TNF a Mouse

What is mouse TNF-α and how does it differ from human TNF-α?

Mouse TNF-α is a multifunctional proinflammatory cytokine belonging to the tumor necrosis factor superfamily. It exists as a membrane-anchored form and can form biologically active trimers. Unlike human TNF-α, the naturally-occurring mouse form is glycosylated, although non-glycosylated recombinant TNF-α exhibits comparable biological activity . Mouse and human TNF-α share approximately 79% amino acid sequence identity and demonstrate cross-reactivity between species . This relatively high homology enables certain experimental approaches using human reagents in mouse models, though species-specific differences must be considered when translating findings.

The molecular weight of mouse TNF-α is approximately 17 kDa, and it appears as a single band on SDS-PAGE under non-reducing conditions, despite existing as multimers of two, three, or five noncovalently linked units in its native state .

Which cell types produce TNF-α in mice?

In mice, TNF-α is produced by multiple cell types including:

• Neutrophils

• Activated lymphocytes

• Macrophages

• Natural killer (NK) cells

• Lymphokine-activated killer (LAK) cells

• Astrocytes

• Endothelial cells

• Smooth muscle cells

• Certain transformed cells

This broad expression pattern underscores the diverse biological roles of TNF-α in immune regulation, inflammatory responses, and tissue homeostasis. The cell-specific production of TNF-α can be differentially regulated, as demonstrated in the hTNF.LucBAC reporter mouse model, where various cell types show distinct responses to different stimuli .

What are the primary biological functions of TNF-α in mice?

TNF-α functions as a master regulator of inflammatory responses in mice with several key roles:

• Immune cell regulation: TNF-α activates macrophages and other immune cells during infection and inflammation .

• Anti-microbial defense: It is crucial for bacterial clearance, as demonstrated in Rhodococcus aurantiacus infection models where TNF-α-deficient mice showed impaired bacterial elimination .

• Granuloma formation: TNF-α is essential for proper granulomatous inflammation, with TNF-α^-/- mice failing to form granulomas in response to bacterial infection .

• Inflammatory response modulation: TNF-α regulates the balance of pro-inflammatory cytokines, particularly with IL-6, creating a negative feedback loop where they mutually regulate each other's production .

• Neuroprotection: Knockout studies in mice have suggested neuroprotective functions of TNF-α under certain conditions .

• Cytolysis or cytostasis: TNF-α can cause death or growth inhibition of certain transformed cells and demonstrates synergistic effects with other cytokines .

How does TNF-α contribute to Alzheimer's disease pathogenesis in mouse models?

TNF-α has emerged as a significant factor in Alzheimer's disease (AD) pathogenesis, with complex roles evidenced in various mouse models. In 5XFAD mice, which develop AD-like pathology, TNF-α modulates amyloid burden through several mechanisms:

• Peripheral-central inflammation connection: Studies using 5XFAD mice crossed with TNF^ΔARE/+ mice (which have deletion of the 3'UTR of endogenous TNF-α) demonstrate that increased peripheral TNF-α levels can significantly influence brain pathology, reducing amyloid deposition despite unchanged brain TNF-α levels .

• Microglial activation: Elevated peripheral TNF-α induces microglial activation in the brain, enhancing phagocytic clearance of amyloid without altering APP levels or processing enzymes .

• Immune cell infiltration: In 5XFAD/TNF^ΔARE/+ mice, there is increased infiltration of peripheral leukocytes and perivascular macrophages into the brain, which contributes to altered amyloid dynamics .

• Synaptic effects: Despite reduced amyloid burden, elevated TNF-α is associated with synaptic degeneration, indicating complex, potentially biphasic effects on neuronal health .

In 3xTgAD mouse models, soluble TNF signaling has been identified as a critical mediator of neuroinflammation's effects on early (pre-plaque) AD pathology, suggesting that specific targeting of soluble TNF may offer therapeutic potential .

What is the relationship between TNF-α and other cytokines in inflammatory regulation?

