Molecular signatures in prion disease: altered death receptor pathways in a mouse model.

BACKGROUND: Prion diseases are transmissible and fatal neurodegenerative diseases characterized by accumulation of misfolded prion protein isoform (PrPSc), astrocytosis, microgliosis, spongiosis, and neurodegeneration. Elevated levels of cell membrane associated PrPSc protein and inflammatory cytokines hint towards the activation of death receptor (DR) pathway/s in prion diseases. Activation of DRs regulate, either cell survival or apoptosis, autophagy and necroptosis based on the adaptors they interact. Very little is known about the DR pathways activation in prion disease. DR3 and DR5 that are expressed in normal mouse brain were never studied in prion disease, so also their ligands and any DR adaptors. This research gap is notable and investigated in the present study. METHODS: C57BL/6J mice were infected with Rocky Mountain Laboratory scrapie mouse prion strain. The progression of prion disease was examined by observing morphological and behavioural abnormalities. The levels of PrP isoforms and GFAP were measured as the marker of PrPSc accumulation and astrocytosis respectively using antibody-based techniques that detect proteins on blot and brain section. The levels of DRs, their glycosylation and ectodomain shedding, and associated factors warrant their examination at protein level, hence western blot analysis was employed in this study. RESULTS: Prion-infected mice developed motor deficits and neuropathology like PrPSc accumulation and astrocytosis similar to other prion diseases. Results from this research show higher expression of all DR ligands, TNFR1, Fas and p75NTR but decreased levels DR3 and DR5. The levels of DR adaptor proteins like TRADD and TRAF2 (primarily regulate pro-survival pathways) are reduced. FADD, which primarily regulate cell death, its level remains unchanged. RIPK1, which regulate pro-survival, apoptosis and necroptosis, its expression and proteolysis (inhibits necroptosis but activates apoptosis) are increased. CONCLUSIONS: The findings from the present study provide evidence towards the involvement of DR3, DR5, DR6, TL1A, TRAIL, TRADD, TRAF2, FADD and RIPK1 for the first time in prion diseases. The knowledge obtained from this research discuss the possible impacts of these 16 differentially expressed DR factors on our understanding towards the multifaceted neuropathology of prion diseases and towards future explorations into potential targeted therapeutic interventions for prion disease specific neuropathology.

Functional metagenomic analysis reveals potential inflammatory triggers associated with genetic risk for autoimmune disease.

To assess functional differences between the microbiomes of individuals with autoimmune risk-associated human leukocyte antigen (HLA) genetics and autoimmune protection-associated HLA, we performed a metagenomic analysis of stool samples from 72 infants in the All Babies in Southeast Sweden general-population cohort and assessed haplotype-peptide binding affinities. Infants with risk-associated HLA DR3-DQ2.5 and DR4-DQ8 had a higher abundance of known pathogen-associated molecular patterns and virulence related genes than infants with protection-associated HLA DR15-DQ6.2. However, there was limited overlap in the type of inflammatory trigger between risk groups. Supported by a high Firmicutes/Bacteroides ratio and differentially abundant flagellated species, genes related to the synthesis of flagella were prominent in those with HLA DR3-DQ2.5. However, this haplotype had a significantly lower likelihood of binding affinity to flagellin peptides. O-antigen biosynthesis genes were significantly correlated with the risk genotypes and absent from protective genotype association, supported by the differential abundance of gram-negative bacteria seen in the risk-associated groups. Genes related to vitamin B biosynthesis stood out in higher abundance in infants with HLA DR3-DQ2.5/DR4-DQ8 heterozygosity compared to those with autoimmune-protective genetics. Prevotella species and genus were significantly abundant in all infant groups with high risk for autoimmune disease. The potential inflammatory triggers associated with genetic risk for autoimmunity have significant implications. These results suggest that certain HLA haplotypes may be creating the opportunity for dysbiosis and subsequent inflammation early in life by clearing beneficial microbes or not clearing proinflammatory microbes. This HLA gatekeeping may prevent genetically at-risk individuals from benefiting from probiotic therapies by restricting the colonization of those beneficial bacteria.

