Serum decoy receptor 3 (DcR3): a promising biomarker for atopic asthma in children.

Bronchial asthma is a common chronic inflammatory disease with high prevalence and morbidity, particularly in school-aged children. Decoy receptor 3 (DcR3) is a soluble decoy receptor that belongs to the tumor necrosis factor receptor superfamily and has been reported to be elevated in several allergic and inflammatory diseases. This study was designed to determine the role of DcR3 in pediatric asthma. The serum DcR3 levels were analyzed in 85 subjects (60 pediatric patients with bronchial asthma and 25 age- and sex-matched healthy control children) using the enzyme-linked immunosorbent assay technique. Patients with asthma had higher serum DcR3 levels than healthy control subjects (p = 0.007). In the atopic group of patients with asthma, the serum DcR3 levels were inversely correlated with the asthma control test score (R = - 0.392, p = 0.039). Overall, DcR3 could be a promising biomarker of atopic asthma, specifically in pediatric patients.

Immune landscape of human prostate cancer: immune evasion mechanisms and biomarkers for personalized immunotherapy.

BACKGROUND: Despite recent advances in cancer immunotherapy, the efficacy of these therapies for the treatment of human prostate cancer patients is low due to the complex immune evasion mechanisms (IEMs) of prostate cancer and the lack of predictive biomarkers for patient responses. METHODS: To understand the IEMs in prostate cancer and apply such understanding to the design of personalized immunotherapies, we analyzed the RNA-seq data for prostate adenocarcinoma from The Cancer Genome Atlas (TCGA) using a combination of biclustering, differential expression analysis, immune cell typing, and machine learning methods. RESULTS: The integrative analysis identified eight clusters with different IEM combinations and predictive biomarkers for each immune evasion cluster. Prostate tumors employ different combinations of IEMs. The majority of prostate cancer patients were identified with immunological ignorance (89.8%), upregulated cytotoxic T lymphocyte-associated protein 4 (CTLA4) (58.8%), and upregulated decoy receptor 3 (DcR3) (51.6%). Among patients with immunologic ignorance, 41.4% displayed upregulated DcR3 expression, 43.26% had upregulated CTLA4, and 11.4% had a combination of all three mechanisms. Since upregulated programmed cell death 1 (PD-1) and/or CTLA4 often co-occur with other IEMs, these results provide a plausible explanation for the failure of immune checkpoint inhibitor monotherapy for prostate cancer. CONCLUSION: These findings indicate that human prostate cancer specimens are mostly immunologically cold tumors that do not respond well to mono-immunotherapy. With such identified biomarkers, more precise treatment strategies can be developed to improve therapeutic efficacy through a greater understanding of a patient's immune evasion mechanisms.

Association of Tumor Necrosis Like factor 1 A (TL1A) and its Decoy Receptor (DcR3) with The Disease Activity and Autoantibody Production in Rheumatoid Arthritis Patients.

Different cytokines play roles in the pathogenesis and tissue damage of Rheumatoid Arthritis (RA) including, Tumor necrosis factor superfamily (TNFSF) and their receptors particularly TNF-like ligand 1A (TL1A), and its decoy receptor DcR3. This study included 150 subjects, of them 50 patients having Rheumatoid Arthritis (RA), 50 patients with Osteoarthritis (OA), and 50 normal controls. Clinical examination was done and data was collected from patient's sheets, routine laboratory investigations included, rheumatoid factor (RF) antibody, anti-cyclic citrullinated peptide (anti-CCP) antibody, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP). Disease activity score 28 was calculated and used to measure the activity of RA. Serum and synovial fluid (SF) TL1A and DcR3 levels were measured by (ELISA), while IL-17 was measured in supernatant fluid of PBMC culture after stimulation with recombinant human (rh) TL1A. Results showed significantly higher levels of TL1A and its decoy receptor DcR3 in RA patients than the other two groups. It was also found that TL1A is significantly related to the disease activity and enhances IL-17 production after stimulation of PBMC. These results can guide scientists to the future substitutions in the way of treatment of various inflammatory and autoimmune diseases.

