Description
TSLP: A key cytokine in inflammation and its therapeutic intervention
- Gene Information: Thymic stromal lymphopoietin (TSLP) is a protein-coding gene located on chromosome 5q22.1. It encodes a hemopoietic cytokine that is a member of the interleukin 7-like cytokine family.
- Protein Expression: TSLP is primarily expressed by activated epithelial cells (lung, gut), skin keratinocytes, and fibroblasts. Two main isoforms exist: the short form (sfTSLP) is constitutively expressed and plays a homeostatic role, while the long form (lfTSLP) is induced during inflammation.
- Signaling Pathway: TSLP exerts its effects by binding to a high-affinity heterodimeric receptor complex composed of the TSLP receptor chain (TSLPR) and the IL-7 receptor alpha chain (IL-7Rα).
- Therapeutic Inhibition: By blocking TSLP binding to its receptor, tezepelumab inhibits downstream inflammation and improves clinical outcomes, including reduced serum IgE levels, decreased airway eosinophils, reduced mucus production, and lowered cytokine secretion.
IL-4/IL-4Rα: The core signaling pathway in type 2 inflammation and therapeutic intervention
- Gene Information: Interleukin-4 is a cytokine encoded by the IL4 gene, located on chromosome 5q31.1. It belongs to the four-helix bundle cytokine family. Interleukin-4 receptor subunit alpha is a transmembrane protein encoded by the IL4R gene, located on chromosome 16p12.1. It serves as the essential, high-affinity binding chain for both IL-4 and IL-13 signaling.
- Protein Expression: IL-4 is primarily secreted by activated immune cells driving allergic responses, including Th2 cells, mast cells, basophils, and eosinophils. IL-4Rα is widely and constitutively expressed across a diverse range of cell types. It is found on hematopoietic cells (B cells, T cells, macrophages) as well as non-hematopoietic cells (airway epithelial cells, smooth muscle cells, and fibroblasts), making it a ubiquitous sensor for type 2 inflammation.
- Signaling Pathway: Type I Receptor Complex (IL-4 Specific): Formed by the assembly of IL-4Rα and the common gamma chain (γc). This complex is predominantly expressed on hematopoietic cells. Type II Receptor Complex (IL-4/IL-13 Shared): Formed by the assembly of IL-4Rα and IL-13Rα1. This complex is widely expressed on non-hematopoietic barrier tissues. When IL-4 binds to IL-4Rα, it recruits either γc or IL-13Rα1, cross-activating downstream JAK1/JAK3 (Type I) or JAK1/TYK2 (Type II) kinases. This triggers the phosphorylation, dimerization, and nuclear translocation of STAT6 to drive the transcription of pro-inflammatory genes.
- Therapeutic Inhibition: Because IL-4Rα is the shared and indispensable subunit for both IL-4 and IL-13 signaling, it represents a master therapeutic target. By utilizing monoclonal antibodies such as dupilumab to specifically block IL-4Rα, therapies can simultaneously shut down both Type I and Type II receptor signaling. This dual inhibition effectively halts downstream type 2 inflammation, leading to profound clinical improvements, including: reduced serum IgE levels (by blocking B-cell class switching), decreased tissue eosinophilia (by suppressing eotaxin and VCAM-1 expression), diminished airway remodeling and mucus hypersecretion, and lowered systemic secretion of downstream type 2 cytokines.
IL-5/IL-5Rα: A key cytokine-receptor axis in eosinophilic inflammation and its therapeutic intervention
- Gene Information: Interleukin 5 (IL5) is a protein-coding gene located on chromosome 5q31.1, encoding a cytokine that acts as a primary growth and differentiation factor for eosinophils. Its specific receptor alpha subunit, IL5RA (CD125), is encoded by a gene situated on chromosome 3p26.2.
- Protein Expression: IL-5 is predominantly produced and secreted as a disulfide-linked homodimeric glycoprotein by activated T helper 2 cells, mast cells, and type 2 innate lymphoid cells (ILC2s). IL-5Rα is selectively and highly expressed on the surface of eosinophils, basophils, and their bone marrow progenitor cells.