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 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.

Proinsulin peptide promotes autoimmune diabetes in a novel HLA-DR3-DQ2-transgenic murine model of spontaneous disease.

AIMS/HYPOTHESIS: The molecular basis for the pathological impact of specific HLA molecules on autoimmune diseases such as type 1 diabetes remains unclear. Recent natural history studies in children have indicated a link between specific HLA genotypes and the first antigenic target against which immune responses develop. We set out to examine this link in vivo by exploring the diabetogenicity of islet antigens on the background of a common diabetes-associated HLA haplotype. METHODS: We generated a novel HLA-transgenic mouse model that expresses high-risk genes for type 1 diabetes (DRB1*03:01-DQA1*05:01-DQB1*02:01) as well as human CD80 under the rat insulin promoter and human CD4, on a C57BL/6 background. Adjuvanted antigen priming was used to reveal the diabetogenicity of candidate antigens and peptides. RESULTS: HLA-DR3-DQ2+huCD4+IA/IE-/-RIP.B7.1+ mice spontaneously developed autoimmune diabetes (incidence 46% by 35 weeks of age), accompanied by numerous hallmarks of human type 1 diabetes (autoantibodies against GAD65 and proinsulin; pancreatic islet infiltration by CD4+, CD8+ B220+, CD11b+ and CD11c+ immune cells). Disease was markedly accelerated and had deeper penetrance after adjuvanted antigen priming with proinsulin (mean onset 11 weeks and incidence 100% by 20 weeks post challenge). Moreover, the diabetogenic effect of proinsulin located to the 15-residue B29-C11 region. CONCLUSIONS/INTERPRETATION: Our study identifies a proinsulin-derived peptide region that is highly diabetogenic on the HLA-DR3-DQ2 background using an in vivo model. This approach and the peptide region identified may have wider implications for future studies of human type 1 diabetes.

TNFSF/TNFRSF cytokine gene expression in sickle cell anemia: Up-regulated TNF-like cytokine 1A (TL1A) and its decoy receptor (DcR3) in peripheral blood mononuclear cells and plasma.

BACKGROUND: Sickle cell anemia (SCA), a disorder with an important inflammatory component, where vasoocclusion is major contributor to the disease pathophysiology. Pro-inflammatory cytokines play an important regulatory role in the process of inflammation. We investigated the expression TL1A/DR3/DcR3 cytokine signaling pathway in peripheral blood mononuclear cells (PBMC) and their corresponding plasma levels in SCA subjects who presented with acute painful episodes. MATERIALS AND METHODS: PBMC were isolated from the blood of SCA subjects and normal healthy controls. RNA isolated from PBMC was used for real time gene expression of TL1A/DR3/DcR3. Gene expression was compared in subgroups within SCA subjects with co-inherited fetal hemoglobin (HbF) or alpha-globin gene deletions. Plasma prepared from blood was used for determination of TL1A/DR3/DcR3 proteins by ELISA assays. RESULTS: In the PBMC of SCA subjects, expression of TL1A and DcR3 is elevated, while DR3 expression is lowered in comparison to normal control PBMC. In SCA subjects with HbF > 10%, TL1A/DcR3 expression is lower, while HbF < 10% is associated with increased TL1A/DcR3 expression. Moreover, subjects with HbF > 10% appear to have significantly fewer pain episodes in comparison to those with HbF < 10%. Deletion of alpha-globin genes appears to have no significant effect on TL1A/DR3/DcR3 expression. Circulating levels of TL1A, DR3 and DcR3 in plasma were significantly elevated in SCA subjects. CONCLUSIONS: Elevated TL1A and DcR3 expression in PBMC of SCA subjects during painful vasoocclusive crisis, suggest an altered TL1A expression may contribute to the pathophysiology of vasoocclusive crisis in SCA. HbF > 10% appears to moderate TL1A elevation, while HbF < 10% exacerbates TL1A/DcR3 responses. Furthermore, subjects with HbF > 10% have significantly lower pain episodes reported as compared to subjects with HbF < 10%.