Subgrouping breast cancer patients based on immune evasion mechanisms unravels a high involvement of transforming growth factor-beta and decoy receptor 3.

In the era of immunotherapy and personalized medicine, there is an urgent need for advancing the knowledge of immune evasion in different cancer types and identifying reliable biomarkers that guide both therapy selection and patient inclusion in clinical trials. Given the differential immune responses and evasion mechanisms in breast cancer, we expect to identify different breast cancer groups based on their expression of immune-related genes. For that, we used the sequential biclustering method on The Cancer Genome Atlas RNA-seq breast cancer data and identified 7 clusters. We found that 77.4% of the clustered tumor specimens evade through transforming growth factor-beta (TGF-ß) immunosuppression, 57.7% through decoy receptor 3 (DcR3) counterattack, 48.0% through cytotoxic T-lymphocyte-associated protein 4 (CTLA4), and 34.3% through programmed cell death-1 (PD-1). TGF-ß and DcR3 are potential novel drug targets for breast cancer immunotherapy. Targeting TGF-ß and DcR3 may provide a powerful approach for treating breast cancer because 57.7% of patients overexpressed these two molecules. Furthermore, triple-negative breast cancer (TNBC) patients clustered equally into two subgroups: one with impaired antigen presentation and another with high leukocyte recruitment but four different evasion mechanisms. Thus, different TNBC patients may be treated with different immunotherapy approaches. We identified biomarkers to cluster patients into subgroups based on immune evasion mechanisms and guide the choice of immunotherapy. These findings provide a better understanding of patients' response to immunotherapies and shed light on the rational design of novel combination therapies.

Dendritic cells engineered to secrete anti-DcR3 antibody augment cytotoxic T lymphocyte response against pancreatic cancer in vitro.

AIM: To investigate the enhanced cytotoxic T lymphocyte responses against pancreatic cancer (PC) in vitro induced by dendritic cells (DCs) engineered to secrete anti-DcR3 monoclonal antibody (mAb). METHODS: DCs, T lymphocytes and primary PC cells were obtained from PC patients. DCs were transfected with a designed humanized anti-DcR3 monoclonal antibody heavy and light chain mRNA and/or total tumor RNA (DC-tumor-anti-DcR3 RNA or DC-total tumor RNA) by using electroporation technology. The identification, concentration and function of anti-DcR3 mAb secreted by DC-tumor-anti-DcR3 RNA were determined by western blotting and enzyme-linked immunosorbent assay. After co-culturing of autologous isolated PC cells with target DCs, the effects of secreting anti-DcR3 mAb on RNA-DCs' viability and apoptosis were assessed by MTT assay and flow cytometry. Analysis of enhanced antigen-specific immune response against PC induced by anti-DcR3 mAb secreting DCs was performed using a 51Cr releasing test. T cell responses induced by RNA-loaded DCs were analyzed by measuring cytokine levels, including IFN-γ, IL-10, IL4, TNF-α and IL-12. RESULTS: The anti-DcR3 mAb secreted by DCs reacted with recombinant human DcR3 protein and generated a band with 35 kDa molecular weight. The secreting mAb was transient, peaking at 24 h and becoming undetectable after 72 h. After co-incubation with DC-tumor-anti-DcR3 RNA for designated times, the DcR3 level in the supernatant of autologous PC cells was significantly down-regulated (P < 0.05). DCs secreting anti-DcR3 mAb could improve cell viability and slow down the apoptosis of RNA-loaded DCs, compared with DC-total tumor RNA (P < 0.01). The anti-DcR3 mAb secreted by DC-tumor-anti-DcR3 RNA could enhance the induction of cytotoxic T lymphocytes (CTLs) activity toward RNA-transfected DCs, primary tumor cells, and PC cell lines, compared with CTLs stimulated by DC-total tumor RNA or control group (P < 0.05). Meanwhile, the antigen-specific CTL responses were MHC class I-restricted. The CD4+ T cells and CD8+ T cells incubated with anti-DcR3 mAb secreting DCs could produce extremely higher level IFN-γ and lower level IL4 than those incubated with DC-total tumor RNA or controls (P < 0.01). CONCLUSION: DCs engineered to secrete anti-DcR3 antibody can augment CTL responses against PC in vitro, and the immune-enhancing effects may be partly due to their capability of down-regulating DC apoptosis and adjusting the Th1/Th2 cytokine network.

