Targeting TNFRSF25 by agonistic antibodies and multimeric TL1A proteins co-stimulated CD8+ T cells and inhibited tumor growth.

BACKGROUND: Tumor necrosis factor receptor superfamily 25 (TNFRSF25) is a T-cell co-stimulatory receptor. Expression of its ligand, TNF-like cytokine 1A (TL1A), on mouse tumor cells has been shown to promote tumor regression. This study aimed to develop TNFRSF25 agonists (both antibodies (Abs) and TL1A proteins) and to investigate their potential antitumor effects. METHODS: Anti-mouse TNFRSF25 (mTNFRSF25) Abs and multimeric TL1A proteins were generated as TNFRSF25 agonists. Their agonism was assessed in luciferase reporter and T-cell co-stimulation assays, and their antitumor effects were evaluated in syngeneic mouse tumor models. TNFRSF25 expression within the tumor microenvironment and the effects of an anti-mTNFRSF25 agonistic Ab on tumor-infiltrating T cells were evaluated by flow cytometry. Cell depletion assays were used to identify the immune cell types that contribute to the antitumor effect of the anti-mTNFRSF25 Ab. The Fc gamma receptor (FcγR) dependence of TNFRSF25 agonists was assessed in an in vivo T-cell expansion model and a mouse tumor model using Fc variants and FcγR-deficient mice. RESULTS: TNFRSF25 agonists exhibited antitumor effects in syngeneic mouse tumor models without causing observed side effects. We identified an anti-mTNFRSF25 agonistic Ab, 1A6-m1, which exhibited greater antitumor activity than a higher affinity anti-TNFRSF25 Ab which engages an overlapping epitope with 1A6-m1. 1A6-m1 activated CD8+ T cells and antigen-specific T cells, leading to tumor regression; it also induced long-term antitumor immune memory. Although activating TNFRSF25 by 1A6-m1 expanded splenic regulatory T (Treg) cells, it did not influence intratumoral Treg cells. Moreover, 1A6-m1's antitumor effects required the engagement of both inhibitory FcγRIIB and activating FcγRIII. Replacing 1A6-m1's CH1-hinge region with that of human IgG2 (h2) conferred enhanced antitumor effects. Finally, we also generated multimeric human and mouse TL1A fusion proteins as TNFRSF25 agonists, and they co-stimulated CD8+ T cells and reduced tumor growth, even in the absence of Fc-FcγR interactions. CONCLUSION: Our data demonstrates the potential of activating TNFRSF25 by Abs and multimeric TL1A proteins for cancer immunotherapy and provides insights into their development astherapeutics.

Anti-TL1A monoclonal antibody modulates the dysregulation of Th1/Th17 cells and attenuates granuloma formation in sarcoidosis by inhibiting the PI3K/AKT signaling pathway.

Sarcoidosis is a systemic granulomatous disease characterized by non-caseating epithelioid cell granulomas. One of its immunological hallmarks is the differentiation of CD4 + naïve T cells into Th1/Th17 cells, accompanied by the release of numerous pro-inflammatory cytokines. The TL1A/DR3 signaling pathway plays a crucial role in activating effector lymphocytes, thereby triggering pro-inflammatory responses. The primary aim of this investigation was to scrutinize the impact of anti-TL1A monoclonal antibody on the dysregulation of Th1/Th17 cells and granuloma formation in sarcoidosis. Initially, the abnormal activation of the TL1A/DR3 signaling pathway in pulmonary tissues of sarcoidosis patients was confirmed using qPCR and immunohistochemistry techniques. Subsequently, employing a murine model of sarcoidosis, the inhibitory effects of anti-TL1A monoclonal antibody on the TL1A/DR3 signaling pathway in sarcoidosis were investigated through qPCR, immunohistochemistry, and Western blot experiments. The influence of anti-TL1A monoclonal antibody on granulomas was assessed through HE staining, while their effects on sarcoidosis Th1/Th17 cells and associated cytokine mRNA levels were evaluated using flow cytometry and qPCR, respectively. Immunofluorescence and Western blot experiments corroborated the inhibitory effects of anti-TL1A monoclonal antibody on the aberrant activation of the PI3K/AKT signaling pathway in sarcoidosis. The findings of this study indicate that the TL1A/DR3 signaling pathway is excessively activated in sarcoidosis. Anti-TL1A monoclonal antibody effectively inhibit this abnormal activation in sarcoidosis, thereby alleviating the dysregulation of Th1/Th17 cells and reducing the formation of pulmonary granulomas. This effect may be associated with the inhibition of the downstream PI3K/AKT signaling pathway. Anti-TL1A monoclonal antibody hold promise as a potential novel therapeutic intervention for sarcoidosis.

