TNFSF14 Mouse

What is TNFSF14 and what is its structure in mouse models?

TNFSF14, also known as LIGHT (lymphotoxin-like, exhibits inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes), is a member of the TNF superfamily. In mice, the LIGHT gene encodes a 239 amino acid type II transmembrane glycoprotein consisting of a 37 amino acid N-terminal cytoplasmic domain, a 21 amino acid transmembrane region, and a 181 amino acid extracellular domain. Like other TNF family members, functional LIGHT is assembled as a homotrimer. Mouse LIGHT shares approximately 71% amino acid sequence identity with its human counterpart. While LIGHT exists primarily as a membrane-bound protein, a soluble form can be generated through proteolytic processing at a metalloprotease cleavage site present in the protein structure .

Which receptors does mouse TNFSF14 interact with and how?

Mouse TNFSF14 primarily interacts with two receptors: lymphotoxin beta receptor (LTβR) and herpes virus entry mediator (HVEM). The signaling network is complex, as these receptors can also bind other ligands. Research indicates that LIGHT signaling through LTβR, rather than HVEM, plays a critical role in the progression of DSS-induced colitis, as demonstrated by LTβR-deficient mice exhibiting a more severe disease phenotype. This suggests receptor-specific outcomes in inflammatory conditions despite both receptors binding to the same ligand . The interaction between LIGHT and its receptors initiates downstream signaling cascades that regulate various biological processes, including inflammation and tissue homeostasis.

What are the primary methodological approaches for detecting TNFSF14 in mouse samples?

Several methodological approaches can be employed to detect TNFSF14 in mouse samples:

• Flow Cytometry: Using specific antibodies like the monoclonal rat IgG antibody (clone #906909) that recognizes mouse LIGHT/TNFSF14. This approach is particularly effective for detecting LIGHT expression on cell surfaces, as demonstrated in transfected cell lines .

• Direct ELISA: Specific antibodies can detect mouse LIGHT/TNFSF14 in ELISA-based assays, allowing for quantification in biological samples .

• Immunohistochemistry: Though not explicitly mentioned in the search results, antibodies suitable for flow cytometry are often adaptable for immunohistochemical staining.

When performing flow cytometry, approximately 0.25 μg of antibody per 10^6 cells is recommended for optimal detection. Controls should include irrelevant transfectants and control antibody staining to properly set quadrant markers and ensure specificity .

How does TNFSF14 function in inflammatory bowel disease mouse models?

Despite TNFSF14 being pro-inflammatory in several contexts, it surprisingly plays a protective role in mouse models of colitis. LIGHT-deficient mice exhibit more severe disease pathogenesis in DSS-induced colitis models. The protective mechanism involves a complex signaling network between LIGHT, its receptors, and competing ligands.

These findings suggest a nuanced regulatory mechanism where the balance between multiple ligands competing for the same receptor determines disease outcomes. When designing colitis studies, researchers should consider not only LIGHT expression but also the complex interplay between LIGHT, LTαβ, and their receptors.

What is the role of TNFSF14 in renal fibrosis pathogenesis?

TNFSF14 has been identified as a critical contributor to renal fibrosis development. Studies using the unilateral ureteral obstruction (UUO) mouse model demonstrate that TNFSF14 levels are significantly increased in both UUO-induced renal fibrotic mice and in patients with fibrotic nephropathy compared to controls .

Functionally, TNFSF14 deficiency leads to marked reduction in:

• Renal fibrosis lesions

• Inflammatory cytokine expression in UUO mice

The mechanism appears to involve sphingosine kinase 1 (Sphk1), a critical molecule in fibrotic nephropathy. TNFSF14 knockout mice undergoing UUO surgery show remarkably reduced Sphk1 levels. In vitro experiments confirm this relationship, as recombinant TNFSF14 administration markedly upregulates Sphk1 expression in primary mouse renal tubular epithelial cells (mTECs) .

This research highlights TNFSF14 as a potential therapeutic target for renal fibrosis, as blocking its function could potentially mitigate fibrotic progression. When designing experiments to study renal fibrosis, researchers should consider measuring both membrane-bound and soluble forms of TNFSF14, as both may contribute to disease pathology.

How do TNFSF14 expression patterns change during inflammatory responses in mouse models?

In UUO-induced renal fibrosis, both TNFSF14 and its receptors HVEM and LTβR are rapidly upregulated . Similarly, in inflammatory contexts, not only is membrane-bound LIGHT increased, but the soluble form of LIGHT is also elevated through proteolytic processing. This soluble form has been confirmed to play important roles in liver inflammation and DSS-induced colitis .