The TNF-TNFR Family of Co-signal Molecules.

Costimulatory signals initiated by the interaction between the tumor necrosis factor (TNF) ligand and cognate TNF receptor (TNFR) superfamilies promote clonal expansion, differentiation, and survival of antigen-primed CD4+ and CD8+ T cells and have a pivotal role in T-cell-mediated adaptive immunity and diseases. Accumulating evidence in recent years indicates that costimulatory signals via the subset of the TNFR superfamily molecules, OX40 (TNFRSF4), 4-1BB (TNFRSF9), CD27, DR3 (TNFRSF25), CD30 (TNFRSF8), GITR (TNFRSF18), TNFR2 (TNFRSF1B), and HVEM (TNFRSF14), which are constitutive or inducible on T cells, play important roles in protective immunity, inflammatory and autoimmune diseases, and tumor immunotherapy. In this chapter, we will summarize the findings of recent studies on these TNFR family of co-signaling molecules regarding their function at various stages of the T-cell response in the context of infection, inflammation, and cancer. We will also discuss how these TNFR co-signals are critical for immune regulation and have therapeutic potential for the treatment of T-cell-mediated diseases.

Control of Regulatory T Cells by Co-signal Molecules.

Foxp3-expressing regulatory T cells (Tregs) perform a vital function in the maintenance of immune homeostasis. A large part of Treg suppressive function is derived from their ability to control and restrict the availability of co-signal molecules to other T cells. However, Tregs themselves also depend on many of the same co-signals for their own homeostasis, making this a complex system of feedback. In this chapter, we discuss the critical role of co-signaling in Treg cell biology.

Oestrogen-deficiency induces bone loss by modulating CD14+ monocyte and CD4+ T cell DR3 expression and serum TL1A levels.

BACKGROUND: Oestrogen-deficiency induced by menopause is associated with reduced bone density and primary osteoporosis, resulting in an increased risk of fracture. While the exact etiology of menopause-induced primary osteoporotic bone loss is not fully known, members of the tumour necrosis factor super family (TNFSF) are known to play a role. Recent studies have revealed that the TNFSF members death receptor 3 (DR3) and one of its ligands, TNF-like protein 1A (TL1A) have a key role in secondary osteoporosis; enhancing CD14+ peripheral blood mononuclear cell (PBMC) osteoclast formation and bone resorption. Whether DR3 and TL1A contribute towards bone loss in menopause-induced primary osteoporosis however, remains unknown. METHODS: To investigate this we performed flow cytometry analysis of DR3 expression on CD14+ PBMCs isolated from pre- and early post-menopausal females and late post-menopausal osteoporotic patients. Serum levels of TL1A, CCL3 and total MMP-9 were measured by ELISA. In vitro osteoclast differentiation assays were performed to determine CD14+ monocyte osteoclastogenic potential. In addition, splenic CD4+ T cell DR3 expression was investigated 1 week and 8 weeks post-surgery, using the murine ovariectomy model. RESULTS: In contrast to pre-menopausal females, CD14+ monocytes isolated from post-menopausal females were unable to induce DR3 expression. Serum TL1A levels were decreased approx. 2-fold in early post-menopausal females compared to pre-menopausal controls and post-menopausal osteoporotic females; no difference was observed between pre-menopausal and late post-menopausal osteoporotic females. Analysis of in vitro CD14+ monocyte osteoclastogenic potential revealed no significant difference between the post-menopausal and post-menopausal osteoporotic cohorts. Interestingly, in the murine ovariectomy model splenic CD4+ T cell DR3 expression was significantly increased at 1 week but not 8 weeks post-surgery when compared to the sham control. CONCLUSION: Our results reveals for the first time that loss of oestrogen has a significant effect on DR3; decreasing expression on CD14+ monocytes and increasing expression on CD4+ T cells. These data suggest that while oestrogen-deficiency induced changes in DR3 expression do not affect late post-menopausal bone loss they could potentially have an indirect role in early menopausal bone loss through the modulation of T cell activity.