TL1A priming induces a multi-cytokine Th9 cell phenotype that promotes robust allergic inflammation in murine models of asthma.

Multi-cytokine-producing Th9 cells secrete IL-9 and type 2 cytokines and mediate mouse and human allergic inflammation. However, the cytokines that promote a multi-cytokine secreting phenotype have not been defined. Tumor necrosis factor superfamily member TL1A signals through its receptor DR3 to increase IL-9. Here we demonstrate that TL1A increases expression of IL-9 and IL-13 co-expressing cells in murine Th9 cell cultures, inducing a multi-cytokine phenotype. Mechanistically, this is linked to histone modifications allowing for increased accessibility at the Il9 and Il13 loci. We further show that TL1A alters the transcription factor network underlying expression of IL-9 and IL-13 in Th9 cells and increases binding of transcription factors to Il9 and Il13 loci. TL1A-priming enhances the pathogenicity of Th9 cells in murine models of allergic airway disease through the increased expression of IL-9 and IL-13. Lastly, in both chronic and memory-recall models of allergic airway disease, blockade of TL1A signaling decreases the multi-cytokine Th9 cell population and attenuates the allergic phenotype. Taken together, these data demonstrate that TL1A promotes the development of multi-cytokine Th9 cells that drive allergic airway diseases and that targeting pathogenic T helper cell-promoting cytokines could be an effective approach for modifying disease.

The ever-expanding role of cytokine receptor DR3 in T cells.

Death Receptor 3 (DR3) is a cytokine receptor of the Tumor Necrosis Factor receptor superfamily that plays a multifaceted role in both innate and adaptive immunity. Based on the death domain motif in its cytosolic tail, DR3 had been proposed and functionally affirmed as a trigger of apoptosis. Further studies, however, also revealed roles of DR3 in other cellular pathways, including inflammation, survival, and proliferation. DR3 is expressed in various cell types, including T cells, B cells, innate lymphocytes, myeloid cells, fibroblasts, and even outside the immune system. Because DR3 is mainly expressed on T cells, DR3-mediated immune perturbations leading to autoimmunity and other diseases were mostly attributed to DR3 activation of T cells. However, which T cell subset and what T effector functions are controlled by DR3 to drive these processes remain incompletely understood. DR3 engagement was previously found to alter CD4 T helper subset differentiation, expand the Foxp3+ Treg cell pool, and maintain intraepithelial γδ T cells in the gut. Recent studies further unveiled a previously unacknowledged aspect of DR3 in regulating innate-like invariant NKT (iNKT) cell activation, expanding the scope of DR3-mediated immunity in T lineage cells. Importantly, in the context of iNKT cells, DR3 ligation exerted costimulatory effects in agonistic TCR signaling, unveiling a new regulatory framework in T cell activation and proliferation. The current review is aimed at summarizing such recent findings on the role of DR3 on conventional T cells and innate-like T cells and discussing them in the context of immunopathogenesis.

Tumor necrosis factor-like cytokine 1A plays a role in inflammatory bowel disease pathogenesis.

