TNFRSF14 Mouse

What is TNFRSF14 and what are its alternative names in scientific literature?

TNFRSF14 (Tumor Necrosis Factor Receptor Superfamily Member 14) is a type I transmembrane glycoprotein belonging to the TNF receptor superfamily. In scientific literature, it is also known as Herpesvirus Entry Mediator (HVEM), CD270, Another TRAF-Associated Receptor (ATAR), TNF Receptor-like molecule (TR2), and HveA. This receptor plays important biological roles in HSV infection, T cell proliferation, tumor immunity, transplantation immunity, inflammatory responses, and autoimmune diseases . It was originally identified from the TNFR family EST database by Montgomery in 1996 and named for its ability to regulate the entry of herpes simplex virus (HSV)-1 into human cells .

What is the structural composition of mouse TNFRSF14 protein?

Mouse TNFRSF14/HVEM protein has a well-characterized structure consisting of 275 amino acid residues. It contains a 36 amino acid signal peptide, a 170 amino acid extracellular domain featuring three cysteine-rich domains (CRDs), a 24 amino acid transmembrane region, and a 45 amino acid cytoplasmic tail with a TRAF interaction domain . Unlike some TNFRSF members, HVEM lacks a death domain in its intracellular region but has a PXQT short peptide that binds to multiple TNFR-related factors, which transmit signals intracellularly and activate NF-κB and AP1 transcription factors . The functional extracellular domain spans from Gln39 to Val207 in the amino acid sequence, and the protein's molecular mass is approximately 45.6 kDa .

What are the binding partners of mouse TNFRSF14 and their biological implications?

TNFRSF14 interacts with multiple ligands that induce different cellular responses:

• TNFSF14 (LIGHT): A TNF family member that co-stimulates T cells and promotes inflammation. Interaction of HVEM with LIGHT is crucial for T cell activation and inflammatory processes .

• BTLA (B and T Lymphocyte Attenuator): HVEM triggers inhibitory signaling in effector T cells and regulatory T cells as a ligand of BTLA .

• CD160: Another binding partner that influences immune cell function.

• HSV-gD: The envelope glycoprotein D of Herpes Simplex Virus, which facilitates viral entry into target cells .

• LT-α (Lymphotoxin alpha): A TNF family cytokine that can bind to TNFRSF14.

These diverse interactions make TNFRSF14 a unique immune regulator capable of both costimulatory and inhibitory functions depending on the specific ligand engagement and cellular context .

How is TNFRSF14 expression regulated in different mouse tissues and cell types?

TNFRSF14 expression varies across mouse tissues and cell types, with distinct regulatory patterns:

• Highest expression is found on naïve, memory, and regulatory T cells, with expression declining during T cell activation .

• Present at low levels on most resting leukocytes .

• Expressed on mast cells, where it can enhance IgE-mediated signaling and influence allergic responses .

• Found in peripheral blood T cells, B cells, and monocytes, as well as various tissues enriched in lymphoid cells .

Expression of TNFRSF14 is dynamically regulated during immune responses, suggesting its role in fine-tuning immune reactions. Interestingly, the HVEM gene is located at chromosome 1p36.22-36.3 in humans with a DNA size of 7.46 KB and total mRNA length of 1.7 KB . The mouse ortholog shares significant homology and functional similarity.

How does TNFRSF14 contribute to asthma pathology in mouse models?

TNFRSF14 significantly contributes to asthma pathology through multiple mechanisms:

• TNFRSF14 expression specifically on mast cells is necessary for the full development of multiple features of asthma pathology in mice .

• In OVA-induced and house dust mite (HDM)-induced asthma models, studies with both genetic deletion of Tnfrsf14 and neutralizing antibodies demonstrate that TNFRSF14 is required for:

  • Development of airway hyperreactivity (AHR)

  • Airway inflammation with inflammatory cell infiltration

  • Airway remodeling (structural changes in airways)

  • Production of antigen-specific IgE and IgG1 antibodies

Mechanistically, TNFRSF14 enhances IgE-dependent mast cell activation, leading to increased degranulation and release of inflammatory mediators. Additionally, TNFSF14 (LIGHT), the ligand for TNFRSF14, supports TH2 cell generation and longevity, further contributing to allergic inflammation . These findings highlight TNFRSF14 as a potential therapeutic target in asthma.

