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 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 endogenous mouse 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.

• Mouse IL33 was detected exclusively in wild-type C57BL/6 mice
• Human IL33 was detected in homozygous B-hIL33/hTSLP/hTSLPR mice, but not in wild-type mice.

Strain-specific IL33 expression evaluated by ELISA in wild-type C57BL/6 mice and homozygous B-hIL33/hTSLP/hTSLPR mice. Calcipotriol (MC903), dissolved in ethanol, was topically applied to the ears of wild-type C57BL/6 mice and homozygous B-hIL33/hTSLP/hTSLPR mice for 7 days (male, 6-week-old, n = 3). Mouse and human IL33 levels in ear tissue homogenates were quantified by ELISA (mouse IL33, R&D Systems M3300; human IL33, R&D Systems D3300B).

• Mouse TSLP was detected exclusively in wild-type C57BL/6 mice
• Human TSLP was detected in homozygous B-hIL33/hTSLP/hTSLPR mice, but not in wild-type mice.

Strain-specific TSLP expression evaluated by ELISA in wild-type C57BL/6 mice and homozygous B-hIL33/hTSLP/hTSLPR mice. Calcipotriol (MC903), dissolved in ethanol, was topically applied to the ears of wild-type C57BL/6 mice and homozygous B-hIL33/hTSLP/hTSLPR mice for 7 days (male, 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/6 mice, but not in homozygous B-hIL33/hTSLP/hTSLPR mice.
• Human TSLPR was exclusively detectable in homozygous B-hIL33/hTSLP/hTSLPR mice, but not in wild-type C57BL/6 mice.

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 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).

• The percentages of T cells, B cells, NK cells, DCs, neutrophils, monocytes, and macrophages in homozygous B-hIL33/hTSLP/hTSLPR mice were similar to those in C57BL/6JNifdc.
• Humanization of IL33, TSLP and TSLPR does not affect normal immune cell development or splenic distribution.

Analysis of leukocyte subpopulations by flow cytometry in immune organs and blood. Splenocytes, peripheral blood, and lymph nodes were isolated from female C57BL/6JNifdc and B-hIL33/hTSLP/hTSLPR mice (female, 9-week-old, n = 3). Single live cells were gated on the CD45⁺ population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

• The proportions of CD4⁺ T cells, CD8⁺ T cells, and Tregs in homozygous B-hIL33/hTSLP/hTSLPR mice were comparable to those in C57BL/6JNifdc.
• Humanization of TSLP, TSLPR, and IL7R does not affect normal T cell development, differentiation, or splenic distribution.

Analysis of T-cell subpopulations by flow cytometry in immune organs and blood. Splenocytes, peripheral blood, and lymph nodes were isolated from female C57BL/6JNifdc and B-hIL33/hTSLP/hTSLPR mice (female, 9-week-old, n = 3). Single live cells were gated on the CD3⁺ T-cell population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

Growth curve of B-hIL33/hTSLP/hTSLPR mice. Eight-week-old mice were grouped by sex (10 males and 10 females). Body weight was measured weekly for 12 weeks on the same day each week. The minimum and maximum body weights shown in the table were calculated from the mean ± SD.

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

Complete blood count (CBC) of B-hIL33/hTSLP/hTSLPR mice. Values are expressed as mean ± SD.

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

Complete blood count (CBC) of B-hIL33/hTSLP/hTSLPR mice. Values are expressed as mean ± SD.

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

Complete blood count (CBC) of B-hIL33/hTSLP/hTSLPR mice. Values are expressed as mean ± SD.

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

Complete blood count (CBC) of B-hIL33/hTSLP/hTSLPR mice. Values are expressed as mean ± SD.

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

Complete blood count (CBC) of B-hIL33/hTSLP/hTSLPR mice. Values are expressed as mean ± SD.

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

Complete blood count (CBC) of B-hIL33/hTSLP/hTSLPR mice. Values are expressed as mean ± SD.

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

Complete blood count (CBC) of B-hIL33/hTSLP/hTSLPR mice. Values are expressed as mean ± SD.

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

Blood biochemical parameters of B-hIL33/hTSLP/hTSLPR mice are shown. Values are expressed as mean ± SD.

