TSLP Mouse

What is TSLP and what are its key functions in mouse models?

Thymic Stromal Lymphopoietin (TSLP) is a cytokine initially identified in the conditioned medium of a mouse thymic stromal cell line that promoted B cell development. In mice, TSLP can co-stimulate growth of thymocytes and mature T cells, and support B lymphopoiesis. While mouse IL-7 facilitates the development of B220+/IgM- pre-B cells, TSLP promotes the development of B220+/IgM+ B cells . More recently, TSLP has been implicated in allergic inflammatory responses, serving as a key mediator in the development of atopic dermatitis (AD) and asthma in mouse models .

How does mouse TSLP expression compare to human TSLP?

Mouse and human TSLP share approximately 43% amino acid sequence identity. Human TSLP consists of a 159 amino acid residue precursor protein with a 28 amino acid signal sequence. Six of the seven cysteine residues involved in intramolecular disulfide bond formation in mouse TSLP are conserved in human TSLP . While both mediate allergic inflammation, human TSLP preferentially stimulates myeloid cells, inducing T cell-attracting chemokines from monocytes and enhancing CD11c+ dendritic cell maturation, showing some functional differences from mouse TSLP .

How is TSLP expression regulated in mouse skin and other tissues?

TSLP expression can be triggered by skin barrier defects. In mice with Notch signaling deficiency in keratinocytes, TSLP is significantly upregulated in the skin . This endogenous skin-derived TSLP can enter the systemic circulation, creating elevated serum TSLP levels. While TSLP expression has been detected in many tissues in humans, with the highest expression in heart, liver, testis, and prostate , in experimental mouse models, skin-derived TSLP can have systemic effects even without local expression in other organs such as the lung .

What is the composition of the TSLP receptor complex in mice?

The TSLP receptor complex in mice consists of a heterodimer of IL-7 receptor alpha (IL7Rα) and the TSLP-specific receptor chain (TSLPR, also called CRLM-2). TSLPR is a member of the hemopoietin receptor family most closely related to the common gamma chain (γc) . Both components are required for functional TSLP signaling, as demonstrated by the abolition of TSLP-evoked responses in IL7Rα-deficient mice .

What signaling pathways are activated downstream of TSLP receptor in neurons?

In sensory neurons, TSLP receptor activation leads to phospholipase C (PLC) signaling, which couples to TRPA1 channel activation. This is similar to MrgprC11 coupling to TRPA1, but distinct from MrgprA3 which uses Gβγ signaling to activate TRPA1. TSLP signaling in neurons triggers both release of Ca²⁺ from intracellular stores and subsequent Ca²⁺ influx . Pharmacological inhibition of PLC using U73122 significantly reduces TSLP-evoked neuronal activation and subsequent itch behaviors, confirming the importance of this pathway .

How do researchers distinguish between TSLP effects on immune cells versus neurons?

Researchers use several approaches to distinguish direct TSLP effects on neurons from immune cell-mediated effects:

• Cell-specific knockout models: Comparing TSLP-evoked responses in mice lacking specific immune cell populations (e.g., RAG1-/-, NOD SCID for T and B cells; Kit(W-sh) for mast cells) versus wild-type mice .

• Temporal analysis: Acute TSLP-evoked behaviors (within minutes) likely represent direct neuronal effects, while longer-term responses (hours to days) may involve immune cell activation and cytokine production .

• In vitro calcium imaging: Direct application of TSLP to isolated DRG neurons while monitoring calcium responses can confirm direct neuronal activation independent of immune cells .

• Immunohistochemistry: Co-localization studies of TSLP receptors with neuronal markers like PGP9.5 in skin nerve endings help establish the neuronal expression of receptors .

How does TSLP evoke itch behaviors in mouse models?

TSLP directly activates a subset of sensory neurons through TSLP receptors expressed on these neurons. Upon intradermal injection of TSLP into the cheek of mice, robust scratching behaviors are observed within 5 minutes (latency to scratch = 4.1 ± 0.3 min), indicating an acute itch response . This itch behavior requires functional TSLP receptors and TRPA1 channels, as demonstrated by the significant reduction in scratching in IL7Rα-deficient mice and complete abolishment in TRPA1-deficient mice .

What distinguishes TSLP-activated sensory neurons from other itch-responsive neurons?

TSLP activates an undescribed subset of sensory neurons that is largely distinct from those responding to other pruritogens. Specifically:

• TSLP activates approximately 5-6% of sensory neurons in mouse dorsal root ganglia (DRG) .

• Most TSLP-responsive neurons are insensitive to other itch compounds like histamine, chloroquine, and BAM8-22 .

• TSLP-responsive neurons require TRPA1 but not TRPV1 channels, whereas some other itch pathways depend on TRPV1 .