High Incidence of Celiac Disease in a Long-term Study of Adolescents With Susceptibility Genotypes.

BACKGROUND & AIMS: Little is known about the incidence of celiac disease in the general population of children in the United States. We aimed to estimate the cumulative incidence of celiac disease in adolescents born in the Denver metropolitan area. METHODS: We collected data on HLA-DR, DQ genotypes of 31,766 infants, born from 1993 through 2004 at St. Joseph's Hospital in Denver, from the Diabetes Autoimmunity Study in the Young. Subjects with susceptibility genotypes for celiac disease and type 1 diabetes were followed up for up to 20 years for development of tissue transglutaminase autoantibodies (tTGA). Outcomes were the development of celiac disease autoimmunity (CDA) or celiac disease. CDA was defined as persistence of tTGA for at least 3 months or development of celiac disease. Celiac disease was defined based on detection of Marsh 2 or greater lesions in biopsy specimens or persistent high levels of tTGA. For each genotype, the cumulative incidence of CDA and celiac disease were determined. To estimate the cumulative incidence in the Denver general population, outcomes by each genotype were weighted according to the frequency of each of these genotypes in the general population. RESULTS: Of 1339 subjects followed up, 66 developed CDA and met criteria for celiac disease and 46 developed only CDA. Seropositivity for tTGA resolved spontaneously, without treatment, in 21 of the 46 subjects with only CDA (46%). The estimated cumulative incidence for CDA in the Denver general population at 5, 10, and 15 years of age was 2.4%, 4.3%, and 5.1%, respectively, and incidence values for celiac disease were 1.6%, 2.8%, and 3.1%, respectively. CONCLUSIONS: In a 20-year prospective study of 1339 children with genetic risk factors for celiac disease, we found the cumulative incidence of CDA and celiac disease to be high within the first 10 years. Although more than 5% of children may experience a period of CDA, not all children develop celiac disease or require gluten-free diets.

TNFSF15 inhibits VEGF-stimulated vascular hyperpermeability by inducing VEGFR2 dephosphorylation.

Vascular hyperpermeability is critical in ischemic diseases, including stroke and myocardial infarction, as well as in inflammation and cancer. It is well known that the VEGF-VEGFR2 signaling pathways are pivotal in promoting vascular permeability; however, counterbalancing mechanisms that restrict vascular permeability to maintain the integrity of blood vessels are not yet fully understood. We report that TNF superfamily member 15 (TNFSF15), a cytokine largely produced by vascular endothelial cells and a specific inhibitor of the proliferation of these same cells, can inhibit VEGF-induced vascular permeability in vitro and in vivo, and that death receptor 3 (DR3), a cell surface receptor of TNFSF15, mediates TNFSF15-induced dephosphorylation of VEGFR2. Src homology region 2 domain-containing phosphatase-1 (SHP-1) becomes associated with DR3 upon TNFSF15 interaction with the latter. In addition, a protein complex consisting of VEGFR2, DR3, and SHP-1 is formed in response to the effects of TNFSF15 and VEGF on endothelial cells. It is plausible that this protein complex provides a structural basis for the molecular mechanism in which TNFSF15 induces the inhibition of VEGF-stimulated vascular hyperpermeability.-Yang, G.-L., Zhao, Z., Qin, T.-T., Wang, D., Chen, L., Xiang, R., Xi, Z., Jiang, R., Zhang, Z.-S., Zhang, J., Li. L.-Y. TNFSF15 inhibits VEGF-stimulated vascular hyperpermeability by inducing VEGFR2 dephosphorylation.

Expression of death receptor 3 (DR3) on peripheral blood mononuclear cells of patients with psoriasis vulgaris.