The binding of tumor necrosis factor-like cytokine 1A (TL1A) to death receptor 3 (DR3) plays an important role in the interaction between dendritic cells (DCs) and T cells and contributes to intestinal inflammation development. However, the mechanism by which DCs expressing TL1A mediate helper T (Th) cell differentiation in the intestinal lamina propria (LP) during the pathogenesis of inflammatory bowel disease remains unclear. In this study, we found that TL1A/DR3 promoted Th1 and Th17 cell differentiation in T-T and DC-T cell interaction-dependent manners. TL1A-deficient CD4+ T cells failed to polarize into Th1/Th17 cells and did not cause colonic inflammation in a T cell transfer colitis model. Notably, TL1A was located in the cytoplasm and nuclei of DCs, positively regulated the DC-specific ICAM-grabbing nonintegrin/RAF1/nuclear factor κB signaling pathway, enhanced the antigen uptake ability of DCs, and promoted TLR4-mediated DC activation, inducing naive CD4+ T cell differentiation into Th1 and Th17 cells. Our work reveals that TL1A plays a regulatory role in inflammatory bowel disease pathogenesis.

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.

The cytokine receptor DR3 identifies and promotes the activation of thymic NKT17 cells.

Invariant natural killer T (iNKT) cells correspond to a population of thymus-generated T cells with innate-like characteristics and effector functions. Among the various iNKT subsets, NKT17 is the only subset that produces the proinflammatory cytokine IL-17. But, how NKT17 cells acquire this ability and what would selectively trigger their activation remain incompletely understood. Here, we identified the cytokine receptor DR3 being specifically expressed on thymic NKT17 cells and mostly absent on other thymic iNKT subsets. Moreover, DR3 ligation promoted the in vivo activation of thymic NKT17 cells and provided costimulatory effects upon agonistic α-GalCer stimulation. Thus, we identified a specific surface marker for thymic NKT17 cells that triggers their activation and augments their effector functions both in vivo and in vitro. These findings provide new insights for deciphering the role and function of murine NKT17 cells and for understanding the development and activation mechanisms of iNKT cells in general.

Meloxicam Inhibits Apoptosis in Neurons by Deactivating Tumor Necrosis Factor Receptor Superfamily Member 25, Leading to the Decreased Cleavage of DNA Fragmentation Factor Subunit α in Alzheimer's Disease.

Neuronal apoptosis is considered to be a critical cause of Alzheimer's disease (AD). Recently, meloxicam has shown neuroprotective effects; however, the inherent mechanisms are highly overlooked. Using APP/PS1 transgenic (Tg) mice as in vivo animal models, we found that meloxicam inhibits apoptosis in neurons by deactivating tumor necrosis factor receptor superfamily member 25 (TNFRSF25), leading to the suppression of the expression of fas-associated protein with death domain (FADD) and the cleavage of DNA fragmentation factor subunit α (DFFA) and cysteine aspartic acid protease-3 (caspase 3) via ß-amyloid protein (Aß)-depressing mechanisms. Moreover, the meloxicam treatment blocked the effects of ß-amyloid protein oligomers (Aßo) on stimulating the synthesis of tumor necrosis factor α (TNF-α) and TNF-like ligand 1A (TL1A) in neuroblastoma (N) 2a cells. TNF-α and TL1A induce apoptosis in neurons via TNFR- and TNFRSF25-dependent caspase 3-activating mechanisms, respectively. Knocking down the expression of TNFRSF25 blocked the effects of TL1A on inducing apoptosis in neurons by deactivating the signaling cascades of FADD, caspase 3, and DFFA. Consistently, TNFRSF25 shRNA blocked the effects of Aßo on inducing neuronal apoptosis, which was corroborated by the efficacy of meloxicam in inhibiting Aßo-induced neuronal apoptosis. By ameliorating neuronal apoptosis, meloxicam improved memory loss in APP/PS1 Tg mice.

Role of TL1A in Inflammatory Autoimmune Diseases: A Comprehensive Review.

