Gene targeting strategy for B-hIL36R/hIL1RACP mice. The chimeric IL1RL2 CDS was inserted after the 5’UTR of mouse Il1rl2 gene in B-hIL36R/hIL1RACP mice. The insertion disrupts the endogenous murine Il1rl2 gene, resulting in the absence of mouse transcripts. The chimeric IL1RAP CDS was inserted after the 5’UTR of mouse Il1rap gene in B-hIL36R/hIL1RACP mice. The insertion disrupts the endogenous murine Il1rap gene, resulting in the absence of mouse transcripts.

Strain specific analysis of IL36R gene expression in wild type (WT) mice, B-hIL36R mice and B-hIL36R/hIL1RACP mice by RT-PCR. Human IL36R mRNA was detectable only in lung of homozygous B-hIL36R mice (H/H) and homozygous B-hIL36R/hIL1RACP mice (H/H;H/H) but not in WT mice (+/+). The positive band was confirmed to be correct by sequencing.

Strain specific IL1RACP expression analysis in homozygous B-hIL36R/hIL1RACP mice by flow cytometry. Monocytes, macrophages and neutrophils were collected from wild type (WT) C57BL/6 mice (+/+) and homozygous B-hIL36R/hIL1RACP mice (H/H;H/H), and analyzed by flow cytometry with species-specific anti-hIL1RACP antibody. Human IL1RACP was exclusively detectable in homozygous B-hIL36R/hIL1RACP mice (H/H;H/H) but not in WT mice (+/+).

Homozygous B-hIL36R/hIL1RACP mice were induced ear skin inflammation with injection of human IL36g cytokine (2ug/25uL per dose) intradermal in ear skin, pre-treated with IL-36 pathway blocker Spesolimab i.v. (10 mg/kg), and ear skin thickness was measured. The results demonstrated that the human IL-36g cytokine significantly induced ear skin inflammation in B-hIL36R/hIL1RACP mice, characterized by ear skin thickening. Additionally, the IL-36 pathway blocker Spesolimab was effective in alleviating the skin inflammation. Values are expressed as mean ± SEM. The data was obtained from a partner.

Complete blood count (CBC). Blood from female C57BL/6 and B-hIL36R/hIL1RACP mice (n=8, 8 week-old) was collected and analyzed for CBC. There was no differences among any measurement between C57BL/6 and B-hIL36R/hIL1RACP mice, indicating that introduction of IL36R and IL1RACP in place of its mouse counterpart do not change blood cell composition and morphology. Values are expressed as mean ± SEM.

Blood biochemistry tests of B-hIL36R/hIL1RACP mice. Serum from the C57BL/6 and B-hIL36R/hIL1RACP mice (n=8, 8 week-old) was collected and analyzed for levels of ALT and AST. There was no differences on either measurement between C57BL/6 and B-hIL36R/hIL1RACP mice, indicating that introduction of IL36R and IL1RACP in place of its mouse counterpart do not change ALT and AST levels or health of liver. Values are expressed as mean ± SEM.