- Signaling Pathway: IL-5 exerts its biological effects by binding with high affinity to the extracellular domain of IL-5Rα. This interaction recruits the shared signal-transducing common beta subunit to form a functional heterodimeric receptor complex, which subsequently activates the downstream intracellular JAK2/STAT5 and MAPK signaling cascades to promote eosinophil survival, maturation, and activation.
- Therapeutic Inhibition: By blocking this cytokine-receptor axis, targeted therapeutics inhibit downstream eosinophilic inflammation and improve clinical outcomes. Anti-IL-5 monoclonal antibodies (such as mepolizumab or reslizumab) neutralize the soluble ligand, while anti-IL-5Rα antibodies (such as benralizumab) target the receptor to induce antibody-dependent cell-mediated cytotoxicity (ADCC), drastically lowering airway and blood eosinophil counts in patients with severe eosinophilic asthma.
Targeting Strategy
TSLP
- Exons 1-5 of the mouse Tslp gene, which encode the entire protein (from ATG to stop codon), are replaced with the corresponding human sequences.
- The endogenous mouse promoter, 5′ UTR, and 3′ UTR regions are retained, allowing human TSLP expression to be driven by the native mouse Tslp promoter, while endogenous mouse Tslp transcription and translation are abolished.
TSLPR
- A chimeric CDS encoding the human TSLPR extracellular and transmembrane domains fused to the mouse TSLPR cytoplasmic domain, followed by the mouse 3′ UTR and stop codon, is inserted immediately downstream of the mouse Tslpr signal peptide to replace part of exon 2 of the endogenous Tslpr gene.
- Expression of the chimeric TSLPR protein is driven by the native mouse Tslpr promoter, while endogenous mouse Tslpr transcription and translation are disrupted.
IL4
- Exons 1-4 of the mouse Il4 gene, which encode the full coding sequences of human IL4 gene, including the promoter, 5’UTR and 3’UTR, are replaced with the corresponding human sequences.
- The human promoter, 5′ UTR, and 3′ UTR regions are replaced, allowing human IL4 expression to be driven by the human IL4 promoter, while endogenous mouse Il4 transcription and translation areabolished.
IL4RA
- Exons 4-7 of mouse Il4ra gene that encode extracellular domain are replaced by human counterparts in B- hTSLP/hTSLPR/hIL4/hIL4RA/hIL5/hIL5RA mice.
- The promoter, 5’UTR, and 3’UTR region of the mouse gene are retained. The human IL4RA expression is driven by endogenous mouse Il4ra promoter, while mouse Il4ra gene transcription and translation will be disrupted.
IL5
- Exons 1-4, including 3’UTR of the mouse IL5 gene that encodes the full length protein, are replaced with the corresponding human sequences.
- The endogenous mouse promoter and 5′ UTR regions are retained, allowing human IL5 expression to be driven by the native mouse Il5 promoter, while endogenous mouse Il5 transcription andtranslation are abolished.
IL5RA
- A chimeric CDS encoding the human IL5RA extracellular domain fused to the mouse IL5RA transmembrane and cytoplasmic domain, followed by the mouse 3′ UTR and stop codon, is insertedimmediately downstream of the mouse Il5 signal peptide to replace part of exon 5 of the endogenous Il5r gene.
- Expression of the chimeric IL5RA protein is driven by the native mouse Il5ra promoter, while endogenous mouse Il5ra transcription and translation are disrupted.B-hTSLP/hTSLPR/hIL4/hIL4RA/hIL5/hIL5RA mice were obtained by mating B-hTSLP/hTSLPR mice(121269) with B hIL4/hIL4RA/hIL5/hIL5RA mice (112844).
TSLP Protein Expression Analysis in Ear Homogenates
Strain-specific TSLP expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hTSLP/hTSLPR/hIL4/hIL4RA/hIL5/hIL5RA mice by ELISA. Ear tissues were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTSLP/hTSLPR/hIL4/hIL4RA/hIL5/hIL5RA mice (H/H;H/H;H/H;H/H;H/H;H/H) (female, 8-week-old, n = 3) following topical application of MC903 (0.2 mmol/L, 0.2 mL) to the ears for 6 days. The expression levels of mouse and human TSLP were analyzed by ELISA (mouse TSLP ELISA kit: BioLegend, 434107; human TSLP ELISA kit: BioLegend, 434207). Mouse TSLP was exclusively detectable in wild-type C57BL/6JNifdc mice but not in homozygous B-hTSLP/hTSLPR/hIL4/hIL4RA/hIL5/hIL5RA mice. Human TSLP was exclusively detectable in homozygous B-hTSLP/hTSLPR/hIL4/hIL4RA/hIL5/hIL5RA mice but not in wild-type C57BL/6JNifdc mice. Values are expressed as mean ± SEM.
