Basic Information
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Gene targeting strategy
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Gene targeting strategy for B-hIL17A mice. Exons 1-3 of mouse Il17a gene that encode the extracellular domain were replaced by human IL17A exons 1-3 in B-hIL17A mice.
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mRNA expression analysis
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Species-specific IL17A gene expression analysis in wild-type and humanized B-hIL17A mice by RT-PCR. Mouse Il17a mRNA was detected in splenocytes isolated from wild-type C57BL/6 (+/+) mice, while human IL17A mRNA was detected in homozygous B-hIL17A (H/H) mice.
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Protein expression analysis
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Species-specific IL17A protein expression analysis in wild–type and humanized B-hIL17A mice. Mouse IL17A protein was detected in wild-type mice, while human IL17A protein was detected in B-hIL17A mice.
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Analysis of leukocyte cell subpopulations in B-hIL17A mice
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Analysis of spleen leukocyte subpopulations. Splenocytes were isolated from wild-type and B-hIL17A mice (n=3) and analyzed by flow cytometry to assess leukocyte subpopulations as indicated. As shown, leukocyte subpopulations in homozygous B-hIL17A mice are similar to those in wild-type mice, indicating that development of T, NK, Monocyte, DC and macrophage cells are not affected by the knock-in of hIL17A.
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Analysis of T cell subpopulation in B-hIL17A mice
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Analysis of spleen T cell subpopulations. Spleen lymphocytes were isolated from wild-type C57BL/6 and B-hIL17A mice (n=3), and analyzed by flow cytometry to profile T cell subpopulations. As shown above, the T cell subpopulations in homozygous B-hIL17A mice are similar to those in wild-type mice, indicating that T cell development is not affected by the introduction of hIL17A.
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Blood tests
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Blood routine test
Complete blood counts (CBC). Blood from the C57BL/6 and B-hIL17Amice (n=3) was collected and analyzed. As shown above, there are no significant differences in terms of CBC between C57BL/6 and B-hIL17A mice.
Blood biochemical test
Blood biochemistry test. Serum ALT/AST levels of C57BL/6 and B-hIL17A mice were tested (n=3). Results indicate that there are no significant differences in ALT/AST levels between C57BL/6 and B-hIL17A mice.
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Disease modeling using B-hIL17A mice
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MS/EAE model
Schematic of EAE model
Experimental Autoimmune Encephalomyelitis (EAE) is an induced demyelinating disease model that closely resembles the progression and symptoms of the human neurological disease Multiple Sclerosis (MS).
Clinical score of EAE model
EAE induction by MOG35–55/CFA Emulsion PTX in 10-week-old B-hIL17A mice. Data are expressed as mean ± SEM from a typical experiment (n = 5 except n = 4 for B-hIL17A-F PBS). MOG: myelin-oligodendrocyte glycoprotein; PTX: pertussis toxin. F: female; M: male.
H&E staining and IHC staining in mouse EAE model
Local inflammation of the CNS in B-hIL17A mice (female，n=5) during EAE. On day 45 after MOG/CFA and PTx immunization, spinal cords were removed. The tissue sections were stained with H&E(A,B) and IHC (C,D)(Green, MBP; Blue, DAPI). The sections at the lumbar level are shown. The results showed that the infiltration of inflammatory cells in the MOG group was significantly increased, and the myelin protein was greatly reduced.
IL17A+ cells in lymph node of EAE model
IL-17 was primarily produced by CD4 Th17 cells during the development of EAE. To detect IL-17 production, cells from lymph node of B-hIL17A mice（female，n=5） immunized with MOG/CFA were stimulated for 6 hours by PMA and ionomycin in the presence of brefeldin A. IL-17-producing cells were analyzed by FACS, along with IFNg. The percentage of IL-17+CD3+CD4+ T cells in CD3+CD4+ T cells was increased in response to MOG immunization in B-hIL17A mice (left panel). So was percentage of IFNg+ T cells.
IL17A+ cells in CNS of EAE model
IL-17 was primarily produced by CD4 Th17 cells during the development of EAE. To detect IL-17 production, cells from CNS(Brain cell) of B-hIL17A mice（female，n=5） immunized with MOG/CFA were stimulated for 6 hours by PMA and ionomycin in the presence of brefeldin A. IL-17-producing cells were analyzed by FACS, along with IFNg. The percentage of IL-17+CD3+CD4+ T cells in CD3+CD4+ T cells was increased in response to MOG immunization in B-hIL17A mice (left panel). So was percentage of IFNg+ T cells.
hIL17A Ab efficacy evaluation（B-hIL17A mice）
hIL17A Ab efficacy evaluation（B-hIL17A mice）
EAE induction by MOG35–55/CFA Emulsion PTX in B-hIL17A mice. The clinical score and body weight was collected after the treatment of anti-hIL17A antibody. After treatment of anti-hIL17A antibody, the clinical score level was much lower than the control in homozygous B-hIL17A mice.Data are expressed as mean ± SEM from a typical experiment (n = 6). MOG: myelin-oligodendrocyte glycoprotein; PTX: pertussis toxin.
Imiquimod-Induced Psoriasis Model
IMQ-induced psoriasis model
Experimental schedule for induction of psoriasis-like skin lesions in B-hIL17A mice.
Mice at 10 week-old of age received a daily topical of commercially available IMQ cream on the shaved back for 8 consecutive days to induce psoriasis-like skin lesions. Severity of skin inflammation was daily scored and back skin was collected at the endpoint. IMQ: imiquimod.
IMQ-induced skin inflammation in B-hIL17A mice phenotypically resembles psoriasis.
Mice (female, 10 week-old, n=5) were scored daily for up to 6 days for body weight and clinical signs of skin inflammation following treatment with imiquimod (IMQ) cream. Mice in each group were treated with different dose of ixekizumab produced in house. Doses are shown in legend. (A) Phenotypical presentation of mouse back skin after 6 days of treatment. (B) Body weight changes during treatment. (C-D) Erythema and scaling score of the back was scored daily on a scale from 0 to 4. Additionally, the cumulative score (erythema plus scaling) is depicted. Values are expressed as mean ± SEM.
Mice (female, 8 week-old, n=6) were scored daily for up to 6 days for body weight and clinical signs of skin inflammation following treatment with imiquimod (IMQ) cream. Mice in each group were treated with different doses of Secukinumab (commercial drug). (A) Experimental schedule for induction of psoriasis-like skin lesions in B-hIL17A mice. (B) Phenotypical presentation of mouse back skin at day0 and day 5. (C) Body weight changes during treatment. (D-E) Erythema and scaling score of the back was scored daily. Additionally, the cumulative score (erythema plus scaling) is depicted. Values are expressed as mean ± SEM.
Dose dependent effects of antibodies on keratinocyte proliferation and inflammatory cell infiltration in IMQ induced psoriasis-like skin lesions in B-hIL17A mice. Back skin was collected at the endpoint and stained with Hematoxylin and eosin (H&E). (A) H&E staining of the back skin. (B) Histological changes were scored. (C) Epidermal thickness of the mice. Results indicated that therapeutic effects of Secukinumab (commercial drug) on psoriasis-like skin lesions in B-hIL17A mice were dose dependent, confirming that B-hIL17A mice provide a powerful model for in vivo evaluation of anti-human IL17A antibodies. Values are expressed as mean ± SEM.