BACKGROUND: A series of previous reports indicated that tumour necrosis factor-like ligand 1A (TL1A) and its receptor death receptor 3 (DR3) are involved in the pathogenesis of psoriasis vulgaris (PV), which is a common chronic skin disease accompanied by a number of comorbidities, although their exact roles remain unclear. Our previous studies demonstrated that serum TL1A levels were substantially elevated in patients with PV, but the detection of DR3 expression in peripheral blood mononuclear cells (PBMCs) of patients with PV had not been reported. Therefore, we detected DR3 expression on CD4+, CD8+, CD14+ and CD19+ PBMCs of patients with PV, atopic dermatitis (AD) and healthy volunteers. METHODS: Blood samples were collected from participants with PV before and after treatment. Then, PBMCs from patients with PV were isolated. The Psoriasis Area Severity Index (PASI) was used to assess severity in patients with PV. The DR3 on CD4+, CD8+, CD14+ and CD19+ PBMCs were detected by flow cytometry analysis. Pearson's correlation analysis was then used to investigate the relationship between DR3 expression and PASI scores in patients with PV. RESULTS: Comparing with the healthy volunteers and patients with AD, the percentage of DR3-expressing on CD8+ and CD14+ PBMCs in patients with PV was elevated, but the percentage of DR3-expressing on CD8+ and CD14+ cells decreased after anti-inflammatory treatment, which was correlated with PASI scores. CONCLUSIONS: Taken together, these findings suggest that DR3 may play a key role in the pathogenesis of PV.

Integration of VDR genome wide binding and GWAS genetic variation data reveals co-occurrence of VDR and NF-κB binding that is linked to immune phenotypes.

BACKGROUND: The nuclear hormone receptor superfamily acts as a genomic sensor of diverse signals. Their actions are often intertwined with other transcription factors. Nuclear hormone receptors are targets for many therapeutic drugs, and include the vitamin D receptor (VDR). VDR signaling is pleotropic, being implicated in calcaemic function, antibacterial actions, growth control, immunomodulation and anti-cancer actions. Specifically, we hypothesized that the biologically significant relationships between the VDR transcriptome and phenotype-associated biology could be discovered by integrating the known VDR transcription factor binding sites and all published trait- and disease-associated SNPs. By integrating VDR genome-wide binding data (ChIP-seq) with the National Human Genome Research Institute (NHGRI) GWAS catalog of SNPs we would see where and which target gene interactions and pathways are impacted by inherited genetic variation in VDR binding sites, indicating which of VDR's multiple functions are most biologically significant. RESULTS: To examine how genetic variation impacts VDR function we overlapped 23,409 VDR genomic binding peaks from six VDR ChIP-seq datasets with 191,482 SNPs, derived from GWAS-significant SNPs (Lead SNPs) and their correlated variants (r 2 > 0.8) from HapMap3 and the 1000 genomes project. In total, 574 SNPs (71 Lead and 503 SNPs in linkage disequilibrium with Lead SNPs) were present at VDR binding loci and associated with 211 phenotypes. For each phenotype a hypergeometric test was used to determine if SNPs were enriched at VDR binding sites. Bonferroni correction for multiple testing across the 211 phenotypes yielded 42 SNPs that were either disease- or phenotype-associated with seven predominately immune related including self-reported allergy; esophageal cancer was the only cancer phenotype. Motif analyses revealed that only two of these 42 SNPs reside within a canonical VDR binding site (DR3 motif), and that 1/3 of the 42 SNPs significantly impacted binding and gene regulation by other transcription factors, including NF-κB. This suggests a plausible link for the potential cross-talk between VDR and NF-κB. CONCLUSIONS: These analyses showed that VDR peaks are enriched for SNPs associated with immune phenotypes suggesting that VDR immunomodulatory functions are amongst its most important actions. The enrichment of genetic variation in non-DR3 motifs suggests a significant role for the VDR to bind in multimeric complexes containing other transcription factors that are the primary DNA binding component. Our work provides a framework for the combination of ChIP-seq and GWAS findings to provide insight into the underlying phenotype-associated biology of a given transcription factor.

