the expression of VEGI and DR3 in both OS and HMVE cells, without inducing DcR3 secretion. In addition, it was observed that the combination of VPA and Hy significantly enhanced the inhibitory effect on vascular tube formation by VEGI/DR3 autocrine and paracrine pathways. Furthermore, the VEGI/VEGF­A immune complex was pulled down by immunoprecipitation. Taken together, these findings suggest that DNA methyltransferase and histone deacetylase inhibitors not only have the potential to induce the re­expression of tumor suppressor genes in cancer cells, but also exert anti­angiogenic effects, via enhancement of the VEGI/DR3 pathway and VEGI/VEGF­A interference.

Death Receptor 3 Signaling Controls the Balance between Regulatory and Effector Lymphocytes in SAMP1/YitFc Mice with Crohn's Disease-Like Ileitis.

Death receptor 3 (DR3), a member of the tumor necrosis factor receptor (TNFR) superfamily, has been implicated in regulating T-helper type-1 (TH1), type-2 (TH2), and type-17 (TH17) responses as well as regulatory T cell (Treg) and innate lymphoid cell (ILC) functions during immune-mediated diseases. However, the role of DR3 in controlling lymphocyte functions in inflammatory bowel disease (IBD) is not fully understood. Recent studies have shown that activation of DR3 signaling modulates Treg expansion suggesting that stimulation of DR3 represents a potential therapeutic target in human inflammatory diseases, including Crohn's disease (CD). In this study, we tested a specific DR3 agonistic antibody (4C12) in SAMP1/YitFc (SAMP) mice with CD-like ileitis. Interestingly, treatment with 4C12 prior to disease manifestation markedly worsened the severity of ileitis in SAMP mice despite an increase in FoxP3+ lymphocytes in mesenteric lymph node (MLN) and small-intestinal lamina propria (LP) cells. Disease exacerbation was dominated by overproduction of both TH1 and TH2 cytokines and associated with expansion of dysfunctional CD25-FoxP3+ and ILC group 1 (ILC1) cells. These effects were accompanied by a reduction in CD25+FoxP3+ and ILC group 3 (ILC3) cells. By comparison, genetic deletion of DR3 effectively reversed the inflammatory phenotype in SAMP mice by promoting the expansion of CD25+FoxP3+ over CD25-FoxP3+ cells and the production of IL-10 protein. Collectively, our data demonstrate that DR3 signaling modulates a multicellular network, encompassing Tregs, T effectors, and ILCs, governing disease development and progression in SAMP mice with CD-like ileitis. Manipulating DR3 signaling toward the restoration of the balance between protective and inflammatory lymphocytes may represent a novel and targeted therapeutic modality for patients with CD.

Nintedanib effects on delaying cancer progression and decreasing COX-2 and IL-17 in the prostate anterior lobe in TRAMP mice.

Prostate cancer is the most prevalent type of cancer in men around the world. Due to its high incidence, new therapies have been evaluated, including drugs capable of inhibiting the FGF/VEGF pathways, as Nintedanib. The aim herein was to evaluate the Nintedanib therapeutic effects on morphology and COX-2 and IL-17 levels in the prostate anterior lobe in different grades of the tumor progression in TRAMP mice. Animals were treated with Nintedanib at a dose of 10 mg/kg/day in initial and intermediate grades of tumor development. At the end of treatment, the prostate anterior lobe was collected and submitted to morphological, immunohistochemical and Western Blotting analyses. The results showed that Nintedanib delayed the prostate carcinogenesis progression, with over 20% of reduction in frequency of tissue injuries, particularly in the group treated from 12 to 16 weeks of age. Also, decreased COX-2 and IL-17 levels were observed in both groups treated with Nintedanib in the prostate anterior lobe. Thus, we concluded that Nintedanib was effective in delaying tumor progression and, despite not directly acting on inflammation, Nintedanib may adversely affect inflammatory pathways, favoring prostate cancer delay.

A variant of death-receptor 3 associated with rheumatoid arthritis interferes with apoptosis-induction of T cell.

Rheumatoid arthritis (RA) is a chronic polyarthritis of unknown etiology. To unravel the molecular mechanisms in RA, we performed targeted DNA sequencing analysis of patients with RA. This analysis identified a variant of the death receptor 3 (DR3) gene, a member of the family of apoptosis-inducing Fas genes, which contains four single-nucleotide polymorphisms (SNPs) and a 14-nucleotide deletion within exon 5 and intron 5. We found that the deletion causes the binding of splicing regulatory proteins to DR3 pre-mRNA intron 5, resulting in a portion of intron 5 becoming part of the coding sequence, thereby generating a premature stop codon. We also found that this truncated DR3 protein product lacks the death domain and forms a heterotrimer complex with wildtype DR3 that dominant-negatively inhibits ligand-induced apoptosis in lymphocytes. Myelocytes from transgenic mice expressing the human DR3 variant produced soluble truncated DR3, forming a complex with TNF-like ligand 1A (TL1A), which inhibited apoptosis induction. In summary, our results reveal that a DR3 splice variant that interferes with ligand-induced T cell responses and apoptosis may contribute to RA pathogenesis.

DNA methylation polymerase chain reaction (PCR) array of apoptosis-related genes in pleomorphic adenomas of the salivary glands.