When designing experiments to track TNFSF14 expression during inflammation, researchers should:

• Monitor both membrane-bound and soluble forms of LIGHT

• Track receptor expression changes (HVEM and LTβR)

• Consider cell-specific expression patterns

• Establish appropriate time points, as expression can change rapidly during disease progression

What experimental considerations are important when using TNFSF14 antibodies in mouse studies?

When using antibodies against mouse TNFSF14 in research, several methodological considerations are critical:

• Antibody Specificity: Ensure the antibody specifically detects mouse LIGHT/TNFSF14 with minimal cross-reactivity. Antibodies like clone #906909 have been validated for specificity in direct ELISAs .

• Proper Reconstitution and Storage: Lyophilized antibodies should be reconstituted at 0.5 mg/mL in sterile PBS. Storage conditions significantly impact antibody functionality:

  • 12 months at -20 to -70°C as supplied

  • 1 month at 2 to 8°C under sterile conditions after reconstitution

  • 6 months at -20 to -70°C under sterile conditions after reconstitution

• Freeze-Thaw Cycles: Use a manual defrost freezer and avoid repeated freeze-thaw cycles to maintain antibody integrity .

• Optimal Dilutions: For flow cytometry applications, recommended usage is 0.25 μg per 10^6 cells, though optimal dilutions should be determined by each laboratory for each specific application .

• Appropriate Controls: Include proper controls when detecting TNFSF14, such as irrelevant transfectants and control antibody staining to set accurate quadrant markers in flow cytometry experiments .

How does the TNFSF14/LTαβ/LTβR signaling network function in experimental colitis models?

The TNFSF14/LTαβ/LTβR signaling network plays a complex role in experimental colitis that extends beyond simple pro- or anti-inflammatory effects. Research using DSS-induced colitis in mice has revealed several key insights:

These findings suggest a complex compensatory mechanism within the signaling network, where the balance between multiple ligands (LIGHT and LTαβ) competing for the same receptor (LTβR) determines disease outcomes. When one ligand is absent, the signaling balance through shared receptors is disrupted, potentially explaining the paradoxical effects observed in various knockout models.

Researchers investigating this pathway should consider genetic approaches that allow for conditional or inducible deletion of pathway components in specific cell types, as the cellular source of these molecules may significantly impact disease outcomes.

What are the optimal storage and handling conditions for TNFSF14 reagents?

To maintain the integrity and activity of TNFSF14 reagents such as antibodies and recombinant proteins, proper storage and handling are essential:

For antibodies against mouse LIGHT/TNFSF14:

• Store lyophilized antibodies at -20 to -70°C for up to 12 months from the date of receipt

• After reconstitution at 0.5 mg/mL in sterile PBS, store at 2-8°C for up to 1 month or at -20 to -70°C for up to 6 months under sterile conditions

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles that can degrade antibody quality

For experiments involving flow cytometry:

• Calculate the appropriate amount of antibody (0.25 μg per 10^6 cells is recommended)

• Include proper controls (irrelevant transfectants and control antibody staining) to set accurate quadrant markers

• Follow established protocols for membrane protein staining to maximize signal-to-noise ratio

Following these guidelines will ensure consistent and reliable results when working with TNFSF14 in experimental settings.

How can researchers effectively study the dual roles of TNFSF14 in different inflammatory contexts?

TNFSF14 exhibits context-dependent roles in inflammation, acting as pro-inflammatory in some settings while playing protective roles in others, such as in colitis models. To effectively study these dual roles, researchers should consider the following methodological approaches:

• Tissue-specific conditional knockout models: Generate mice with tissue-specific deletion of TNFSF14 or its receptors to determine cell-specific contributions to disease pathology.

• Temporal expression studies: Analyze TNFSF14 expression at different time points during disease progression to identify critical windows for intervention.

• Soluble vs. membrane-bound LIGHT analysis: Distinguish between these two forms of TNFSF14, as they may have different biological activities. Use specific assays to detect the soluble form in body fluids and membrane-bound form on cell surfaces .

• Receptor-specific studies: Use receptor-blocking antibodies or receptor-specific knockout models to distinguish between LIGHT signaling through HVEM versus LTβR .

• Combinatorial knockout approaches: As seen with the LIGHT/LTαβ double knockout studies, removing multiple components of the signaling network can reveal compensatory mechanisms that single knockout models might miss .