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

What is the molecular structure and function of human TNFSF15?

Human TNFSF15, also known as Vascular Endothelial Growth Inhibitor (VEGI), is encoded by a gene located on chromosome 9q32 spanning approximately 18,850 bases . The mature TNFSF15 protein consists of 192 amino acids organized into three distinct functional domains: a TNF homology domain for receptor binding, a coiled-coil stalk region for trimerization, and an intracellular N-terminal region . It functions as a type II transmembrane protein with a single transmembrane domain, a small cytoplasmic domain, and an extracellular C-terminus . TNFSF15 acts as a cytokine that binds to TNFRSF25 (DR3) and the decoy receptor TNFRSF21/DR6, activating NF-kappaB and MAP kinases to regulate immune responses .

How is TNFSF15 expressed in human tissues and what regulates its expression?

TNFSF15 demonstrates a distinct expression pattern across human tissues:

• Predominantly expressed in endothelial cells

• Notably absent in B and T lymphocytes

• Expression is inducible by TNF and IL-1α stimulation

• Gene expression varies by tissue type and can be affected by genetic polymorphisms

• Expression levels fluctuate in disease states, showing reduction in certain asthma phenotypes and elevation in some autoimmune conditions

For experimental design considerations, researchers should account for tissue-specific expression patterns and inducible nature of TNFSF15 when selecting appropriate biological systems and stimulation conditions.

What signaling pathways does TNFSF15 activate in different cell types?

Upon binding to its receptors, TNFSF15 activates several key signaling pathways with cell type-specific outcomes:

In macrophages specifically, TNFSF15:DR3 interactions amplify pattern-recognition-receptor (PRR)-initiated signaling through TRADD/FADD/MALT-1 and caspase-8-mediated pathways, leading to autocrine IL-1 secretion . In contrast, in retinal cells, TNFSF15 inhibits GSDME-dependent pyroptosis by directly interacting with GSDME to prevent its cleavage by caspase-3 .

What is the relationship between TNFSF15 and its receptors?

TNFSF15 interacts with two primary receptors that mediate distinct biological effects:

• Death Receptor 3 (DR3/TNFRSF25): The main functional receptor predominantly expressed on activated T cells and macrophages. Binding to DR3 initiates downstream signaling cascades critical for immune responses . Through this interaction, TNFSF15 promotes T-cell activation, proliferation, and cytokine generation .

• Decoy receptor TNFRSF21/DR6: Functions as a regulatory receptor that can bind TNFSF15 without initiating the full spectrum of downstream signaling .

For effective experimental analysis of TNFSF15 function, researchers should verify receptor expression in their cellular models using flow cytometry or western blotting. The interaction between TNFSF15 and DR3 is essential for T-cell immune responses in T-cell-mediated autoimmune diseases , while in macrophages, this interaction enhances PRR-initiated signaling and cytokine secretion .

What are the optimal conditions for handling recombinant TNFSF15 in laboratory settings?

For maximum experimental reproducibility and protein activity when working with recombinant TNFSF15:

• Storage: Store lyophilized protein at -20°C; avoid repeated freeze/thaw cycles

• Reconstitution: Reconstitute in sterile PBS to a concentration of 1 mg/mL

• Working solutions: Prepare fresh dilutions in appropriate buffers for each experiment

• Temperature considerations: Maintain at 4°C during short-term experimental procedures

• Activity verification: Include functional assays to confirm protein activity in each experiment

Researchers should document handling procedures in detail to improve reproducibility across studies. When reconstituting the protein, allow complete dissolution through gentle rotation rather than vigorous vortexing to preserve the protein structure and function.

What cell models are most appropriate for studying different TNFSF15 functions?

Selection of appropriate cell models depends on the specific TNFSF15 function under investigation:

When designing experiments, researchers should:

• Verify receptor expression in selected cell lines before conducting experiments

• Include appropriate positive and negative controls

• Consider using primary cells when possible for greater physiological relevance

• Account for potential species differences when translating findings from animal models

How can researchers effectively measure TNFSF15 activity in experimental systems?

Several complementary methodological approaches can be employed to assess TNFSF15 activity:

• Signaling pathway activation:

  • Western blotting for phosphorylated NF-κB, MAPK, or PI3K components

  • Luciferase reporter assays for transcription factor activation

  • Phospho-flow cytometry for single-cell signaling analysis

• Cytokine production assessment:

  • ELISA or multiplex assays for secreted cytokines (IL-1, IL-6, TNF-α)

  • qRT-PCR for cytokine gene expression changes

  • Intracellular cytokine staining and flow cytometry

• Functional assays:

  • Endothelial cell proliferation inhibition assays

  • Apoptosis detection methods (Annexin V/PI staining, TUNEL assay)

  • Pyroptosis assessment in relevant cell types (GSDME cleavage, LDH release)

• Protein-protein interaction studies:

  • Co-immunoprecipitation of TNFSF15 with receptors or targets like GSDME

  • Surface plasmon resonance for binding kinetics analysis

  • Proximity ligation assays for visualizing interactions in situ

For comprehensive functional characterization, researchers should employ multiple complementary assays and include appropriate controls for each experimental approach.