TNF-α operates within a complex cytokine network, with particularly important regulatory relationships with IL-6. Research using Rhodococcus aurantiacus infection models has revealed:

• Reciprocal regulation: TNF-α and IL-6 negatively regulate each other's production. TNF-α^-/- mice show enhanced IL-6 production in response to bacterial stimulation, while treatment with recombinant TNF-α reduces IL-6 secretion .

• Cytokine balance effects on survival: TNF-α^-/- mice exhibit high mortality rates following R. aurantiacus infection, associated with elevated IL-6 levels. Administration of recombinant TNF-α attenuates IL-6 production and improves survival .

• Experimental evidence of mutual regulation:

  • Anti-TNF-α treatment increases IL-6 production by wild-type mouse cells

  • Anti-IL-6 treatment increases TNF-α production

  • Treatment of TNF-α^-/- cells with rTNF-α decreases IL-6 secretion

This balance between TNF-α and other cytokines, particularly IL-6, appears critical for controlling inflammatory responses and determining disease outcomes.

How do genetic modifications of TNF-α in mice affect phenotypes in disease models?

Genetic manipulation of TNF-α in mice produces distinct phenotypes that illuminate its role in various pathologies:

The TNF^ΔARE/+ model is particularly valuable for studying peripheral inflammation effects on central nervous system pathology, as these mice develop systemic inflammatory conditions reminiscent of human autoimmune diseases while allowing cross-breeding with neurological disease models like 5XFAD .

The consequences of these genetic modifications demonstrate that proper TNF-α regulation is essential for:

• Balanced inflammatory responses

• Effective pathogen clearance

• Appropriate tissue remodeling during inflammation

• Prevention of autoimmunity

What techniques are available for quantifying mouse TNF-α in biological samples?

Several validated methodologies exist for measuring mouse TNF-α in research settings:

• ELISA (Enzyme-Linked Immunosorbent Assay):

  • The most common method for quantifying TNF-α in serum, plasma, urine, or cell culture medium

  • Typically employs a sandwich technique with capture and detector antibodies specific to mouse TNF-α

  • Can detect both natural and recombinant mouse TNF-α with high specificity

  • Sensitivities can reach the picogram/mL range

  Protocol considerations:

  • Sample preparation is critical (proper dilution, removal of particulates)

  • Standard curves must be prepared with recombinant mouse TNF-α

  • Detection range varies by kit but typically spans 15-2000 pg/mL

• Reporter systems:

  • The hTNF.LucBAC transgenic mouse model expresses luciferase under control of the human TNF locus

  • Allows real-time monitoring of TNF expression through luciferase activity measurement

  • Enables screening of compounds that modulate TNF synthesis

• Flow cytometry:

  • For detection of membrane-bound TNF-α or intracellular TNF-α in specific cell populations

  • Requires cell permeabilization for intracellular detection

• RT-qPCR:

  • Measures TNF-α mRNA expression rather than protein levels

  • Useful for studying transcriptional regulation

When selecting a quantification method, researchers should consider the biological matrix, required sensitivity, and whether protein or gene expression information is needed.

How can TNF-α signaling be manipulated in mouse models?

Researchers have several approaches available for modulating TNF-α signaling in mouse experimental systems:

• Genetic approaches:

  • TNF-α knockout mice (TNF-α^-/-): Complete absence of TNF-α expression

  • TNF^ΔARE/+ mice: Enhanced peripheral TNF-α due to 3'UTR deletion

  • Conditional knockouts: Cell-specific or inducible TNF-α deletion

• Pharmacological inhibition:

  • Recombinant dominant-negative TNF variants (e.g., XENP345)

    • Selectively inhibits soluble TNF signaling

    • Can be administered via osmotic minipumps for localized delivery to specific brain regions

  • Anti-TNF-α antibodies

• Viral vector delivery:

  • Lentiviral vectors expressing dominant-negative TNF (Lenti-DN-TNF)