What is the role of TNFRSF14 in mouse models of viral infection?

TNFRSF14 plays dual roles in viral infection models:

• As the herpesvirus entry mediator (HVEM), TNFRSF14 facilitates the entry of herpes simplex virus (HSV)-1 into cells by binding to the viral glycoprotein D (gD) .

• Soluble forms of TNFRSF14 (HVEM-Ig) can exert significant antiviral effects against HSV-1 infection in vivo by competing with cellular HVEM for binding to viral gD .

• Beyond viral entry, TNFRSF14 signaling influences immune responses to viral infections by modulating T cell activation and function, affecting inflammatory responses, and influencing the balance between protective immunity and immunopathology .

Understanding this dual role is critical for developing targeted therapeutic approaches for viral infections. The receptor's name "Herpesvirus Entry Mediator" reflects its historical identification as a portal for HSV infection .

How does TNFRSF14 interact with the FcεRI pathway in mast cells?

TNFRSF14 interacts with the FcεRI pathway in mast cells through several mechanisms:

• TNFSF14:TNFRSF14 interactions enhance IgE-mediated mast cell signaling and mediator production .

• Engagement of TNFSF14 by TNFRSF14 on the mast cell surface enhances IgE-dependent aggregation of FcεRI .

• In experiments with mouse bone marrow-derived cultured mast cells (BMCMCs), exposure to both DNP-HSA-A650 (antigen) and TNFSF14 dramatically increases both the number and area of individual clusters formed by FcεRI and TNFSF14 on the plasma membrane surface .

• This clustering effect potentiates downstream signaling events, resulting in increased secretion of both pre-stored and de novo synthesized mast cell mediators .

These findings demonstrate that TNFRSF14 functions as a critical co-receptor that can amplify IgE-dependent mast cell activation, contributing significantly to allergic inflammatory responses and asthma pathology .

What are the validated approaches for studying TNFRSF14 knockout in mice?

Several validated approaches exist for studying TNFRSF14 knockout in mice:

• Conventional gene knockout: Complete deletion of the Tnfrsf14 gene to generate Tnfrsf14-/- mice. These mice develop normally but show diminished responses in models of inflammatory diseases, including asthma .

• Neutralizing antibody administration: As an alternative to genetic deletion, anti-TNFRSF14 neutralizing antibodies can be administered after antigen sensitization to block TNFRSF14 function in specific experimental contexts .

• Bone marrow chimeras: Engrafting bone marrow from Tnfrsf14-/- mice into irradiated wild-type recipients to study the role of TNFRSF14 in hematopoietic cells.

• Adoptive transfer models: Particularly useful for studying mast cell-specific functions of TNFRSF14, involving the engraftment of mast cells that either do or do not express TNFRSF14 into genetically mast cell-deficient mice .

These approaches allow researchers to dissect the cell type-specific and context-dependent roles of TNFRSF14 in various physiological and pathological settings, with particular utility in understanding allergic inflammation and asthma models .

What experimental protocols are optimal for studying TNFRSF14-mediated mast cell activation?

Optimal experimental protocols for studying TNFRSF14-mediated mast cell activation include:

• Bone marrow-derived mast cell (BMMC) culture: Isolating bone marrow cells from mice and culturing them with IL-3 and SCF to generate a pure population of mast cells, which express similar levels of FcεRIα and CD117 (KIT) regardless of TNFRSF14 expression .

• IgE sensitization and challenge: Sensitizing mast cells overnight with anti-DNP monoclonal mouse IgE antibody, then challenging with DNP-HSA (antigen) in the presence or absence of soluble TNFSF14 to assess the costimulatory effect of TNFRSF14 signaling .