TL1A, also called TNFSF15, is a member of tumor necrosis factor family. It is expressed in different immune cell, such as monocyte, macrophage, dendritic cell, T cell and non-immune cell, for example, synovial fibroblast, endothelial cell. TL1A competitively binds to death receptor 3 or decoy receptor 3, providing stimulatory signal for downstream signaling pathways, and then regulates proliferation, activation, apoptosis of and cytokine, chemokine production in effector cells. Recent findings showed that TL1A was abnormally expressed in autoimmune diseases, including rheumatoid arthritis, inflammatory bowel disease, psoriasis, primary biliary cirrhosis, systemic lupus erythematosus and ankylosing spondylitis. In vivo and in vitro studies further demonstrated that TL1A was involved in development and pathogenesis of these diseases. In this study, we comprehensively discussed the complex immunological function of TL1A and focused on recent findings of the pleiotropic activity conducted by TL1A in inflammatory autoimmune disease. Finish of the study will provide new ideas for developing therapeutic strategies for these diseases by targeting TL1A.

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.

Aqueous metabolome of tissue-specific conditional Pten-knockout mouse prostate cancer and TRAMP neuroendocrine carcinoma.

BACKGROUND: Metabolic reprograming is now a recognized hallmark of cancer. The prostate-specific phosphatase and tensin homolog deleted on chromosome 10 (Pten) gene-conditional knockout (KO) mouse carcinogenesis model is highly desirable for studying prostate cancer biology and prevention due to its close resemblance of primary molecular defects and histopathological features of human prostate cancer. We have recently published macromolecular profiling of this model by proteomics and transcriptomics, denoting a preeminence of inflammation and myeloid suppressive immune cell features. Here, we performed metabolomic analyses of Pten-KO prostate versus wild type (WT) counterpart for discernable changes in the aqueous metabolites and contrasted to those in the TRAMP neuroendocrine carcinoma (NECa). METHODS: Three matched pairs of tissue-specific conditional Pten-KO mouse prostate and WT prostate of litter/cage-mates at 20-22 weeks of age and three pairs of TRAMP NECa versus WT (28-31 weeks) were profiled for their global aqueous metabolite changes, using hydrophilic interaction liquid chromatography-tandem mass spectrometry. RESULTS: The Pten-KO prostate increased purine nucleotide pools, cystathionine, and both reduced and oxidized glutathione (GSH, GSSG), and gluconate/glucuronate species in addition to cholesteryl sulfate and polyamine precursor ornithine. On the contrary, Pten-KO prostate contained diminished pools of glycolytic intermediates and phosphorylcholine derivatives, select amino acids, and their metabolites. Bioinformatic integration revealed a significant shunting of glucose away from glycolysis-citrate cycle and glycerol-lipid genesis to pentose phosphate cycle for NADPH/GSH/GSSG redox and pentose moieties for purine and pyrimidine nucleotides, and glycosylation/glucuronidation. Implicit arginine catabolism to ornithine was consistent with immunosuppression in Pten-KO model. While also increased in cystathionine-GSH/GSSG, purine, and pyrimidine nucleotide pools and glucuronidation at the expense of glycolysis-citrate cycle, the TRAMP NECa increased abundance of many amino acids, methyl donor S-adenosyl-methionine, and intermediates for phospholipids without increasing cholesteryl sulfate or ornithine. CONCLUSIONS: The aqueous metabolomic patterns in Pten-KO prostate and TRAMP NECa shared similarities in the greater pools of cystathionine, GSH/GSSG redox pair, and nucleotides and shunting away from glycolysis-citrate cycle in both models. Remarkable metabolic distinctions between them included metabolisms of many amino acids (protein synthesis; arginine-ornithine/immune suppression) and cholesteryl sulfate and methylation donor for epigenetic regulations.

Roles for ADAM17 in TNF-R1 Mediated Cell Death and Survival in Human U937 and Jurkat Cells.