    • Can be stereotactically injected into specific brain regions

    • Provides local inhibition of TNF signaling

  • Adeno-associated virus (AAV) vectors for TNF-α overexpression or knockdown

• Recombinant TNF-α administration:

  • Commercially available mouse recombinant TNF-α (≥95% purity)

  • Typical potency: 0.01-0.5 ng/mL EC₅₀

  • Can be administered systemically or locally

Example protocol for central TNF modulation:
For hippocampal delivery of TNF inhibitors, stereotactic injection coordinates of AP: -2.0 mm from bregma, ML: -2.0 mm, and DV: -1.6 mm below dura have been validated, with continuous delivery possible via osmotic minipumps .

What stimuli effectively induce TNF-α production in mouse cells for experimental studies?

Different stimuli demonstrate varying effectiveness in inducing TNF-α production depending on the cell type:

The cell-specific response to different stimuli was effectively demonstrated in the hTNF.LucBAC transgenic mouse model, which showed that LPS was the most potent luciferase inducer in macrophages, while TNF-α itself was a strong activator in intestinal organoids .

For LPS stimulation, a validated protocol involves intraperitoneal injection of 0.25 mg/kg LPS (from Escherichia coli O111:B4; 3.0 × 10⁶ E.U./mg) twice weekly for 4-6 weeks .

How can TNF-α reporter mouse models advance research in inflammatory diseases?

TNF-α reporter mouse models offer unique advantages for studying inflammatory processes:

The hTNF.LucBAC transgenic mouse model represents a significant advancement in this area. This model expresses luciferase under the control of the human TNF locus via a bacterial artificial chromosome (BAC) construct .

Key research applications include:

• Real-time visualization of TNF-α expression:

  • Allows non-invasive monitoring of TNF-α transcriptional activity

  • Enables temporal studies of inflammation progression and resolution

• Drug discovery and screening:

  • Provides a platform for screening compounds that modulate TNF-α synthesis

  • Allows for rapid assessment of anti-inflammatory drug candidates

  • Enables dose-response studies for TNF inhibitors

• Cell-specific TNF-α regulation studies:

  • Different cell types show distinct luciferase expression patterns in response to various stimuli

  • Helps identify cell-specific regulatory mechanisms for TNF-α expression

• Correlation with soluble TNF secretion:

  • The transgene-dependent luciferase activity shows positive correlation with endogenous murine soluble TNF secreted into culture medium

  • Provides validation of the reporter as a proxy for actual TNF-α production

This model demonstrated that NF-κB pathway inhibitors and IL-10 downregulate LPS-induced luciferase activity in macrophages, confirming its utility for studying regulatory mechanisms .

What is the significance of TNF-α in neuroinflammation and neurodegenerative models?

TNF-α plays multifaceted roles in neuroinflammation and neurodegeneration:

• Alzheimer's disease models:

  • In 5XFAD mice, peripheral TNF-α modulates brain inflammation and amyloid deposition

  • The 5XFAD/TNF^ΔARE/+ double transgenic model shows:

    • Decreased amyloid deposition

    • Increased microglial activation

    • Enhanced infiltration of peripheral immune cells

    • Synaptic degeneration despite reduced amyloid

• Peripheral-central inflammation communication:

  • Peripheral TNF-α can influence brain pathology without changes in brain TNF-α levels

  • This provides evidence for peripheral TNF-α as a mediator of inflammation between the periphery and brain

• Pre-plaque pathology:

  • Soluble TNF is a critical mediator of neuroinflammation's effects on early AD pathology

  • Inhibition of soluble TNF signaling may provide therapeutic benefit before extensive plaque formation

• Neuroprotective functions:

  • Knockout studies have suggested potential neuroprotective roles for TNF-α under certain conditions

  • This illustrates the complex, context-dependent actions of TNF-α in the CNS

• Therapeutic targeting approaches:

  • Selective inhibition of soluble TNF using dominant-negative TNF variants (XENP345)

  • Lentiviral delivery of dominant-negative TNF to specific brain regions

  • These approaches may offer advantages over complete TNF blockade

The dual nature of TNF-α in neurodegeneration suggests that targeted, rather than complete, inhibition may be more beneficial in neurodegenerative disease contexts.

