Recombinant Human Tumor necrosis factor ligand superfamily member 15 (TNFSF15) (Active)

What is TNFSF15 and what are its primary biological functions?

TNFSF15 (also known as TL1A or VEGI) is a member of the tumor necrosis factor superfamily with multifaceted roles in immune regulation, inflammation, and angiogenesis. Its primary functions include:

• Regulation of vascular homeostasis and inflammatory responses through interactions with its receptor

• Promotion of T-cell activation, proliferation, and generation of multiple cytokines

• Modulation of T-helper cell responses, particularly in promoting the differentiation of Th17 cells, which are crucial in various inflammatory and autoimmune conditions

• Inhibition of angiogenesis by regulating endothelial cell proliferation and survival through downregulation of VEGF production and promotion of apoptosis in vascular endothelial cells

• Mediation of innate immune outcomes in human myeloid-derived cells

This cytokine is emerging as a significant player in immune regulation with therapeutic potential for various inflammatory diseases due to its central role in T-cell-mediated immune responses .

What is the primary receptor for TNFSF15 and how does this signaling axis function?

The primary receptor for TNFSF15 is death receptor 3 (DR3, also known as TNFRSF25). The TNFSF15-DR3 signaling axis functions through the following mechanisms:

• Upon binding to DR3, TNFSF15 promotes T-cell activation, proliferation, and the generation of multiple cytokines

• DR3 is clearly expressed on human macrophages, enabling TNFSF15:DR3 signaling to mediate innate immune outcomes

• This signaling pathway is essential for effective T-cell immune responses, particularly in T-cell-mediated autoimmune diseases

• The interaction activates multiple signaling pathways including MAPK, NF-κB, and PI3K, as evidenced by studies showing that TNFSF15 knockdown significantly attenuates activation of these pathways

• TNFSF15:DR3 signaling enhances cytokine secretion upon stimulation of pattern-recognition-receptors, mycobacterial components, and live bacteria

This receptor-ligand interaction represents a critical immunoregulatory mechanism that influences both innate and adaptive immunity, positioning it as a potential therapeutic target for immune modulation .

How is TNFSF15 expression regulated during immune responses?

TNFSF15 expression is tightly regulated through several mechanisms:

• It is primarily expressed in immune cells such as macrophages and T cells

• Expression can be induced by pro-inflammatory stimuli, suggesting a positive feedback mechanism during inflammatory responses

• NOD2 stimulation increases both surface and soluble TNFSF15 protein levels

• TNFSF15 induction occurs transcriptionally, with the canonical TNFSF15 147-bp isoform peaking 4 hours after MDP (muramyl dipeptide) treatment

• Surface TNFSF15 can be processed to a soluble form through the action of TACE (TNF-α converting enzyme)

• NOD2 regulation of TACE affects TNFSF15 processing, creating a complex regulatory network

This dynamic regulation of TNFSF15 expression and processing allows for precise control of its biological activities during various immune challenges and inflammatory conditions .

How is recombinant human TNFSF15 produced for research applications?

Recombinant human TNFSF15 for research applications is typically produced through the following methods:

• Plasmid expression in mammalian cells, which ensures proper protein folding and post-translational modifications

• The process often involves inserting a gene segment that codes for specific amino acid residues (e.g., 72-251aa) of human TNFSF15

• Co-expression with tags such as N-terminal 10xHis-Avi-tag for purification and detection purposes

• Quality control measures include SDS-PAGE for purity assessment (typically >95%)

• Endotoxin testing using LAL assay, with acceptable levels being below 1.0 EU/μg

• Functional validation through ELISA, demonstrating specific antibody binding with defined EC50 values

These production methods ensure consistent quality and biological activity of recombinant TNFSF15, which is critical for reliable experimental outcomes in research settings .

What analytical techniques are recommended for measuring TNFSF15 levels in clinical samples?

Several analytical techniques have been validated for measuring TNFSF15 levels in clinical samples:

• Enzyme-linked immunosorbent assay (ELISA) is the most commonly used method for measuring TNFSF15 serum levels, as demonstrated in studies of SLE patients

• Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is utilized for genotyping TNFSF15 variants, with verification by direct sequencing for accuracy

• Western blotting can be used to detect TNFSF15 protein expression in cell and tissue lysates, with antibody specificity verified through TNFSF15 knockdown experiments

• Flow cytometry enables detection of surface TNFSF15 on specific cell populations

• Quantitative PCR (qPCR) can measure TNFSF15 transcription, allowing for temporal expression analysis

The selection of an appropriate technique depends on the specific research question, sample type, and required sensitivity and specificity. For clinical biomarker studies, standardized ELISA protocols have shown reliable results in detecting significant differences between patient and control groups .

What experimental models are most suitable for studying TNFSF15 function?