• TSLP receptors are found on approximately 9% of PGP9.5-positive free nerve endings in the skin, representing a specific subset of cutaneous sensory fibers .

What experimental methods are used to quantify TSLP-evoked itch in mice?

Several behavioral assays are employed to quantify TSLP-evoked itch in mice:

• Cheek injection model: TSLP is injected intradermally into the cheek, allowing distinction between itch (scratching with hindlimb) and pain (wiping with forelimb) behaviors .

• Scratching bout quantification: The number of scratching bouts is counted during a defined observation period (typically 30 minutes) following TSLP injection .

• Pharmacological intervention: The effects of receptor antagonists, signaling pathway inhibitors, or ion channel blockers on TSLP-evoked scratching are assessed to determine the molecular mechanisms involved .

• Genetic knockout models: TSLP-evoked behaviors are compared between wild-type mice and mice lacking specific components of the TSLP signaling pathway (e.g., IL7Rα-deficient mice) or downstream effectors (e.g., TRPA1-deficient mice) .

What are the main types of TSLP mouse models available for research?

Several TSLP mouse models have been developed for investigating its role in allergic inflammation:

• K14-TSLPtg mice: These mice overexpress TSLP specifically in keratinocytes under the control of the keratin 14 promoter. On an inbred background, they develop spontaneous AD-like skin inflammation, while on an outbred background, they maintain high serum TSLP levels without skin inflammation .

• SPC-TSLP mice: These mice express a lung-specific TSLP transgene under the control of the surfactant protein C promoter. They develop a spontaneous and progressive asthma-like disease when combined with antigenic stimulation .

• Skin barrier-deficient mice: Mice with skin-specific deletion of Notch signaling components develop skin barrier defects that lead to elevated TSLP production and systemic TSLP increase .

• TSLPR and IL7Rα knockout mice: These mice lack functional TSLP receptors and are used to study the requirement of TSLP signaling in various disease models .

How do researchers induce controlled TSLP expression in mouse models?

Researchers employ several strategies to induce controlled TSLP expression:

• Tissue-specific promoters: Using tissue-specific promoters like K14 (skin keratinocytes) or SPC (lung epithelium) to drive TSLP expression in specific organs .

• Inducible expression systems: Employing tetracycline-regulated or tamoxifen-inducible systems to control the timing and level of TSLP expression.

• Topical MC903 (calcipotriol): Application of vitamin D3 analog to mouse skin induces TSLP expression in keratinocytes, mimicking atopic dermatitis.

• Barrier disruption: Physical or chemical disruption of the skin barrier (e.g., tape-stripping or detergent treatment) can induce endogenous TSLP expression.

• Allergen exposure: Sensitization and challenge with allergens like house dust mite extract or ovalbumin can trigger TSLP expression in epithelial tissues.

What are the key phenotypic differences between skin-specific and lung-specific TSLP transgenic mice?

The phenotypic differences between skin-specific and lung-specific TSLP transgenic mice include:

• Skin-specific K14-TSLPtg mice:

  • On inbred backgrounds: Develop spontaneous AD-like skin inflammation with epidermal thickening, dermal infiltration by mast cells and eosinophils, and elevated serum IgE levels .

  • On outbred backgrounds: Maintain high serum TSLP levels without developing skin pathology, but remain susceptible to asthma-like responses upon allergen challenge .

  • Demonstrate progression from skin inflammation to airway hyperresponsiveness (the "atopic march") .

• Lung-specific SPC-TSLP mice:

  • Develop a progressive asthma-like disease characterized by airway inflammation .

  • Require antigenic stimulation for full disease development, as TSLP alone causes only a weak innate response insufficient for complete airway inflammatory disease .

  • Do not develop skin inflammation, as TSLP expression is confined to the lung .

How does skin-derived TSLP contribute to lung inflammation in mouse models?

Skin-derived TSLP can enter the systemic circulation and sensitize the lung airways to inhaled allergens, even in the absence of direct lung TSLP expression. This mechanism has been demonstrated in both barrier-defective mice and K14-TSLPtg mice on an outbred background . These mice maintain high systemic TSLP levels and develop severe allergic lung inflammation when challenged with allergens like ovalbumin, despite the absence of detectable TSLP in their bronchoalveolar lavage fluid . Importantly, this occurs without requiring concurrent skin lesions, supporting the model that systemic TSLP is sufficient to predispose mice to allergic lung inflammation .

What experimental protocols are used to study the atopic march in TSLP mouse models?

Researchers use several experimental protocols to study the atopic march:

• Two-phase sensitization and challenge:

  • First phase: Induction of skin barrier defects or TSLP overexpression in the skin.

  • Second phase: Intranasal or aerosol challenge with allergens like ovalbumin (OVA) without prior sensitization .