What concentration ranges of recombinant TNFSF15 are effective for different experimental endpoints?

Optimal TNFSF15 concentrations vary based on the specific biological readout:

Researchers should perform dose-response and time-course studies to determine optimal concentrations and time points for their specific experimental system. Additionally, comparing recombinant TNFSF15 effects to physiological levels observed in relevant disease contexts can provide important translational insights.

How is TNFSF15 implicated in inflammatory bowel disease pathogenesis?

TNFSF15 has been identified as a significant risk factor in inflammatory bowel disease (IBD) through multiple mechanisms:

• Genetic associations:

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

  • Disease risk polymorphisms (e.g., rs6478108 A) are associated with increased TNFSF15 expression

  • Risk variants enhance pattern-recognition-receptor-induced signaling and cytokine secretion

• Mechanistic contributions:

  • Enhanced TNFSF15:DR3 signaling amplifies PRR responses in macrophages

  • TACE-induced TNFSF15 cleavage leads to soluble TNFSF15 production

  • Soluble TNFSF15 promotes TRADD/FADD/MALT-1 and caspase-8-mediated IL-1 secretion

  • This creates a pro-inflammatory feedback loop in intestinal myeloid cells

Experimental approaches to investigate these associations include genotyping IBD cohorts for TNFSF15 variants, performing ex vivo studies with patient-derived cells, and using animal models with relevant TNFSF15 mutations or gene expression alterations.

What role does TNFSF15 play in autoimmune disorders?

TNFSF15 demonstrates complex roles in autoimmune disorders, with substantial evidence from systemic lupus erythematosus (SLE) research:

• SLE associations:

  • The TNFSF15 rs4979462 gene variant significantly increases SLE risk in female subjects (OR = 2.6, 95% CI = 1.1–6.3)

  • The T-variant correlates with serositis and thrombotic manifestations

  • Serum TNFSF15 levels are elevated in SLE patients compared to healthy controls

  • TNFSF15 serum levels correlate with SLE disease activity (p = 0.012)

• Immunomodulatory mechanisms:

  • TNFSF15-TNFRSF25 signaling promotes T-cell activation and proliferation

  • This pathway is essential for T-cell-mediated autoimmune responses

  • Through DR3 binding, TNFSF15 stimulates the generation of multiple cytokines

These findings suggest TNFSF15 could serve as both a biomarker for disease activity and a potential therapeutic target in autoimmune disorders. Methodological approaches for further investigation include genotyping using PCR-RFLP with verification by direct sequencing, measurement of serum TNFSF15 using ELISA, and correlation analysis with clinical parameters.

How does TNFSF15 contribute to diabetic retinopathy protection?

Recent research has revealed a protective role for TNFSF15 in diabetic retinopathy (DR):

This protective mechanism represents a potential therapeutic avenue for diabetic retinopathy treatment through targeting the TNFSF15-GSDME interaction. Experimental approaches for investigating this pathway include high glucose culture models of retinal cells, assessment of pyroptosis markers, co-immunoprecipitation studies, and functional studies with TNFSF15 overexpression or knockdown.

What is the relationship between TNFSF15 and asthma pathophysiology?

Emerging evidence suggests TNFSF15 may play a role in asthma development through its effects on angiogenesis:

• Genetic associations:

  • Four SNPs in the TNFSF15 gene are associated with childhood-onset asthma in British white participants

  • The asthma-associated rs7856856 variant (T>C) is linked to reduced TNFSF15 gene expression

• Proposed mechanism:

  • TNFSF15 functions as an inhibitor of vascular endothelial cell growth factor

  • Decreased TNFSF15 levels might contribute to increased angiogenesis in asthmatic patients

  • Angiogenesis is an important event in airway inflammation and tissue remodeling in asthma

• Gene interaction network:

  • Genetic interactions are predicted between TNFSF15 with RORA, GATA3, and SLC8A1

  • Among 340 genes reported for asthma, 24 genes relate to cardiovascular system development

  • 14 of these genes associate with vasculature development, including 7 with angiogenesis

These findings suggest that reduced TNFSF15 expression could promote asthma development through loss of angiogenesis inhibition. Future research directions include functional studies evaluating the effect of TNFSF15 on airway remodeling and investigating whether serum TNFSF15 levels could serve as a marker for persistent asthma development.

How does TNFSF15 interact with pattern recognition receptors in inflammatory responses?

