IL33: A key cytokine in inflammation and its therapeutic intervention

• Gene Information: Interleukin 33 (IL33) is a protein-coding gene located on chromosome 9p24.1. It encodes an alarmin cytokine that belongs to the interleukin-1 (IL-1) family.
• Protein Expression: IL-33 is constitutively expressed in the nuclei of endothelial cells, epithelial cells (lung, gut), and skin keratinocytes. It acts as an intracellular nuclear factor under steady state but is released as a full-length active cytokine upon cell damage or mechanical stress.
• Signaling Pathway: Extracellular IL-33 exerts its effects by binding to a heterodimeric receptor complex consisting of the specific receptor ST2 (also known as IL-1RL1) and the co-receptor IL-1 receptor accessory protein (IL-1RAcP).
• Therapeutic Inhibition: By blocking the IL-33/ST2 signaling pathway, targeted therapeutics (such as anti-IL-33 or anti-ST2 monoclonal antibodies) inhibit downstream type 2 and non-type 2 inflammation, leading to reduced tissue eosinophils, lowered airway hyperresponsiveness, and decreased secretion of pro-inflammatory 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.

IL33

•  Exons 2-8 of mouse IL33 gene that encode the whole molecule (ATG to STOP codon) were replaced by human counterparts in B-hIL33/hTSLP/hTSLPR plus Mice.
• The promoter, 5’UTR and 3’UTR region of the mouse gene are retained. allowing human IL33 expression to be driven by the native mouse IL33 promoter, while endogenousmouse IL33 transcription and translation are abolished.

 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.

IL7R

• Exons 1-6 of mouse Il7r gene that encode extracellular domain are replaced by human counterparts in B-hIL4/hIL4RA/hTSLP/hTSLPR plus mice. 
• The promoter and 5’UTR region of the mouse gene are retained. The human IL7R expression is driven by endogenous mouse IL7R promoter, while mouse IL7R gene transcription and translation will be disrupted.
• B-hIL33/hTSLP/hTSLPR plus mice were obtained by breeding B-hTSLP/hTSLPR mice plus with B-hIL33 mice.

Strain-specific IL33 expression evaluated by ELISA in wild-type C57BL/6 mice and homozygous B-hIL33/hTSLP/hTSLPR plus mice. Lung tissue homogenate were collected from wild-type mice and homozygous B-hIL33/hTSLP/hTSLPR plus mice (female, 6-week-old, n = 3). Mouse and human IL33 levels in lung tissue homogenates were quantified by ELISA (mouse IL33, R&D Systems M3300; human IL33, R&D Systems D3300B).

Strain-specific TSLP expression evaluated by ELISA in wild-type C57BL/6 mice and homozygous B-hIL33/hTSLP/hTSLPR plus mice. Calcipotriol (MC903), dissolved in ethanol, was topically applied to the ears of wild-type C57BL/6 mice and homozygous B-hIL33/hTSLP/hTSLPR plus 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 and human TSLPR expression analysis in bone marrow.Bone marrow cells were collected from wild-type C57BL/6 mice and homozygous B-hIL33/hTSLP/hTSLPR plus mice. TSLPR expressionon BMDCs was analyzed by flow cytometry using anti-mouse TSLPR antibody (Biolegend, 300312) and anti-human TSLP Rantibody (Biolegend, 100312).

Mouse and human IL7R expression analysis in spleen.Splenocytes were collected from wild-type C57BL/6 mice and homozygous B-hIL33/hTSLP/hTSLPR plus mice. IL7R expression on splenocytes was analyzed by flow cytometry using anti-mouse IL7R antibody (Biolegend, 135011) and anti-human IL7R antibody (Biolegend, 351303).

Mouse and human IL7R expression analysis in spleen. Splenocytes were collected from wild-type C57BL/6 mice and homozygous B-hIL33/hTSLP/hTSLPR plus mice. IL7R expression on splenocytes was analyzed by flow cytometry using anti-mouse IL7R antibody (Biolegend, 135011) and anti-human IL7R antibody (Biolegend, 351303).