Signaling via death receptor family members such as TNF-R1 mediates pleiotropic biological outcomes ranging from inflammation and proliferation to cell death. Pro-survival signaling is mediated via TNF-R1 complex I at the cellular plasma membrane. Cell death induction requires complex IIa/b or necrosome formation, which occurs in the cytoplasm. In many cell types, full apoptotic or necroptotic cell death induction requires the internalization of TNF-R1 and receptosome formation to properly relay the signal inside the cell. We interrogated the role of the enzyme A disintegrin and metalloprotease 17 (ADAM17)/TACE (TNF-α converting enzyme) in death receptor signaling in human hematopoietic cells, using pharmacological inhibition and genetic ablation. We show that in U937 and Jurkat cells the absence of ADAM17 does not abrogate, but rather increases TNF mediated cell death. Likewise, cell death triggered via DR3 is enhanced in U937 cells lacking ADAM17. We identified ADAM17 as the key molecule that fine-tunes death receptor signaling. A better understanding of cell fate decisions made via the receptors of the TNF-R1 superfamily may enable us, in the future, to more efficiently treat infectious and inflammatory diseases or cancer.

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.

Use of Post-transplant Cyclophosphamide Treatment to Build a Tolerance Platform to Prevent Liquid and Solid Organ Allograft Rejection.

Corneal transplantation (CT) is the most frequent type of solid organ transplant (SOT) performed worldwide. Unfortunately, immunological rejection is the primary cause of graft failure for CT and therefore advances in immune regulation to induce tolerance remains an unmet medical need. Recently, our work and others in pre-clinical studies found that cyclophosphamide (Cy) administered after ("post-transplant," PTCy) hematopoietic stem cell transplantation (HSCT), i.e., liquid transplants is effective for graft vs. host disease prophylaxis and enhances overall survival. Importantly, within the past 10 years, PTCy has been widely adopted for clinical HSCT and the results at many centers have been extremely encouraging. The present studies found that Cy can be effectively employed to prolong the survival of SOT, specifically mouse corneal allografts. The results demonstrated that the timing of PTCy administration is critical for these CT and distinct from the kinetics employed following allogeneic HSCT. PTCy was observed to interfere with neovascularization, a process critically associated with immune rejection of corneal tissue that ensues following the loss of ocular "immune privilege." PTCy has the potential to delete or directly suppress allo-reactive T cells and treatment here was shown to diminish T cell rejection responses. These PTCy doses were observed to spare significant levels of CD4+ FoxP3+ (Tregs) which were found to be functional and could readily receive stimulating signals leading to their in vivo expansion via TNFRSF25 and CD25 agonists. In total, we posit future studies can take advantage of Cy based platforms to generate combinatorial strategies for long-term tolerance induction.

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.

Role of Myeloid-Derived Suppressor Cells in High-Dose-Irradiated TRAMP-C1 Tumors: A Therapeutic Target and an Index for Assessing Tumor Microenvironment.