Human group3 innate lymphoid cells express DR3 and respond to TL1A with enhanced IL-22 production and IL-2-dependent proliferation.

Death receptor 3 (DR3, TNFRSF25) is expressed by activated lymphocytes and signaling by its ligand, TL1A, enhances cytokine expression and proliferation. Recent studies show that DR3 is also present on murine type 2 innate lymphoid cells (ILC2s). Here, we show that DR3 is expressed by IL-22-producing human group 3 innate lymphoid cells (ILC3s). Stimulation of ILC3s with exogenous TL1A alone had no impact on cytokine production or proliferation. Addition of TL1A to IL-1ß + IL-23 significantly enhanced the amount IL-22 produced by ILC3s as well as the percentage IL-22- and IL-8-producing cells. Addition of TL1A to IL-1ß + IL-23 also augmented ILC3 proliferation. Mechanistically, this occurred through the upregulation of CD25 and responsiveness to IL-2 stimulation. The combination of TL1A, IL-1ß+ IL-23, and IL-2 expanded ILC3s while IL-1ß+ IL-23 did not increase proliferation above controls. After 2 weeks of expansion, ILC3s maintained their phenotype, transcription factor expression, and function (IL-22 production). These findings identify DR3 as a costimulatory molecule on ILC3s that could be exploited for ex vivo expansion and clinical use.

Tumour necrosis factor superfamily member 15 (Tnfsf15) facilitates lymphangiogenesis via up-regulation of Vegfr3 gene expression in lymphatic endothelial cells.

Lymphangiogenesis is essential in embryonic development but is rare in adults. It occurs, however, in many disease conditions including cancers. Vascular endothelial growth factor-C/D (VEGF-C/D) and VEGF receptor-3 (Vegfr3) play a critical role in the regulation of lymphangiogenesis. We investigated how the VEGF-C/Vegfr3 signalling system is regulated by tumour necrosis factor superfamily member 15 (Tnfsf15), an endothelium-derived cytokine. We report here that Tnfsf15, which is known to induce apoptosis in vascular endothelial cells, can promote lymphatic endothelial cell (LEC) growth and migration, stimulate lymphangiogenesis, and facilitate lymphatic circulation. Treatment of mouse LECs with Tnfsf15 results in up-regulation of Vegfr3 expression; this can be inhibited by gene silencing of death domain-containing receptor-3 (DR3; Tnfrsf25), a cell surface receptor for Tnfsf15, with siRNA, or by blocking Tnfsf15-DR3 interaction with a Tnfsf15 neutralizing antibody, 4-3H. Additionally, Tnfsf15/DR3 signalling pathways in LECs include activation of NF-κB. Tnfsf15-overexpressing transgenic mice exhibit a marked enhancement of lymph drainage; this is confirmed by treatment of wild-type mice with intraperitoneal injection of recombinant Tnfsf15. Moreover, systemic treatment of pregnant Tnfsf15 transgenic mice with 4-3H leads to inhibition of embryonic lymphangiogenesis. Our data indicate that Tnfsf15, a cytokine produced largely by endothelial cells, facilitates lymphangiogenesis by up-regulating Vegfr3 gene expression in LECs, contributing to the maintenance of the homeostasis of the circulatory system. This finding also suggests that Tnfsf15 may be of potential value as a therapeutic tool for the treatment of lymphoedema.

HLA-DRB1*03 as a risk factor for microalbuminuria in same duration of type 1 diabetes: a case control study.

