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 Lowered systemic secretion of downstream type 2 cytokines.

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.

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 are abolished.

IL4RA

• Exons 4-7 of mouse IL4RA gene that encode extracellular domain are replaced by human counterparts in B-hIL4/hIL4RA/hTSLP/hTSLPR mice.
• The promoter and 5’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.

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.

• Mouse IL4 was detected exclusively in wild-type C57BL/6JNifdc mice
• Human IL4 was detected in homozygous B-hIL4/hIL4RA/hTSLP/hTSLPR mice, but not in wild-type mice.

Strain-specific IL4 expression evaluated by ELISA in wild-type C57BL/6JNifdc mice and homozygous B-hIL4/hIL4RA/hTSLP/hTSLPR mice. Serum was collected from wild-type C57BL/6JNifdc mice (male, n=3, 9-week-old) and homozygous B-hIL4/hIL4RA/hTSLP/hTSLPR mice (male, n=3, 12-week-old) stimulated with anti-mCD3ε (BioXCell, BE0001-1) in vivo for 2 hrs. Expression level of mouse IL-4 and human IL-4 were analyzed by ELISA (Mouse IL-4 ELISA Kit : Biolegend, 431104; Human IL-4 ELISA Kit: Biolegend, 430304) .

• Mouse IL4RA was detected exclusively in wild-type C57BL/6JNifdc mice
• Human IL4RA was detected in homozygous B-hIL4/hIL4RA/hTSLP/hTSLPR mice, but not in wild-type mice.

Strain specific IL4RA expression evaluated in wild-type C57BL/6JNifdc mice and homozygous B-hIL4/hIL4RA/hTSLP/hTSLPR mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice(+/+) and homozygous B-hIL4/hIL4RA/hTSLP/hTSLPR mice (H/H; H/H; H/H;H/H) stimulated with anti-CD3ε in vivo for 24 hrs, and protein expression was analyzed with anti-mouse IL4RA antibody (Biolegend, 144804)  and anti-human IL4RA antibody (Biolegend, 355006) by flow cytometry.

• Mouse TSLP was detected exclusively in wild-type C57BL/6JNifdc mice
• Human TSLP was detected in homozygous B-hIL4/hIL4RA/hTSLP/hTSLPR mice, but not in wild-type mice.

Strain-specific TSLP expression evaluated by ELISA in wild-type C57BL/6JNifdc mice and homozygous B-hIL4/hIL4RA/hTSLP/hTSLPR mice. Calcipotriol (MC903), dissolved in ethanol, was topically applied to the ears of wild-type C57BL/6JNifdc mice, homozygous B-hIL4/hIL4RA/hTSLP/hTSLPR mice for 7 days (female, 6-week-old, n = 3). Mouse and human TSLP levels in ear tissue homogenates were quantified by ELISA (mouse TSLP, BioLegend 434107; human TSLP, BioLegend 434207).

• Mouse TSLPR was exclusively detectable in wild-type C57BL/6JNifdc mice, but not in homozygous B-hIL4/hIL4RA/hTSLP/hTSLPR mice.
• Human TSLPR was exclusively detectable in homozygous B-hIL4/hIL4RA/hTSLP/hTSLPR mice, but not in wild-type C57BL/6JNifdc mice.

Mouse and human TSLPR expression analysis in bone marrow. Bone marrow cells were collected from wild-type C57BL/6JNifdc mice and homozygous B-hIL4/hIL4RA/hTSLP/hTSLPR mice. TSLPR expression on bone marrow cells was analyzed by flow cytometry using anti-mouse TSLPR antibody (Biolegend, 300312) and anti-human TSLPR antibody (Biolegend, 100312).

• No significant differences were observed compared with wild-type mice.

Complete blood count (CBC) of B-hIL4/hIL4RA/hTSLP/hTSLPR mice. Values are expressed as mean ± SD.

• No significant differences were observed compared with wild-type mice.

Blood biochemical parameters of B-hIL4/hIL4RA/hTSLP/hTSLPR mice are shown. Values are expressed as mean ± SD.

• No abnormalities were observed.

Organs of female B-hIL4/hIL4RA/hTSLP/hTSLPR mice (8-week-old, n = 10).

• No abnormalities were observed.

Organs of male B-hIL4/hIL4RA/hTSLP/hTSLPR mice (8-week-old, n = 10).

• No abnormalities were observed.

Average weights of major organs in B-hIL4/hIL4RA/hTSLP/hTSLPR mice.

• No obvious abnormalities were observed in any organs examined (brain, heart, lung, liver, spleen, stomach, small intestine, colon, kidney, ovary, uterus , lymph node, thymus and bone marrow).

Histopathological analysis of organs in B-hIL4/hIL4RA/hTSLP/hTSLPR mice. Major organs from B-hIL4/hIL4RA/hTSLP/hTSLPR mice were collected at 8 weeks of age and analyzed by H&E staining (female, n = 10).

• No obvious abnormalities were observed in any organs examined (brain, heart, lung, liver, spleen, stomach, small intestine, colon, kidney, ovary, uterus , lymph node, thymus and bone marrow).

Histopathological analysis of organs in B-hIL4/hIL4RA/hTSLP/hTSLPR mice. Major organs from B-hIL4/hIL4RA/hTSLP/hTSLPR mice were collected at 8 weeks of age and analyzed by H&E staining (male, n = 10).