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

Blood biochemical parameters of B-hIL33/hTSLP/hTSLPR mice are shown. Values are expressed as mean ± SD.

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

Blood biochemical parameters of B-hIL33/hTSLP/hTSLPR mice are shown. Values are expressed as mean ± SD.

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

Blood biochemical parameters of B-hIL33/hTSLP/hTSLPR mice are shown. Values are expressed as mean ± SD.

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

Blood biochemical parameters of B-hIL33/hTSLP/hTSLPR mice are shown. Values are expressed as mean ± SD.

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

Blood biochemical parameters of B-hIL33/hTSLP/hTSLPR mice are shown. Values are expressed as mean ± SD.

• No abnormalities were observed.

The organs of female C57BL/6JNifdc and B-hIL33/hTSLP/hTSLPR mice (32-week-old, n=10).

• No abnormalities were observed.

The organs of male C57BL/6JNifdc and B-hIL33/hTSLP/hTSLPR mice (32-week-old, n=10).

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

Histopathological analysis of organs in B-hIL33/hTSLP/hTSLPR mice. Major organs from B-hIL33/hTSLP/hTSLPR mice were collected at 32 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, testis).

Histopathological analysis of organs in B-hIL33/hTSLP/hTSLPR mice. Major organs from B-hIL33/hTSLP/hTSLPR mice were collected at 32 weeks of age and analyzed by H&E staining (male, n = 10).

Experimental schedule for the induction of asthma and in vivo efficacy of anti-human IL33 antibody and anti-human TSLP antibody in B-hIL33/hTSLP/hTSLPR mice. In the OVA etc.-induced model, animals are administered OVA etc. intranasally to induce asthma models. The anti-human IL33 antibody itepekimab analog and anti-human TSLP antibody Tezepelumab analog (in-house) was administered by intraperitoneal injection (n=8).

• CD45⁺ cells and eosinophils were significantly reduced in the anti–human IL33 antibody and the combination therapy group of itepekimab and tezepelumab treated groups decreased significantly compared with the OVA etc.-induced PBS treated group.

Analysis of immune cells in BALF. B-hIL33/hTSLP/hTSLPR mice (male, 11-week-old, n=8) were immunized with OVA etc. to induce asthma. Anti-human  IL33 antibody (Itepekimab analog, synthesized in-house) and anti-human TSLP antibody (Tezepelumab analog, synthesized in-house) were intraperitoneally injected to B-hIL33/hTSLP/hTSLPR mice. (A&B) The number of CD45+ cells and eosinophils of BALF in the Itepekimab and the combination therapy group of Itepekimab and Tezepelumab treated groups decreased significantly compared with the OVA etc.-induced PBS treated group. Values were expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***P < 0.001. 

• Itepekimab, tezepelumab or the combination therapy group of itepekimab and tezepelumab treated groups showed a significant reduction compared with untreated mice.

Analysis of mouse total IgE in serum. B-hIL33/hTSLP/hTSLPR mice (male, 11-week-old, n=8) were immunized with OVA etc.to induce asthma. Anti-human  IL33 antibody (Itepekimab analog, synthesized in-house) and anti-human TSLP antiboday (Tezepelumab analog, synthesized in-house) were intraperitoneally injected to B-hIL33/hTSLP/hTSLPR mice. Serum was collected at the study endpoint. IgE level was analyzed by ELISA. The results showed that the levels of total IgE in mice treated with Itepekimab, Tezepelumab or the combination therapy group of Itepekimab and Tezepelumab treated groups showed a significant reduction compared with untreated mice. Values were expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***P < 0.001. 

• Itepekimab and the combination therapy group of Itepekimab and Tezepelumab in inflammatory infiltration and mucus secretion in lung tissue was lower than that in untreated mice

H&E staining of asthma-like model in B-hIL33/hTSLP/hTSLPR mice. Lung tissues were collected at the study endpoint and analyzed with H&E staining. The results showed that the group of mice treated with Itepekimab and the combination therapy group of Itepekimab and Tezepelumab in inflammatory infiltration and mucus secretion in lung tissue was lower than that in untreated mice, indicating that B-hIL33/hTSLP/hTSLPR mice provided a powerful preclinical model for in vivo evaluation of anti-human IL33 antibodies 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.