DcR3 suppresses influenza virus-induced macrophage activation and attenuates pulmonary inflammation and lethality.

UNLABELLED: Influenza A virus (IAV) infects macrophages and stimulates innate immunity receptors and sensors to produce proinflammatory cytokines and chemokines, which are responsible for IAV-induced pulmonary inflammation and injury. Decoy receptor 3 (DcR3) is a soluble protein belonging to the tumor necrosis factor receptor superfamily (TNFRSF), and is able to skew macrophage differentiation into an M2 phenotype. We demonstrated that DcR3 attenuated IAV-induced secretion of proinflammatory cytokines and chemokine from macrophages, and mitigated pulmonary infiltration and reduce lethality. Proteome-wide phosphoproteomic mapping revealed that DcR3 not only activated STK10, a negative regulator of cell migration, but also inactivated PKC-α, which are crucial for the activation of ERK and JNK in human macrophages. Furthermore, less pulmonary infiltration with lower levels of proinflammatory cytokines and chemokine in bronchoalveolar lavage fluid (BALF) were observed in DcR3-transgenic mice. Moreover, recombinant DcR3.Fc and heparan sulfate proteoglycan binding domain of DcR3.Fc (HBD.Fc) fusion proteins attenuated weight loss and protected mice from IAV-induced lethality. Thus, DcR3-mediated protection is not only via suppression of proinflammatory cytokine and chemokine release, but also via activation of STK10 to inhibit cell infiltration. DcR3 fusion proteins may become therapeutic agents to protect host from IAV-induced lethality in the future. KEY MESSAGE: • DcR3 suppresses IAV-induced cytokine secretion.• DcR3 inhibits IAV-induced JNK and ERK activation in human macrophages.• DcR3 downregulates TLR3 and 7 expressions in human macrophages.• DcR3 protects mice from IAV-induced lethality.

Decoy receptor 3 suppresses TLR2-mediated B cell activation by targeting NF-κB.

Decoy receptor 3 (DcR3) is a soluble protein in the TNFR superfamily. Its known ligands include Fas ligand, homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes, TNF-like molecule 1A, and heparan sulfate proteoglycans. DcR3 has been reported to modulate the functions of T cells, dendritic cells, and macrophages; however, its role in regulating B cell activation is largely unknown. In this study, we found that the DcR3.Fc fusion protein bound to human and mouse B cells and suppressed the activation of B cells. DcR3.Fc attenuated Staphylococcus aureus, IgM-, Pam(3)CSK(4)-, and LPS-mediated B cell proliferation but did not affect cytokine-induced B cell growth. In the presence of these mitogens, DcR3.Fc did not induce B cell apoptosis, suggesting that DcR3 may inhibit the signal(s) important for B cell activation. Because the combination of Fas.Fc, LT-ßR.Fc (homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes receptor), and DR3.Fc (TNF-like molecule 1A receptor) did not suppress B cell proliferation and because the biological effect of DcR3.Fc on B cells was not blocked by heparin, we hypothesize that a novel ligand(s) of DcR3 mediates its inhibitory activity on B cells. Moreover, we found that TLR2-stimulated NF-κB p65 activation and NF-κB-driven luciferase activity were attenuated by DcR3.Fc. The TLR2-induced cytokine production by B cells was consistently reduced by DcR3. These results imply that DcR3 may regulate B cell activation by suppressing the activation of NF-κB.