Experimental schedule for the induction of asthma and in vivo efficacy of anti-human IL33 antibody, anti-human TSLP antibody and IL33/TSLP BsAb (the antibodies are provided by a client) in B-hIL33/hTSLP/hTSLPR plus mice. In the OVA etc.-induced model, animals are administered OVA etc. intranasally to induce asthma models. The anti-human IL33 antibody, anti-human TSLP antibody and IL33/TSLP BsAb were administered by intraperitoneal injection (n = 6).

Analysis of immune cells in BALF. B-hIL33/hTSLP/hTSLPR plus mice (female, 11-week-old, n = 6) were immunized with OVA, etc., to induce asthma. Anti-human IL33 antibody, anti-human TSLP antibody, and IL33/TSLP BsAbs (antibodies provided by a client) were intraperitoneally injected into B-hIL33/hTSLP/hTSLPR plus mice. (A, B) The number of CD45⁺ cells and eosinophils in the BALF of the Itepekimab-treated group, the Itepekimab and Tezepelumab combination therapy group, and the IL33/TSLP BsAb-treated group decreased significantly compared with the OVA, etc.-induced PBS-treated group. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test. *P < 0.05, P < 0.01, ***P < 0.001.

Analysis of mouse total IgE in serum. B-hIL33/hTSLP/hTSLPR plus mice (female, 11-week-old, n = 6) were immunized with OVA, etc., to induce asthma. Anti-human IL33 antibody, anti-human TSLP antibody, and IL33/TSLP BsAbs (antibodies provided by a client) were intraperitoneally injected into B-hIL33/hTSLP/hTSLPR plus mice. Serum was collected at the study endpoint, and IgE levels were analyzed by ELISA. The results showed that the levels of total IgE in the Itepekimab-treated group, the Itepekimab and Tezepelumab combination therapy group, and the IL33/TSLP BsAb-treated group decreased significantly compared with untreated mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test. *P < 0.05, P < 0.01, ***P < 0.001.

Analysis of mouse TARC in BALF and lung homogenates. B-hIL33/hTSLP/hTSLPR plus mice (female, 11-week-old, n = 6) were immunized with OVA, etc., to induce asthma. Anti-human IL33 antibody, anti-human TSLP antibody, and IL33/TSLP BsAbs (antibodies provided by a client) were intraperitoneally injected into B-hIL33/hTSLP/hTSLPR plus mice. BALF and lung homogenates were collected at the study endpoint, and TARC levels were analyzed by ELISA. The results showed that mouse TARC levels in the Itepekimab-treated group, the Itepekimab and Tezepelumab combination therapy group, and the IL33/TSLP BsAb-treated group were reduced compared with untreated mice. However, no reduction in TARC was observed in the TSLP antibody-treated group. We speculate that this is because even if TSLP signaling is pharmacologically blocked, persistent cellular necrosis and IL-33 release in the tissue can still sustain DC secretion of TARC through the MyD88/NF-κB pathway. This is also why combined or multi-targeted inhibition (e.g., simultaneously targeting TSLP, IL-33, and IL-4Rα) is emerging as a key breakthrough direction in the clinical treatment of some severe type 2 inflammatory conditions. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test. *P < 0.05, P < 0.01, ***P < 0.001.

H&E staining of an asthma-like model in B-hIL33/hTSLP/hTSLPR plus mice. Lung tissues were collected at the study endpoint and analyzed by H&E staining. The results showed that inflammatory and eosinophil infiltration in the lung tissues of the Itepekimab-treated group, the Itepekimab and Tezepelumab combination therapy group, and the IL33/TSLP BsAb-treated group were reduced compared with those in untreated mice, indicating that B-hIL33/hTSLP/hTSLPR plus mice provide a powerful preclinical model for the in vivo evaluation of anti-human IL33 and anti-human TSLP antibodies. Values are expressed as mean ± SEM. Significance was determined by unpaired t-test. *P < 0.05, P < 0.01, ***P < 0.001.