PURPOSE: To investigate the temporal and spatial infiltration of TRAMP-C1 tumors by myeloid-derived suppressor cells (MDSCs) after high-dose radiation therapy (RT), and to explore their effect on tumor growth. METHODS AND MATERIALS: TRAMP-C1 intramuscularly tumors were irradiated with a single dose of 8 Gy or 25 Gy. The dynamics of infiltrated MDSCs and their intratumoral spatial distribution were assessed by immunohistochemistry and flow cytometry. Cytokine levels in the blood and tumor were analyzed by multiplex immunoassay. Mice were injected with anti-Gr-1 antibody to determine whether MDSCs affect tumor growth after RT. RESULTS: CD11b+Gr-1+ MDSCs infiltrated TRAMP-C1 tumors irradiated with 25 Gy, but not 8 Gy, within 4 hours and recruitment persisted for at least 2 weeks. Both CD11b+Ly6G+Ly6C+ polymorphonuclear-MDSCs (PMN-MDSCs) and CD11b+Ly6G-Ly6Chi monocytic-MDSCs (M-MDSCs) were involved. Tumor RT also increased the representation of both MDSC subpopulations in the spleen and peripheral blood. Levels of multiple cytokines were increased in the tumors at 2 weeks, including GM-CSF, G-CSF, CCL-3, CCL-5, CXCL-5, IL-6, IL-17α, and VEGF-a; while G-CSF, IL-6, and TNF-α levels increased in the blood. PMN-MDSCs aggregated in the central necrotic region of the irradiated tumors over time, where they were associated with avascular hypoxia (CD31-PIMO+). MDSCs expressed the proangiogenic factor, matrix metalloproteinase-9, and, within the necrotic area, high levels of arginase-1 and indoleamine 2,3-dioxygenase. Depletion of PMN-MDSCs by Gr-1 antibody increased the efficacy of high-dose RT. CONCLUSIONS: PMN-MDSCs infiltrate TRAMP-C1 tumors after high-dose RT. Their spatial distribution suggests they are involved in the evolution of an intratumoral state of necrosis associated with avascular hypoxia, and their phenotype is consistent with them being immunosuppressive. They appear to promote tumor growth after RT, making them a prime therapeutic target for therapeutic intervention. Assessment of MDSCs and cytokine levels in blood could be an index of the need for such an intervention.

TNFSF15 Promotes Antimicrobial Pathways in Human Macrophages and These Are Modulated by TNFSF15 Disease-Risk Variants.

BACKGROUND & AIMS: TNFSF15 genetic variants leading to increased TNF superfamily member 15 (TNFSF15) expression confer risk for inflammatory bowel disease (IBD), and TNFSF15 is being explored as a therapeutic target in IBD patients. Although the focus for TNFSF15-mediated inflammatory outcomes has been predominantly on its action on T cells, TNFSF15 also promotes inflammatory outcomes in human macrophages. Given the critical role for macrophages in bacterial clearance, we hypothesized that TNFSF15 promotes antimicrobial pathways in human macrophages and that macrophages from TNFSF15 IBD risk carriers with higher TNFSF15 expression have an advantage in these antimicrobial outcomes. METHODS: We analyzed protein expression, signaling, bacterial uptake, and intracellular bacterial clearance in human monocyte-derived macrophages through flow cytometry, enzyme-linked immunosorbent assay, and gentamicin protection. RESULTS: Autocrine/paracrine TNFSF15 interactions with death receptor 3 (DR3) were required for optimal levels of pattern-recognition-receptor (PRR)-induced bacterial clearance in human macrophages. TNFSF15 induced pyruvate dehydrogenase kinase 1-dependent bacterial uptake and promoted intracellular bacterial clearance through reactive oxygen species, nitric oxide synthase 2, and autophagy up-regulation. The TNFSF15-initiated TNF receptor-associated factor 2/receptor-interacting protein kinase 1/RIP3 pathway was required for mitogen-activated protein kinase and nuclear factor-κB activation, and, in turn, induction of each of the antimicrobial pathways; the TNFSF15-initiated Fas-associated protein with death domain/mucosa-associated lymphoid tissue lymphoma translocation protein 1/caspase-8 pathway played a less prominent role in antimicrobial functions, despite its key role in TNFSF15-induced cytokine secretion. Complementation of signaling pathways or antimicrobial pathways restored bacterial uptake and clearance in PRR-stimulated macrophages where TNFSF15:DR3 interactions were inhibited. Monocyte-derived macrophages from high TNFSF15-expressing rs6478108 TT IBD risk carriers in the TNFSF15 region showed increased levels of the identified antimicrobial pathways. CONCLUSIONS: We identify that autocrine/paracrine TNFSF15 is required for optimal PRR-enhanced antimicrobial pathways in macrophages, define mechanisms regulating TNFSF15-dependent bacterial clearance, and determine how the TNFSF15 IBD risk genotype modulates these outcomes.