Decoy receptor 3 attenuates collagen-induced arthritis by modulating T cell activation and B cell expansion.

OBJECTIVE: To investigate the immune-modulated effects of decoy receptor 3 (DCR3) in an experimental model of rheumatoid arthritis (RA). METHODS: We delivered DCR3 plasmid into collagen-induced arthritis (CIA) mice using the hydrodynamic method and evaluated the serum level of DCR3 protein by ELISA. After immunization, we assessed disease severity of arthritis incidence, arthritis scores, paw thickness, and means of arthritic limbs, and used hematoxylin and eosin staining to observe synovial hyperplasia. We analyzed numbers of murine splenocytes and inguinal lymphocyte cells, cell populations, and serum proinflammatory cytokines by flow cytometry. We investigated B cell proliferation by carboxyfluorescein succinimidyl ester assay. We evaluated serum levels of total IgG2a and type II collagen-specific IgG and IgG2a using ELISA. RESULTS: DCR3 expression in sera significantly attenuated disease severity in CIA mice. We found that DCR3 inhibited the volume of inguinal lymph nodes, numbers of CD19+ B cells, and populations of interferon-γ, interleukin 4 (IL-4), IL-17A, and Foxp3-producing CD4+ T cell in vivo. We found that DCR3 inhibited Pam3CSK4 (Toll-like receptor 1/2 ligand)-induced B220+ B cell proliferation in vitro. DCR3 treatment reduced the serum level of IL-6, total IgG2a, and CII-specific IgG2a antibody. CONCLUSION: We postulated that the protective effects of DCR3 in CIA resulted from modulation of the immune system by maintaining the B/T cell balance and decreasing lymphocyte expansion. We suggest DCR3 as a prophylactic and potential therapeutic agent in the treatment of RA.

Decoy strategies: the structure of TL1A:DcR3 complex.

Decoy Receptor 3 (DcR3), a secreted member of the Tumor Necrosis Factor (TNF) receptor superfamily, neutralizes three different TNF ligands: FasL, LIGHT, and TL1A. Each of these ligands engages unique signaling receptors which direct distinct and critical immune responses. We report the crystal structures of the unliganded DcR3 ectodomain and its complex with TL1A, as well as complementary mutagenesis and biochemical studies. These analyses demonstrate that DcR3 interacts with invariant backbone and side-chain atoms in the membrane-proximal half of TL1A which supports recognition of its three distinct TNF ligands. Additional features serve as antideterminants that preclude interaction with other members of the TNF superfamily. This mode of interaction is unique among characterized TNF:TNFR family members and provides a mechanistic basis for the broadened specificity required to support the decoy function of DcR3, as well as for the rational manipulation of specificity and affinity of DcR3 and its ligands.

Decoy receptor 3 protects non-obese diabetic mice from autoimmune diabetes by regulating dendritic cell maturation and function.

Decoy receptor 3 (DcR3), a member of the tumor necrosis factor receptor superfamily, regulates immune responses through competing with receptors of Fas ligand (FasL), LIGHT and TNF-like molecule 1A (TL1A). We have previously demonstrated that transgenic expression of DcR3 in a ß cell-specific manner significantly protects non-obese diabetic (NOD) mice from autoimmune diabetes. In this study, we further investigated the systemic effect of DcR3 in regulating lymphocytes and dendritic cells in NOD mice. Our results demonstrated that both DcR3 plasmid and protein treatments significantly inhibited insulitis and diabetes. Lymphocytes from DcR3.Fc-treated mice revealed less proliferative potential and transferred ameliorated diabetes. By administration of DcR3.Fc in T1 and T2 double transgenic NOD mice expressing human Thy1 or murine Thy1.1 surface marker under IFN-γ or IL-4 promoter control respectively, we observed a remarkable reduction of Th1 and an increase of Th2 immune responses in vivo. Strikingly, in vitro polarization experiments exhibited that not only Th1 but also Th17 cell differentiation was significantly inhibited in splenocytes treated with DcR3.Fc protein. However, this phenomenon was only observed in splenocytes, not in purified CD4(+) T cells, suggesting that DcR3-mediated inhibition of Th1 and Th17 differentiation is not T cell-autonomous and maybe through other cell types such as dendritic cells. Finally, our results demonstrated that DcR3 directly modulates the differentiation and maturation of dendritic cells and subsequently regulates the differentiation and effector function of T cells.