• Confocal microscopy: Visualizing receptor clustering using fluorescently labeled FcεRI (DNP-HSA-A650) and TNFSF14-A594 to observe their co-localization on the plasma membrane and quantify cluster formation .

• Mediator release assays: Measuring degranulation and cytokine production following stimulation with IgE/antigen alone or in combination with TNFSF14 .

These protocols should be performed with appropriate controls, including cells from Tnfrsf14-/- mice to confirm specificity of TNFSF14 binding and signaling. Studies have shown no binding of TNFSF14 on the surface of Tnfrsf14-/- BMCMCs, indicating that TNFSF14 binding is specific for mast cells expressing TNFRSF14 .

How can researchers effectively measure TNFRSF14 expression in mouse tissues?

Researchers can effectively measure TNFRSF14 expression in mouse tissues using several complementary techniques:

• Flow cytometry: Using fluorescently-labeled antibodies against TNFRSF14 (such as Alexa Fluor 350-conjugated anti-TNFRSF14) to quantify protein expression on cell surfaces .

• Western blotting: Detecting TNFRSF14 protein in tissue or cell lysates, with expected molecular mass of approximately 45.6 kDa, though apparent molecular mass in SDS-PAGE appears as 50-60 kDa due to post-translational modifications .

• Immunohistochemistry/Immunofluorescence: Visualizing TNFRSF14 expression in tissue sections to determine anatomical distribution.

• Comparison between wild-type and Tnfrsf14-/- cells: Necessary to confirm antibody specificity and validate expression patterns .

For optimal results, researchers should validate findings using multiple techniques and include appropriate positive and negative controls. It's important to note that TNFRSF14 expression varies across different cell types and can be dynamically regulated during immune responses .

How should researchers design experiments to study TNFRSF14 in asthma models?

When designing experiments to study TNFRSF14 in asthma models, researchers should consider:

• Choice of asthma induction protocols:

  • OVA-induced chronic airway inflammation model: Sensitization with ovalbumin followed by challenge

  • House dust mite (HDM)-induced asthma model: More clinically relevant allergen

• Experimental groups should include:

  • Wild-type controls

  • Tnfrsf14-/- mice

  • Mice treated with TNFRSF14 neutralizing antibodies administered after antigen sensitization

  • For cell-specific studies: Mast cell-deficient mice reconstituted with either TNFRSF14-expressing or TNFRSF14-deficient mast cells

• Key readouts to measure:

  • Airway hyperreactivity measurements

  • Bronchoalveolar lavage fluid analysis for inflammatory cells

  • Histological assessment of airway inflammation and remodeling

  • Measurement of antigen-specific IgE and IgG1 antibodies in plasma

These comprehensive approaches have revealed that TNFRSF14 expression specifically on mast cells significantly contributes to the development of multiple features of asthma pathology in mice, highlighting both the mechanistic insights and therapeutic potential of targeting this pathway .

What are the critical considerations when comparing TNFRSF14 function across different mouse strains?

When comparing TNFRSF14 function across different mouse strains, researchers should consider:

• Genetic background effects: Different inbred mouse strains have distinct immunological phenotypes that may influence TNFRSF14 function and expression levels.

• Disease model susceptibility: Various strains show different susceptibility to asthma, viral infections, and other conditions where TNFRSF14 plays a role.

• Expression pattern differences: The baseline expression of TNFRSF14 and its ligands may vary between strains, affecting experimental outcomes.

• Experimental design considerations:

  • Use of appropriate strain-matched controls

  • Backcrossing genetically modified mice for sufficient generations to ensure genetic homogeneity

  • Detailed reporting of the strain background in publications

Studies have successfully used various mouse strains to investigate TNFRSF14 function, including work with bone marrow-derived mast cells from both wild-type and Tnfrsf14-/- mice that showed similar levels of expression of FcεRIα and CD117 (KIT) but differential responses to TNFSF14 stimulation .

How do researchers address contradictory data on TNFRSF14 function in different experimental contexts?