Various experimental models have proven valuable for investigating TNFSF15 function:

• Human monocyte-derived macrophages (MDM) serve as an excellent model for studying TNFSF15:DR3 signaling in innate immunity

• Cell line models expressing DR3 can be used to study receptor-ligand interactions and downstream signaling events

• RNA interference approaches (siRNA or shRNA) targeting TNFSF15 or DR3 help elucidate the specific roles of this signaling axis

• Case-control studies in human populations are effective for investigating associations between TNFSF15 polymorphisms and disease risk

• TACE inhibition models help distinguish between the roles of membrane-bound versus soluble TNFSF15

• Recombinant protein supplementation experiments can determine if exogenous TNFSF15 rescues phenotypes observed in knockdown models

When designing experiments, it's important to consider that soluble and membrane-bound forms of TNFSF15 may have distinct biological activities, as demonstrated by studies showing that soluble, but not membrane-bound TNFSF15, amplifies NOD2-induced cytokines .

How does TNFSF15 contribute to autoimmune disease pathogenesis?

TNFSF15 plays significant roles in autoimmune disease pathogenesis through several mechanisms:

• In systemic lupus erythematosus (SLE), the TNFSF15 rs4979462 gene variant increases disease risk in female subjects, with a significant association observed between the T-variant and clinical manifestations such as serositis and thrombotic events

• Serum TNFSF15 levels are significantly elevated in SLE patients compared to healthy controls and correlate with disease activity, suggesting its potential role as a biomarker

• The odds ratios for association between TNFSF15 rs4979462 T-variant and SLE in females are substantial (OR = 2.7, 95% CI = 1.2–6.3, p = 0.015), indicating a strong genetic influence

• TNFSF15-TNFRSF25 signaling is essential for effective T-cell immune responses in T-cell-mediated autoimmune diseases

• TNFSF15 promotes differentiation of Th17 cells, which are implicated in various autoimmune conditions

These findings collectively demonstrate that TNFSF15 contributes to autoimmune pathogenesis through both genetic predisposition and immunological mechanisms that promote inflammatory responses .

What is the relationship between TNFSF15 genetic variants and inflammatory bowel disease (IBD)?

The relationship between TNFSF15 genetic variants and IBD has been elucidated through several studies:

• TNFSF15 gene region is one of the 163 identified inflammatory bowel disease risk loci

• Specific polymorphisms (rs3810936, rs7848647, and rs6478108) in the TNFSF15 gene have been associated with a decreased risk of Crohn's disease (CD) across multiple genetic models

• Only the rs3810936 polymorphism appears to have a protective association with ulcerative colitis (UC), suggesting differential genetic influences between CD and UC

• These polymorphisms affect TNFSF15:DR3 signaling, which can enhance pattern-recognition-receptor responses to intestinal microbiota

• Functionally, TNFSF15 polymorphisms may influence intestinal inflammation by altering cytokine production and immune cell activation

Meta-analysis results have confirmed the consistency of these associations across different studies, although heterogeneity was noted for CD but not UC, possibly due to different genetic backgrounds, population stratification, and selection bias .

How does TNFSF15 influence angiogenesis and what are its implications for cancer research?

TNFSF15 exhibits significant anti-angiogenic properties with important implications for cancer research:

• It inhibits angiogenesis by regulating endothelial cell proliferation and survival mechanisms

• TNFSF15 downregulates VEGF production, a key pro-angiogenic factor, thereby limiting new blood vessel formation

• It promotes apoptosis in vascular endothelial cells, further contributing to its anti-angiogenic effects

• These anti-angiogenic properties are particularly significant in cancer contexts, where TNFSF15 can potentially limit tumor growth by preventing the formation of new blood vessels necessary for tumor expansion

• The dual role of TNFSF15 in both immune regulation and angiogenesis makes it an interesting target for cancer immunotherapy research

Understanding the molecular mechanisms by which TNFSF15 regulates angiogenesis could lead to novel therapeutic approaches targeting the tumor microenvironment, potentially complementing existing anti-angiogenic strategies in cancer treatment .

How does TNFSF15 processing affect its biological activity?

TNFSF15 processing significantly impacts its biological activity through several mechanisms:

• Surface TNFSF15 can be enzymatically processed to generate soluble TNFSF15, each form having distinct biological activities

• TACE (TNF-α converting enzyme) plays a crucial role in processing surface TNFSF15 to its soluble form

• TACE inhibition increases surface TNFSF15 levels while decreasing soluble TNFSF15

• Despite increased surface TNFSF15 during TACE inhibition, cytokine secretion decreases, indicating that soluble TNFSF15 is the primary mediator of enhanced cytokine responses

• This processing distinction is functionally significant, as demonstrated by rescue experiments where exogenous soluble recombinant TNFSF15 restores cytokine production during TACE inhibition

• NOD2 stimulation influences this process by increasing both surface and soluble TNFSF15 levels

These findings highlight the importance of post-translational processing in determining TNFSF15 function, with soluble and membrane-bound forms potentially having distinct roles in immune regulation and inflammation .