TNFSF15 plays a crucial role in amplifying pattern recognition receptor (PRR) responses through several mechanisms:

• Enhancement of PRR signaling:

  • TNFSF15:DR3 interactions are critical for amplifying PRR-initiated MAPK/NF-κB/PI3K signaling

  • This leads to increased cytokine secretion in macrophages

  • Human macrophages clearly express DR3, enabling this response

• Molecular mechanism:

  • The process involves TACE-mediated cleavage of TNFSF15 to produce soluble TNFSF15

  • Soluble TNFSF15 then triggers TRADD/FADD/MALT-1 and caspase-8-mediated pathways

  • This ultimately leads to autocrine IL-1 secretion

• PRR types affected:

  • Broad range of pattern-recognition-receptors

  • Responses to mycobacterial components

  • Signaling induced by live bacteria

• Disease relevance:

  • Enhanced in IBD risk variant carriers (rs6478108 A)

  • Contributes to intestinal inflammation

  • Represents a previously unknown mechanism through which TNFSF15:DR3 can contribute to inflammatory conditions

Research approaches to study this interaction include in vitro stimulation of macrophages with PRR ligands with and without TNFSF15, signal transduction analysis, and cytokine measurements using various assays.

What methodologies are effective for examining TNFSF15's role in angiogenesis?

As TNFSF15 is also known as Vascular Endothelial Growth Inhibitor (VEGI), several specialized techniques can assess its anti-angiogenic properties:

• In vitro angiogenesis assays:

  • Endothelial cell proliferation assays

  • Tube formation assays on Matrigel

  • Wound healing/scratch assays

  • Transwell migration and invasion assays

• Ex vivo techniques:

  • Aortic ring assays

  • Choroidal explant sprouting

  • Retinal explant culture

• In vivo approaches:

  • Models of diabetic retinopathy to study TNFSF15's protective effects

  • Laser-induced choroidal neovascularization

  • Tumor xenograft vascularization studies

• Molecular methods:

  • Analysis of angiogenic/anti-angiogenic factor expression

  • VEGF signaling pathway component assessment

  • Endothelial cell gene expression profiling

  • Vessel density quantification in tissue samples

The anti-angiogenic properties of TNFSF15 appear particularly relevant in asthma pathophysiology, where reduced TNFSF15 levels might contribute to increased angiogenesis in asthmatic patients , and in diabetic retinopathy, where TNFSF15 provides protection against vascular damage .

What techniques are effective for studying TNFSF15 polymorphisms and their functional effects?

Investigation of TNFSF15 polymorphisms requires comprehensive methodological approaches:

• Genotyping strategies:

  • PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism)

  • Direct sequencing for verification

  • High-throughput genotyping platforms for population studies

• Functional validation:

  • eQTL analysis to assess expression differences

  • Ex vivo stimulation assays with cells from genotyped individuals

  • Measurement of serum protein levels in different genotype carriers

• Mechanism investigation:

  • For rs7856856 and asthma: Evidence suggests reduced TNFSF15 may promote angiogenesis

  • For rs6478108: Risk-carrier macrophages demonstrate increased TNFSF15 expression and PRR-induced signaling and cytokines

  • For rs4979462: Associated with increased SLE susceptibility and specific clinical manifestations

• Clinical correlation approaches:

  • Case-control studies with appropriate population matching

  • Detailed phenotyping to identify variant-associated clinical features

  • Longitudinal studies to assess impact on disease progression

These genetic association studies provide valuable insights into disease mechanisms and potential personalized therapeutic approaches. When conducting such studies, researchers should consider population stratification, linkage disequilibrium with other variants, and tissue-specific effects.

How can the TNFSF15-DR3 signaling axis be targeted for therapeutic development?

The TNFSF15-DR3 signaling pathway presents several opportunities for therapeutic intervention:

• Potential therapeutic strategies:

  • Recombinant TNFSF15 administration for conditions requiring angiogenesis inhibition or pyroptosis protection

  • TNFSF15-neutralizing antibodies for inflammatory conditions with elevated TNFSF15

  • Small molecule inhibitors of TNFSF15-DR3 interaction

  • Targeted modulation of downstream signaling components

• Disease-specific approaches:

  • For diabetic retinopathy: Enhancing TNFSF15-GSDME interaction to prevent pyroptosis

  • For IBD: Inhibiting TNFSF15-induced amplification of PRR responses

  • For asthma: Potentially replacing deficient TNFSF15 to regulate angiogenesis

  • For SLE: Modulating TNFSF15 levels based on disease activity monitoring

• Experimental validation methodologies:

  • Structure-based drug design targeting the TNFSF15-DR3 interface

  • Cell-based screening assays measuring NF-κB activation

  • Ex vivo testing in patient-derived cells

  • Animal models of specific diseases (colitis, asthma, retinopathy)

Therapeutic development targeting this pathway must consider tissue-specific effects, genetic variation that might affect response, and the balance between beneficial immune modulation and potential adverse effects.