BACKGROUND: Increased urinary albumin excretion rate is the earliest clinical manifestation of diabetic nephropathy. The development of microalbuminuria in patients with type 1 diabetes mellitus (T1D) usually begins 5 to 15 years after the onset of diabetes. The rate of progression of diabetic nephropathy varies considerably among patients and not always can be explained solely by glycaemic control. The evidence suggests that genetic susceptibility may play a role in the development of diabetes microvascular complications, besides the presence of such risk factors as hyperglycaemia, hypertension, dyslipidaemia and smoking. The aim of the study was to evaluate a link between known genetic risk factors for type 1 diabetes mellitus (HLA-DR3/DR4) and microalbuminuria among patients with the same durations of diabetes. METHODS: Ninety-nine patients with T1D at the age 18-35 years were recruited for the study. The urine albumin excretion rate was normal when <30 mg/24 h; microalbuminuria 30-300 mg/24 h. Genotypes were investigated in 39 patients with normal albumin excretion rate and duration of diabetes 13.46 ± 3.72 years and in 60 patients with microalbuminuria and duration of diabetes 15.28 ± 4.08 years (p = 0.11). Genetic typing of DR3 and DR4 antigens successfully was performed for 99 subjects. Statistical analysis was performed using SPSS v. 20.0. RESULTS: Genotyping of 99 patients with T1D was performed: no DR3 and DR4 risk alleles were found in 22 (22.22 %) cases, DR3 alleles were present in 47 (47.48 %) cases, DR4 alleles in 25 (25.25 %) cases, and DR3/DR4 alleles in 5 (5.05 %) cases. The highest 24 h albumin excretion rate was found in patients with DRB1 gene expressed DR3 risk alleles group, the lowest - in patients with DRB1 gene with no expression of both DR3 and DR4 antigen. We confirmed the 1.87 (p = 0.021) increased relative risk for microalbuminuria in patients with DR3/DR3 alleles and same duration of diabetes. The distribution of DR3 and DR4 risk alleles was not associated with cardiovascular autonomic neuropathy both in patients with normal albumin excretion rate and microalbuminuria (1.6 vs 2.1; p = 0.21). CONCLUSIONS: The 1.87 (p = 0.021) increased relative risk for microalbuminuria was found in patients with DR3/DR3 alleles and the same duration of diabetes.

The molecular basis for development of proinflammatory autoantibodies to progranulin.

Recently we identified in a wide spectrum of autoimmune diseases frequently occurring proinflammatory autoantibodies directed against progranulin, a direct inhibitor of TNFR1 & 2 and of DR3. In the present study we investigated the mechanisms for the breakdown of self-tolerance against progranulin. Isoelectric focusing identified a second, differentially electrically charged progranulin isoform exclusively present in progranulin-antibody-positive patients. Alkaline phosphatase treatment revealed this additional progranulin isoform to be hyperphosphorylated. Subsequently Ser81, which is located within the epitope region of progranulin-antibodies, was identified as hyperphosphorylated serine residue by site directed mutagenesis of candidate phosphorylation sites. Hyperphosphorylated progranulin was detected exclusively in progranulin-antibody-positive patients during the courses of their diseases. The occurrence of hyperphosphorylated progranulin preceded seroconversions of progranulin-antibodies, indicating adaptive immune response. Utilizing panels of kinase and phosphatase inhibitors, PKCß1 was identified as the relevant kinase and PP1 as the relevant phosphatase for phosphorylation and dephosphorylation of Ser81. In contrast to normal progranulin, hyperphosphorylated progranulin interacted exclusively with inactivated (pThr320) PP1, suggesting inactivated PP1 to cause the detectable occurrence of phosphorylated Ser81 PGRN. Investigation of possible functional alterations of PGRN due to Ser81 phosphorylation revealed, that hyperphosphorylation prevents the interaction and thus direct inhibition of TNFR1, TNFR2 and DR3, representing an additional direct proinflammatory effect. Finally phosphorylation of Ser81 PGRN alters the conversion pattern of PGRN. In conclusion, inactivated PP1 induces hyperphosphorylation of progranulin in a wide spectrum of autoimmune diseases. This hyperphosphorylation prevents direct inhibition of TNFR1, TNFR2 and DR3 by PGRN, alters the conversion of PGRN, and is strongly associated with the occurrence of neutralizing, proinflammatory PGRN-antibodies, indicating immunogenicity of this alternative secondary modification.

TL1A/DR3 axis involvement in the inflammatory cytokine network during pulmonary sarcoidosis.