IL4 Protein Expression Analysis in Serum
Strain-specific IL-4 expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hTSLP/hTSLPR/hIL4/hIL4RA/hIL5/hIL5RA mice by ELISA. Serum was collected from wild-type C57BL/6JNifdc mice and homozygous B-hTSLP/hTSLPR/hIL4/hIL4RA/hIL5/hIL5RA mice (male, 8-week-old, n = 3) stimulated with anti-mCD3ε (BioXCell, BE0001-1) in vivo for 2 hours. The expression levels of IL-4 were analyzed by ELISA (mouse IL-4 ELISA kit: BioLegend, 431104). Mouse IL-4 was exclusively detectable in wild-type C57BL/6JNifdc mice but not in homozygous B-hTSLP/hTSLPR/hIL4/hIL4RA/hIL5/hIL5RA mice. Human IL-4 was exclusively detectable in homozygous B-hTSLP/hTSLPR/hIL4/hIL4RA/hIL5/hIL5RA mice but not in wild-type C57BL/6JNifdc mice. Values are expressed as mean ± SEM.
TSLPR Protein Expression Analysis in BMDC
Strain-specific TSLPR expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hTSLP/hTSLPR/hIL4/hIL4RA/hIL5/hIL5RA mice by flow cytometry. Bone marrow-derived dendritic cells were produced by culturing bone marrow from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTSLP/hTSLPR/hIL4/hIL4RA/hIL5/hIL5RA mice (H/H;H/H;H/H;H/H;H/H;H/H). Protein expression was analyzed with anti-mouse TSLPR antibody (BioLegend, 151805) and anti-human TSLPR antibody (BioLegend, 322805) by flow cytometry. Mouse TSLPR was exclusively detectable in wild-type C57BL/6JNifdc mice. Human TSLPR was exclusively detectable in homozygous B-hTSLP/hTSLPR/hIL4/hIL4RA/hIL5/hIL5RA mice.
IL5RA Protein Expression Analysis in Bone Marrow
Strain-specific IL5RA expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hTSLP/hTSLPR/hIL4/hIL4RA/hIL5/hIL5RA mice by flow cytometry. Bone marrow was collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTSLP/hTSLPR/hIL4/hIL4RA/hIL5/hIL5RA mice (H/H;H/H;H/H;H/H;H/H;H/H) and analyzed by flow cytometry with species-specific anti-IL5RA antibodies. Mouse IL5RA was exclusively detectable in wild-type C57BL/6JNifdc mice but not in homozygous B-hTSLP/hTSLPR/hIL4/hIL4RA/hIL5/hIL5RA mice. Human IL5RA was exclusively detectable in homozygous B-hTSLP/hTSLPR/hIL4/hIL4RA/hIL5/hIL5RA mice but not in wild-type C57BL/6JNifdc mice.
IL5RA Protein Expression Analysis in Blood
Strain-specific IL5RA expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hTSLP/hTSLPR/hIL4/hIL4RA/hIL5/hIL5RA mice by flow cytometry. Blood was collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTSLP/hTSLPR/hIL4/hIL4RA/hIL5/hIL5RA mice (H/H;H/H;H/H;H/H;H/H;H/H) and analyzed by flow cytometry with species-specific anti-IL5RA antibodies. Mouse IL5RA was exclusively detectable in wild-type C57BL/6JNifdc mice but not in homozygous B-hTSLP/hTSLPR/hIL4/hIL4RA/hIL5/hIL5RA mice. Human IL5RA was exclusively detectable in homozygous B-hTSLP/hTSLPR/hIL4/hIL4RA/hIL5/hIL5RA mice but not in wild-type C57BL/6JNifdc mice.
* When publishing results obtained using this animal model, please acknowledge the source as follows: The animal model [B-hTSLP/hTSLPR/hIL4/hIL4RA/hIL5/hIL5RA mice] (Cat# 114037) was purchased from Biocytogen.