How can contradictory findings about TNFSF15 function be reconciled in research?

Several apparent contradictions exist in TNFSF15 research literature that require careful interpretation:

• Context-dependent effects:

  • TNFSF15 appears protective in diabetic retinopathy by inhibiting pyroptosis but pro-inflammatory in IBD by enhancing PRR signaling

  • Expression levels are increased in SLE but decreased in asthma

  • The same polymorphism may have different effects in different tissues

• Methodological approaches to resolve contradictions:

  • Tissue-specific and cell-type-specific studies to identify context-dependent responses

  • Time-course experiments to capture dynamic changes in TNFSF15 expression and function

  • Dose-response studies, as different concentrations may have opposing effects

  • Development of conditional knockout models to study tissue-specific roles

• Research design considerations:

  • Use multiple experimental systems to validate findings

  • Include appropriate controls for each experimental approach

  • Consider genetic background effects in animal models and human studies

  • Account for environmental factors that may influence TNFSF15 expression

These contradictions likely reflect the complex, context-dependent biology of TNFSF15 rather than experimental inconsistencies. Researchers should consider the specific cellular environment, disease context, and genetic background when interpreting seemingly contradictory results.

What considerations are important when designing experiments combining TNFSF15 with other immune modulators?

Experimental design for combinatorial studies with TNFSF15 requires careful planning:

• Interaction considerations:

  • Potential synergistic or antagonistic effects with other cytokines

  • Receptor competition or cross-regulation

  • Sequential versus simultaneous administration

  • Concentration ratios between TNFSF15 and other factors

• Key combinations to consider:

  • TNFSF15 with TNF-α (both induce and respond to each other)

  • TNFSF15 with IL-1α (induces TNFSF15 expression)

  • TNFSF15 with pattern recognition receptor ligands (enhances responses)

  • TNFSF15 with VEGF (opposing effects on angiogenesis)

• Methodological approaches:

  • Factorial experimental designs to systematically test interactions

  • Response surface methodology to identify optimal combinations

  • Time-course studies with staggered administration

  • Single-cell analysis to identify responding subpopulations

• Controls and analysis:

  • Include single-factor controls in parallel with combination treatments

  • Use appropriate statistical methods to detect interaction effects

  • Consider temporal aspects of signaling pathways

  • Document treatment sequences and concentrations precisely

These considerations are essential for understanding how TNFSF15 functions within the complex network of immune modulators in both physiological and pathological conditions.

What are the best practices for maintaining recombinant TNFSF15 stability and activity?

Maintaining TNFSF15 stability is crucial for experimental reproducibility and reliable results:

• Storage recommendations:

  • Store lyophilized protein at -20°C

  • For reconstituted protein, prepare single-use aliquots to avoid freeze-thaw cycles

  • Avoid repeated freeze/thaw cycles which compromise activity

• Reconstitution best practices:

  • Use sterile PBS for initial reconstitution to 1 mg/mL

  • Allow complete dissolution before use

  • Consider adding carrier protein (0.1-1% BSA) for dilute solutions

  • Filter sterilize if needed using low protein-binding filters

• Activity verification methods:

  • Include positive controls in functional assays

  • Verify protein integrity via SDS-PAGE

  • Confirm specific activity in relevant bioassays before critical experiments

  • Consider lot-to-lot variation when using commercial recombinant proteins

These stability considerations are essential for ensuring reproducible results across experiments and between different research groups studying TNFSF15 functions.

How can researchers distinguish between membrane-bound and soluble TNFSF15 in experimental systems?

The distinct biological activities of membrane-bound versus soluble TNFSF15 require specialized approaches for differentiation:

• Detection methods:

  • Flow cytometry with surface staining for membrane-bound TNFSF15

  • ELISA for soluble TNFSF15 in culture supernatants or biological fluids

  • Western blotting with appropriate antibodies recognizing different forms

  • Immunofluorescence microscopy to visualize cellular localization

• Experimental approaches:

  • Use of TACE/ADAM17 inhibitors to prevent cleavage of membrane-bound TNFSF15

  • Comparison of effects of cell-bound presentation versus soluble protein

  • Creation of cleavage-resistant TNFSF15 mutants

  • Use of specific blocking antibodies against different forms

• Functional discrimination:

  • Soluble TNFSF15 leads to TRADD/FADD/MALT-1 and caspase-8-mediated IL-1 secretion

  • Membrane-bound forms may have distinct signaling properties

  • Different forms may preferentially activate different downstream pathways

Understanding the balance between membrane-bound and soluble TNFSF15 is particularly important in inflammatory conditions where TACE/ADAM17 activity may be altered, affecting the ratio between these forms and consequently modulating immune responses.