What are the key experimental controls when studying TNF-α in mouse models?

Robust TNF-α research requires careful consideration of experimental controls:

• Genetic background controls:

  • When using transgenic models, littermate controls with matched genetic backgrounds are essential

  • For TNF-α knockout studies, heterozygous mice may serve as important intermediate controls

• Stimulus standardization:

  • LPS purity and potency must be standardized (typically reported as endotoxin units/mg)

  • For E. coli O111:B4 LPS, standardized preparations contain approximately 3.0 × 10⁶ E.U./mg

  • Recombinant mouse TNF-α should be verified for purity (≥95%) and endotoxin levels (≤1 EU/μg protein)

• Sample collection timing:

  • TNF-α production follows distinct temporal patterns after stimulation

  • Multiple time points should be assessed for comprehensive understanding

  • Peak circulating levels typically occur 1-2 hours after LPS stimulation

• Vehicle controls:

  • For TNF-α inhibition studies, appropriate vehicle controls are critical

  • For intracranial delivery, vehicle controls have included sterile PBS with 10% glycerol

• Cross-reactivity considerations:

  • Despite 79% homology between mouse and human TNF-α, species-specific reagents should be used when possible

  • Cross-reactivity should be experimentally validated before relying on cross-species reagents

How do storage and handling conditions affect mouse TNF-α stability?

Proper handling of mouse TNF-α is critical for experimental reproducibility:

• Recombinant TNF-α storage:

  • Recommended storage temperature: -20°C

  • Repeated freeze/thaw cycles should be avoided as they may reduce biological activity

  • Working aliquots can minimize freeze/thaw cycles

• Solubility characteristics:

  • Recombinant mouse TNF-α is water-soluble

  • For in vivo administration, sterile PBS with 10% glycerol has been used as a vehicle

• Sample preservation for analysis:

  • For ELISA testing of biological samples:

    • Serum/plasma should be separated promptly and stored at -80°C

    • Protease inhibitors may improve stability in some applications

    • Samples should remain on ice during processing

• Recombinant protein potency:

  • Typical potency for recombinant mouse TNF-α: 0.01-0.5 ng/mL EC₅₀

  • Activity assays should be performed to verify functional integrity after storage

• Endotoxin contamination:

  • Endotoxin levels should be monitored, especially for in vivo applications

  • High-quality recombinant preparations typically contain ≤1 EU/μg protein endotoxin

What emerging approaches show promise for studying TNF-α functions in mouse models?

Several innovative approaches are advancing our understanding of TNF-α biology:

• Single-cell analysis technologies:

  • Single-cell RNA sequencing to identify cell-specific TNF-α production and response patterns

  • Mass cytometry for simultaneous detection of multiple cytokines at the single-cell level

• Advanced genetic modification approaches:

  • CRISPR/Cas9-mediated precise modification of TNF-α regulatory elements

  • Conditional and inducible TNF-α expression systems for temporal control

  • Cell-specific TNF-α modulation to dissect tissue-specific roles

• Reporter mice with enhanced capabilities:

  • Dual reporter systems for simultaneous monitoring of TNF-α and other cytokines

  • Reporter models with improved sensitivity for detecting low-level TNF-α expression

• Intravital imaging:

  • Two-photon microscopy for real-time visualization of TNF-α-expressing cells in vivo

  • Combination with reporter systems for dynamic studies of TNF-α regulation

• Humanized mouse models:

  • Mice with humanized TNF-α systems to improve translational relevance

  • Models that better recapitulate human inflammatory conditions

These emerging approaches promise to provide deeper insights into the complex roles of TNF-α in health and disease, potentially leading to more targeted therapeutic strategies for inflammatory conditions.