Pediatric inflammatory bowel diseases: coming of age.

PURPOSE OF REVIEW: Inflammatory bowel diseases (IBD) comprise a heterogeneous group of distinct intestinal disorders. Here, we discuss the concept of childhood-onset IBD as separate disease forms within a larger multifactorial disease category. RECENT FINDINGS: There are excellent epidemiological data indicating that the incidence of pediatric IBD, mainly Crohn's disease, is still increasing over the last decades, with indicators of more extensive and more severe disease presentations in children compared to adults, also reflected by higher levels of humoral immune responses. Recent genetic scans allowed to identify particular susceptibility genes for pediatric IBD forms, such as IL27 or probably DcR3. Early postnatal onset forms of IBD might reflect monogenetic causes, as suggested with the finding of IL10 signaling defects that may define a new form of IBD. SUMMARY: There are good epidemiological, genetic and clinical data to distinguish different forms of IBD, particularly forms starting early in life. Profound insights in the molecular basis of immune dysregulation in IBD have been gained over the last few years. These recent discoveries will nourish and substantially stimulate the future search for precise cause(s) responsible for life-long intestinal inflammation and it will help to explain the still ongoing rise in incidence in childhood IBD.

Decoy receptor 3 overexpression and immunologic tolerance in hepatocellular carcinoma (HCC) development.

The recently identified decoy receptor 3 (DcR3) inhibits FasL-induced apoptosis by binding to FasL, and it is considered to play a key role in the immune escape system of neoplastic cells. In order to examine the involvement of DcR3 in the immunologic tolerance of hepatocellular carcinoma (HCC), we investigated the amplification and expression of DcR3, FasL, and Fas in an HCC mice model using RT-PCR, western blotting, and ELISA, and analyzed the space-time relationship with various cytokines including the forkhead transcription factor forkhead/winged helix transcription factor gene (Foxp3), CTLA-4, TGF-beta, IL-10, TNF-alpha, and IFN-gamma. The RT-PCR results revealed that Fas expression preceded that of DcR3 during the early phases of tumorigenesis. Thereafter, the expression of DcR3 was up-regulated; however, the expression of Fas was down-regulated and eventually ceased. DcR3 and FasL were expressed and amplified simultaneously in muscle tumor. CTLA-4 expression was earlier than Foxp3, and both CTLA-4 and Foxp3 amplification and expression were consistent with that of DcR3. The results suggest that the elevated levels of DcR3, Foxp3, and CTLA-4 in tissue were positively correlated with tumor growth. The partial tumor immunoregulation inclined to negative modulation, and DcR3 may play an important role in inducing immunologic tolerance.

DcR3 as a diagnostic parameter and risk factor for systemic lupus erythematosus.

In this study, we investigated the diagnostic value of serum death decoy receptor 3 (DcR3) for systemic lupus erythematosus (SLE). The possible pathogenic role of DcR3 in SLE was also assessed. Serum DcR3 levels of 90 SLE patients, 11 patients with rheumatic conditions and 123 healthy controls were determined by ELISA. In all, 43% of the SLE patients, 9% of patients with rheumatic conditions and 2.4% of the normal healthy individuals presented elevated serum DcR3 levels. A higher percentage of DcR3-positive SLE patients, compared with DcR3-negative SLE patients, showed abnormally high serum IgE levels, a surrogate marker of T(h)2-type immune responses. To determine the cause and effect relationship of DcR3 expression and a T(h)2-prone status, we studied young DcR3 transgenic (Tg) mice, whose transgene was driven by an actin promoter. These mice had IL-4 overproduction and augmented serum IgE levels, signs of dominant T(h)2 immune responses. To determine possible SLE pathogenic roles of DcR3, the T-cell-depleted bone marrow of DcR3 Tg mice was transplanted into lethally irradiated syngeneic C57BL/6 female mice. The recipients developed an SLE-like syndrome. They presented anti-dsDNA and anti-nuclear antibodies, along with renal and liver pathology compatible with that of SLE. In total, 90% of Tg bone marrow-transplanted mice, compared with 20% of wild-type bone marrow-transplanted mice, perished within 12 months after the transplantation. Our results showed that serum DcR3 could serve as an additional parameter for SLE diagnosis and that DcR3 secreted from cells of hematopoietic origin was SLE pathogenic in mice.