What signaling pathways are activated by TNFSF15-DR3 interaction?

The TNFSF15-DR3 interaction activates multiple signaling pathways that mediate its biological effects:

• MAPK (Mitogen-Activated Protein Kinase) pathway activation is a key outcome of TNFSF15-DR3 signaling

• NF-κB (Nuclear Factor kappa B) signaling is robustly induced, controlling inflammatory gene expression

• PI3K (Phosphoinositide 3-Kinase) pathway is activated, contributing to cell survival and proliferation signals

• TNFSF15 knockdown significantly attenuates MAPK, NF-κB, and PI3K pathway activation during optimal MDP treatment, indicating its requirement for efficient signal transduction

• Exogenous TNFSF15 synergizes with suboptimal MDP treatment to enhance activation of these signaling pathways

• These signaling events ultimately lead to increased production of pro-inflammatory cytokines

This complex signaling network explains how TNFSF15-DR3 interaction amplifies pattern-recognition receptor responses and contributes to inflammatory conditions when dysregulated .

How does TNFSF15 integrate with pattern recognition receptor (PRR) signaling?

TNFSF15 integrates with pattern recognition receptor signaling through several mechanisms:

• TNFSF15:DR3 enhances signaling and cytokine secretion upon stimulation of a broad range of pattern-recognition-receptors (PRRs)

• NOD2 stimulation (with muramyl dipeptide, MDP) increases both surface and soluble TNFSF15 expression

• TNFSF15 is required for optimal MDP-initiated MAPK, NF-κB, and PI3K activation, starting within the first 15 minutes of stimulation

• Soluble TNFSF15 (rather than membrane-bound) amplifies NOD2-induced cytokine production

• TACE processes TNFSF15 to amplify PRR-induced cytokines, creating a feed-forward loop

• This integration provides a mechanism through which TNFSF15:DR3 can contribute to intestinal inflammation, particularly in response to microbial components

This cross-talk between TNFSF15 and PRR signaling has important implications for understanding inflammatory conditions, especially in contexts like inflammatory bowel disease where inappropriate responses to microbial stimuli contribute to pathogenesis .

What is the potential of TNFSF15 as a biomarker for autoimmune diseases?

TNFSF15 shows promising potential as a biomarker for autoimmune diseases, particularly systemic lupus erythematosus (SLE):

• Median serum TNFSF15 concentration is significantly elevated in SLE patients compared to healthy controls

• TNFSF15 serum levels correlate with SLE disease activity (p = 0.012), suggesting utility as a disease activity marker

• The correlation between TNFSF15 levels and disease activity provides a quantitative measure that could help monitor treatment response

• Standardized ELISA protocols for TNFSF15 measurement have been established, facilitating clinical application

• Combined with genetic information about TNFSF15 variants, serum levels could help stratify patients for personalized treatment approaches

These findings indicate that TNFSF15 could serve as a valuable biological marker for monitoring disease activity in SLE and potentially other autoimmune conditions where this signaling pathway plays a pathogenic role .

How might TNFSF15-targeted therapies be developed for inflammatory diseases?

Development of TNFSF15-targeted therapies for inflammatory diseases could proceed through several strategic approaches:

• Targeting the TNFSF15-DR3 interaction using neutralizing antibodies or soluble receptor decoys to block signaling

• Inhibiting TACE to modulate TNFSF15 processing, though this would need careful consideration as TACE processes multiple surface proteins

• Developing small molecule inhibitors of downstream signaling pathways activated by TNFSF15-DR3 interaction

• Utilizing the protective effect of certain TNFSF15 polymorphisms (like rs3810936, rs7848647, and rs6478108) to guide development of mimetic therapeutics

• Exploring therapeutic applications based on TNFSF15's dual role in immune regulation and angiogenesis

• Considering combination approaches targeting both TNFSF15 and pattern recognition receptor pathways for synergistic effects

As TNFSF15 is being considered for therapy in inflammatory diseases, understanding its complex biology is essential for developing effective and safe therapeutic interventions that modulate rather than completely block its activity .

What genetic screening approaches might be valuable for TNFSF15-associated disease risk assessment?

Several genetic screening approaches could be valuable for assessing TNFSF15-associated disease risk:

• Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with verification by direct sequencing for genotyping TNFSF15 variants

• Screening for specific polymorphisms with known disease associations, such as rs4979462 for SLE risk and rs3810936, rs7848647, and rs6478108 for IBD protection

• Haplotype analysis to increase the power to detect disease associations beyond single SNP analysis

• Genome-wide association studies (GWAS) to identify additional TNFSF15 region variants associated with inflammatory diseases

• Risk stratification based on combined genetic and environmental factors, as gene-environment interactions may modify disease risk

Implementation of these screening approaches could help identify individuals at higher risk for developing TNFSF15-associated inflammatory diseases, potentially enabling earlier intervention and personalized treatment strategies .