• Measurement of airway hyperresponsiveness:

  • Pulmonary function tests to assess airway resistance in response to methacholine.

  • Bronchoalveolar lavage (BAL) to analyze inflammatory cell infiltration.

  • Histological examination of lung tissue for inflammatory changes.

• Analysis of systemic sensitization:

  • Measurement of serum TSLP levels.

  • Quantification of allergen-specific IgE and IgG1 antibodies.

  • Assessment of cytokine profiles in draining lymph nodes.

• Intervention studies:

  • Blocking TSLP signaling with neutralizing antibodies or receptor antagonists.

  • Genetic ablation of TSLP receptors to determine the requirement for TSLP in the atopic march.

What are the advantages and limitations of using TSLP mouse models for atopic march research?

Advantages:

• Recapitulate key aspects of human atopic march, including progression from skin inflammation to airway hyperresponsiveness.

• Allow for genetic manipulation to isolate specific pathways and mechanisms.

• Enable temporal control of TSLP expression and allergen exposure.

• Provide systems to test therapeutic interventions targeting TSLP or its downstream pathways.

• Allow for distinction between local and systemic effects of TSLP.

Limitations:

• Mouse skin differs from human skin in structure and immune composition.

• The time course of atopic march development is accelerated in mouse models compared to humans.

• Some transgenic models may have supraphysiological TSLP levels that don't accurately reflect human disease conditions.

• Genetic background influences disease development, requiring careful consideration of strain selection.

• Most models require artificial allergen sensitization and challenge protocols that may not fully recapitulate natural environmental exposures in humans.

What are the best methods for detecting TSLP expression in mouse tissues?

Several complementary methods are recommended for reliable detection of TSLP expression:

• RT-PCR and qPCR: For detection of TSLP mRNA in tissues and isolated cells. This has been successfully applied to detect TSLP transcripts in mouse and human DRG using RT-PCR .

• ELISA: For quantification of TSLP protein in serum and bronchoalveolar lavage fluid. This is crucial for monitoring systemic TSLP levels in transgenic models .

• Immunohistochemistry: For localization of TSLP protein in tissue sections, though careful antibody validation is essential due to potential cross-reactivity issues .

• In situ hybridization: For visualization of TSLP mRNA expression patterns in intact tissues.

• Reporter mice: TSLP-reporter mouse strains expressing fluorescent proteins under the control of the TSLP promoter can be useful for tracking TSLP-expressing cells in vivo.

• Western blotting: For semi-quantitative assessment of TSLP protein in tissue lysates, though sensitivity may be limited for endogenous expression levels.

How can researchers distinguish between direct and indirect effects of TSLP in mouse models?

Distinguishing direct from indirect TSLP effects requires multiple complementary approaches:

• Cell-type specific receptor knockouts: Delete TSLP receptors from specific cell populations (e.g., neurons, T cells, dendritic cells) to determine which cells are directly responding to TSLP.

• Bone marrow chimeras: Transplant bone marrow from TSLP receptor-deficient mice into wild-type recipients (or vice versa) to separate hematopoietic from non-hematopoietic TSLP responses.

• Ex vivo tissue or cell preparations: Apply TSLP directly to isolated cells or tissues to observe immediate responses independent of secondary mediators.

• Temporal analysis: Early responses (minutes to hours) are more likely to represent direct TSLP effects, while late responses (days) may involve multiple cellular intermediates and secondary mediators .

• Co-localization studies: Perform immunohistochemistry to demonstrate co-expression of TSLP receptors with cell-type specific markers .

• Blocking secondary mediators: Use neutralizing antibodies or antagonists against potential intermediate cytokines or lipid mediators to determine their contribution to TSLP-induced phenotypes.

What controls are essential when conducting experiments with TSLP mouse models?

Essential controls for TSLP mouse research include:

• Genetic background controls: Use age-matched, sex-matched wild-type littermates of the same genetic background as transgenic or knockout mice.

• Receptor component knockouts: Include IL7Rα-deficient and/or TSLPR-deficient mice to confirm specificity of TSLP-dependent phenotypes .

• Vehicle controls: For TSLP injection studies, include appropriate vehicle-injected controls to account for mechanical stimulation effects .

• Negative control tissues: For tissue-specific TSLP transgenic models, analyze tissues not expected to express the transgene (e.g., lung in K14-TSLPtg mice) to confirm specificity of expression .

• Dose-response studies: Test multiple concentrations of TSLP to establish dose-dependent effects and physiologically relevant concentrations.

• Time course experiments: Evaluate phenotypes at multiple time points to distinguish between acute and chronic TSLP effects.

• Immune-deficient controls: Use RAG1-/-, NOD SCID (lacking T and B cells), or Kit(W-sh) mice (lacking mast cells) to determine the requirement for specific immune cell populations in TSLP-induced phenotypes .