Researchers can address contradictory data on TNFRSF14 function through several approaches:

• Context-specific analysis: Explicitly defining the experimental context, including cell types, activation states, and disease models. For example, TNFRSF14 has dual functions (costimulatory and inhibitory) depending on which ligand it interacts with .

• Molecular mechanism dissection: Detailed analysis of signaling pathways activated in different contexts. For instance, while one study reported that bone marrow-derived cultured mouse mast cells (BMCMCs) functionally bind TNFSF14 through TNFRSF3, another study found strong expression of TNFRSF14 but no expression of TNFRSF3 on human mast cell lines and peripheral blood-derived cultured mast cells .

• Technical considerations: Standardization of experimental protocols and validation of reagents across different studies.

• Thorough controls: Including cells from Tnfrsf14-/- mice to confirm specificity of ligand binding and signaling pathways .

By systematically addressing these factors, researchers can reconcile seemingly contradictory data and develop a more nuanced understanding of TNFRSF14's context-dependent functions in immune regulation and disease pathogenesis.

What intracellular signaling pathways are activated by TNFRSF14 in mouse cells?

TNFRSF14 activation in mouse cells triggers several intracellular signaling cascades:

• NF-κB activation: TNFRSF14 contains a PXQT short peptide in its cytoplasmic domain that binds to multiple TNFR-related factors, which then transmit signals that activate the NF-κB transcription factor .

• AP1 transcription factor activation: In parallel with NF-κB, TNFRSF14 signaling leads to AP1 activation .

• Enhanced FcεRI signaling in mast cells: TNFSF14:TNFRSF14 interactions amplify IgE-dependent signaling pathways, leading to increased degranulation and cytokine production .

• Receptor clustering: Engagement of TNFSF14 by TNFRSF14 enhances the IgE-dependent aggregation of FcεRI on the mast cell surface, potentiating downstream signaling events .

These signaling pathways are believed to promote cell survival and mediate inflammatory responses. In mast cells specifically, these pathways lead to enhanced mediator release, contributing to allergic inflammation and asthma pathology .

What therapeutic approaches target TNFRSF14 in mouse disease models?

Several therapeutic approaches targeting TNFRSF14 have shown promise in mouse disease models:

• Neutralizing antibodies: Anti-TNFRSF14 neutralizing antibodies administered after antigen sensitization have been shown to diminish plasma levels of antigen-specific IgG1 and IgE antibodies, reduce airway hyperreactivity, decrease airway inflammation, and limit airway remodeling in asthma models .

• Recombinant proteins: Soluble forms of TNFRSF14 (such as HVEM-Fc fusion proteins) can act as decoy receptors to inhibit interactions with natural ligands. Such proteins have demonstrated antiviral effects against HSV-1 infection in vivo .

• Genetic approaches: Studies using Tnfrsf14-/- mice have provided proof-of-concept for targeting this pathway in inflammatory diseases .

These approaches highlight TNFRSF14 as a potential therapeutic target, particularly in allergic airway diseases where TNFRSF14 expression on mast cells significantly contributes to multiple features of asthma pathology .

How do compensatory mechanisms affect interpretation of TNFRSF14 knockout studies?

Compensatory mechanisms can significantly impact the interpretation of TNFRSF14 knockout studies:

• Redundancy in receptor-ligand interactions: TNFRSF14's ligands (particularly TNFSF14/LIGHT) can bind to alternative receptors. For example, TNFSF14 can also signal through TNFRSF3 (LTβR), potentially compensating for the loss of TNFRSF14 signaling .

• Altered expression of related receptors: Knockout of TNFRSF14 may lead to upregulation of functionally related receptors as a compensatory mechanism.

• Changes in ligand availability: Absence of TNFRSF14 may lead to increased availability of its ligands for binding to alternative receptors.

Despite these potential compensatory mechanisms, studies have clearly demonstrated phenotypic differences in Tnfrsf14-/- mice in various disease models, particularly asthma, indicating that compensation is incomplete . This reinforces the importance of TNFRSF14 in specific immunological contexts and highlights its potential as a therapeutic target.