BACKGROUND: TNF-like ligand 1A (TL1A), a recently recognized member of the TNF superfamily, and its death domain receptor 3 (DR3), firstly identified for their relevant role in T lymphocyte homeostasis, are now well-known mediators of several immune-inflammatory diseases, ranging from rheumatoid arthritis to inflammatory bowel diseases to psoriasis, whereas no data are available on their involvement in sarcoidosis, a multisystemic granulomatous disease where a deregulated T helper (Th)1/Th17 response takes place. METHODS: In this study, by flow cytometry, real-time PCR, confocal microscopy and immunohistochemistry analyses, TL1A and DR3 were investigated in the pulmonary cells and the peripheral blood of 43 patients affected by sarcoidosis in different phases of the disease (29 patients with active sarcoidosis, 14 with the inactive form) and in 8 control subjects. RESULTS: Our results demonstrated a significant higher expression, both at protein and mRNA levels, of TL1A and DR3 in pulmonary T cells and alveolar macrophages of patients with active sarcoidosis as compared to patients with the inactive form of the disease and to controls. In patients with sarcoidosis TL1A was strongly more expressed in the lung than the blood, i.e., at the site of the involved organ. Additionally, zymography assays showed that TL1A is able to increase the production of matrix metalloproteinase 9 by sarcoid alveolar macrophages characterized, in patients with the active form of the disease, by reduced mRNA levels of the tissue inhibitor of metalloproteinase (TIMP)-1. CONCLUSIONS: These data suggest that TL1A/DR3 interactions are part of the extended and complex immune-inflammatory network that characterizes sarcoidosis during its active phase and may contribute to the pathogenesis and to the progression of the disease.

Dynamics of 1α,25-dihydroxyvitamin D3-dependent chromatin accessibility of early vitamin D receptor target genes.

The signaling cascade of the transcription factor vitamin D receptor (VDR) is triggered by its specific ligand 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). In this study we demonstrate that in THP-1 human monocytic leukemia cells 87.4% of the 1034 most prominent genome-wide VDR binding sites co-localize with loci of open chromatin. At 165 of them 1α,25(OH)2D3 strongly increases chromatin accessibility and has at further 217 sites weaker effects. Interestingly, VDR binding sites in 1α,25(OH)2D3-responsive chromatin regions are far more often composed of direct repeats with 3 intervening nucleotides (DR3s) than those in ligand insensitive regions. DR3-containing VDR sites are enriched in the neighborhood of genes that are involved in controling cellular growth, while non-DR3 VDR binding is often found close to genes related to immunity. At the example of six early VDR target genes we show that the slope of their 1α,25(OH)2D3-induced transcription correlates with the basal chromatin accessibility of their major VDR binding regions. However, the chromatin loci controlling these genes are indistinguishable in their VDR association kinetics. Taken together, ligand responsive chromatin loci represent dynamically regulated contact points of VDR with the genome, from where it controls early 1α,25(OH)2D3 target genes.

TL1A increased the differentiation of peripheral Th17 in rheumatoid arthritis.

The interaction between TNF-like protein 1A (TL1A) and its receptors, death receptor-3 (DR3) may be involved in the pathogenesis of rheumatoid arthritis (RA) through the regulation of Th17. Our data here showed that TL1A could significantly promote Th17 differentiation and RORc mRNA expression from naive T cells and enhance IL-17A level in cell supernatant in RA patients. Anti-TNF-α treatment had suppressive effects on TL1A-mediated Th17 differentiation and RORc mRNA expression. In addition, The percentage of peripheral CD4+DR3+T cells of RA was significantly higher than that of healthy controls (HC), and this increased percentage of CD4+DR3+T cells was obviously up-regulated when stimulated with anti-CD3 and anti-CD28 antibody in RA patients. However, anti-CD3 and anti-CD28 antibody stimulation did not increase the percentage of CD4+DR3+IL-17A+T cells in RA patients. These results suggested that TL1A could promote Th17 differentiation in RA via the activation of RORc, and this effect may be mediated by the binding of TL1A with DR3.