OBJECTIVE: The aim of this study was to evaluate the DNA methylation profile in 22 apoptosis-related genes in pleomorphic adenomas (PAs) of the salivary glands, in comparison with normal salivary glands (NSGs), and to address the differences in methylation patterns between smaller and larger tumors. Additionally, we investigated if the hypermethylation of differentially methylated genes between NSGs and PAs impacted the messenger RNA (mRNA) transcription. DESIGN: Twenty-three fresh PA samples and 12 NSG samples were included. The PA samples were divided into 2 groups: PAs with clinical size larger than 2 cm (n = 12) and PAs with clinical size 2 cm or smaller (n = 11). DNA methylation at the promoter region of a panel of 22 genes involved in apoptosis was profiled by using a human apoptosis DNA methylation polymerase chain reaction array, and the transcriptional levels of genes showing differential methylation profiles between PAs and NSGs were assessed. RESULTS: TNFRSF25 and BCL2 L11 were highly methylated in PAs, in comparison with NSGs, irrespective of tumor size. However, no difference could be observed in the mRNA transcription between PAs and NSGs. CONCLUSIONS: Hypermethylation of the proapoptotic genes BCL2 L11 and TNFRSF25 is observed in PA. However, this phenomenon did not impact mRNA transcription.

Death Receptor 3 regulates distinct pathological attributes of acute versus chronic murine allergic lung inflammation.

The Death Receptor 3 (DR3)/Tumour Necrosis Factor-like cytokine 1A (TL1A) axis stimulates effector T cells and type 2 innate lymphocytes (ILC2) that trigger cytokine release and drive disease pathology in several inflammatory and autoimmune diseases, including murine models of acute allergic lung inflammation (ALI). The aim of this study was to elucidate the role of DR3 in chronic ALI compared to acute ALI, using mice genetically deficient in the DR3 gene (DR3ko). Results showed DR3 expression in the lungs of wild-type mice was up-regulated following induction of acute ALI and this increased expression was maintained in chronic disease. DR3ko mice were resistant to cellular accumulation within the alveolar passages in acute, but not chronic ALI. However, DR3ko mice displayed reduced immuno-histopathology and goblet cell hyperplasia; hallmarks of the asthmatic phenotype; in chronic, but not acute ALI. These data suggest DR3 is a potential therapeutic target, involved in temporally distinct aspects of ALI progression and pathogenesis.

Shared αß TCR Usage in Lungs of Sarcoidosis Patients with Löfgren's Syndrome.

Sarcoidosis is a granulomatous disease that primarily affects the lungs and is characterized by an accumulation of CD4+ T cells in the bronchoalveolar lavage (BAL). Previous work has indicated that HLA-DRB1*03:01+ (DR3+) patients diagnosed with the acute form of the disease, Löfgren's syndrome (LS), have an accumulation of CD4+ T cells bearing TCRs using TRAV12-1 (formerly AV2S3). However, the importance of these α-chains in disease pathogenesis and the paired TCRß-chain remains unknown. This study aimed to identify expanded αßTCR pairs expressed on CD4+ T cells derived from the BAL of DR3+ LS patients. Using a deep-sequencing approach, we determined TCRα- and TCRß-chain usage, as well as αßTCR pairs expressed on BAL CD4+ T cells from LS patients. TRAV12-1 and TRBV2 (formerly BV22) were the most expanded V region gene segments in DR3+ LS patients relative to control subjects, and TRAV12-1 and TRBV2 CDR3 motifs were shared among multiple DR3+ LS patients. When assessing αßTCR pairing, TRAV12-1 preferentially paired with TRBV2, and these TRAV12-1/TRBV2 TCRs displayed CDR3 homology. These findings suggest that public CD4+ TCR repertoires exist among LS patients and that these T cells are recognizing the putative sarcoidosis-associated Ag(s) in the context of DR3.

5α-reductase 1 mRNA levels are positively correlated with TRAMP mouse prostate most severe lesion scores.

BACKGROUND: The contribution of 5α-reductase 1 and 5α-reductase 2 to prostate cancer development and progression is not clearly understood. TRAMP mice are a common prostate cancer model, in which 5α-reductase 1 and 5α-reductase 2 expression levels, along with prostate lesions scores, have not been investigated at different time points to further understand prostate carcinogenesis. METHOD/PRINCIPAL FINDINGS: To this end, 8-, 12-, 16-, and 20-week-old male C57BL/6TRAMP x FVB mice prostate most severe and most common lesion scores, 5α-reductase 1 and 5α-reductase 2 in situ hybridization expression, and Ki-67, androgen receptor, and apoptosis immunohistochemistry levels were measured. Levels of these markers were quantified in prostate epithelium, hyperplasia, and tumors sections. Mice developed low- to high-grade prostatic intraepithelial neoplasia at 8 weeks as the most severe and most common lesions, and moderate- and high-grade prostatic intraepithelial neoplasia at 12 and 16 weeks as the most severe lesion in all lobes. Moderately differentiated adenocarcinoma was observed at 20 weeks in all lobes. Poorly differentiated carcinoma was not observed in any lobe until 12-weeks-old. 5α-reductase 1 and 5α-reductase 2 were not significantly decreased in tumors compared to prostate epithelium and hyperplasia in all groups, while proliferation, apoptosis, and androgen receptor were either notably or significantly decreased in tumors compared with prostate epithelium and hyperplasia in most or all groups. Prostate 5αR1 levels were positively correlated with adjusted prostate most severe lesion scores. CONCLUSION: Downregulation of androgen receptor and 5α-reductase 2, along with upregulation of 5α-reductase 1 in tumors may promote prostatic intraepithelial neoplasia and prostate cancer development in TRAMP mice.