[Preparation and identification of monoclonal antibodies against human DcR3].

AIM: To prepare monoclonal antibodies(mAbs) against human DcR3 and identify their characterization. METHODS: BALB/c mice were immunized with purified His-DcR3 protein, and mAbs against DcR3 which prepared by hybridoma technique were purified and identified by their specificity, subtype, titers via ELISA and Western blot. RESULTS: Five hybridoma cell lines secreting mAbs against human DcR3 were obtained, which were determined as IgG1 subtype and ascites titers of five mAbs against DcR3 reached 1x10(-5)-1x10(-7). Five mAbs were proved to recognize His-DcR3 protein specifically, one of which (1B1) could recognize SW480 cell. CONCLUSION: mAbs against DcR3 with high titers and specificity have been prepared and purified successfully, which laid a foundation for the study of DcR3 expression, distribution in tissu and development of ELISA kit.

Apoptosis of dendritic cells induced by decoy receptor 3 (DcR3).

Decoy receptor 3 (DcR3) is a soluble decoy receptor belonging to the tumor necrosis factor receptor (TNFR) superfamily, and its expression is not only up-regulated in cancer cells derived from various cell lineages, but also correlates with overall survival of patients with cancer. It has been shown that DcR3 sensitize cells of hematopoietic origin to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis; therefore, we asked whether DcR3 down-regulated host immunity by inducing immune cell apoptosis. We demonstrate that DcR3 induces dendritic cell (DC) apoptosis by activating PKC-delta and JNK subsequently to up-regulate DR5 to recruit Fas-associated death domain (FADD) to propagate the apoptotic signals. The association of FADD with DR5 results in the formation of death-inducing signaling complex (DISC) to trigger the downstream apoptotic signaling cascade. PKC-delta is activated via cross-linking of heparan sulfate proteoglycan (HSPG) on DCs, because recombinant protein containing the heparin-binding domain (HBD) of DcR3 and the Fc portion of IgG(1), the HBD.Fc fusion protein, is also able to trigger DC apoptosis. This provides the first evidence that cross-linking of HSPG on DCs can activate PKC-delta to induce DC apoptosis via the formation of DR5 DISC, and elucidates a novel mechanism of DcR3-mediated immunosuppression.

Correlation between expression of DcR3 on tumor cells and sensitivity to FasL.

To investigate the correlation between sensitivity to Fas ligand (FasL) and expression level of decoy receptor 3 (DcR3) on tumor cell surface, Fas/DcR3 mRNA expression was detected by RT-PCR. Anti-DcR3 mAb was used to detect expression level of DcR3 on surface of tumor cells by flow cytometry. Caspase-8, caspase-9, caspase-3, Bcl-2 expressions were analyzed by Western blot, respectively. Sensitivity to apoptosis induced by FasL was determined by Annexin V apoptosis kit. The expressions of DcR3 on the surface of tumor cells from high to low were approximately 35.3% in BGC823 cells, and 21.6% in MCF-7 cells, respectively. The apoptotic rates induced by FasL from low to high were 15.6% in BGC823 cells, and 58.2% in MCF-7 cells, respectively. There was a significant correlation between the expression levels of DcR3 with FasL-inducing apoptosis.