IL-17A: A key pro-inflammatory cytokine in autoimmune skin and joint diseases

• Gene Information: The IL17A gene (also known as CTLA8) is a protein-coding gene located on chromosome 6p12.2. This cytokine is the founding member of the IL-17 family and primarily signals through a heterodimeric receptor complex composed of IL-17RA and IL-17RC.
• Protein Expression: IL-17A is predominantly produced by specialized CD4+ T cells Th17 cells), as well as various innate immune cells, innate lymphoid cells (ILC3s), and neutrophils. It exists primarily as a secreted, disulfide-linked homodimer (IL-17A/A) or as a heterodimer with IL-17F (IL-17A/F).
• Signaling Pathway: Upstream IL-17A production is driven by the IL-23/Th17 axis. Downstream IL-17A binds to the IL-17RA/IL-17RC complex on target cells (e.g., keratinocytes), recruiting Act1 to activate NF-κB and MAPKs.
• Therapeutic Inhibition: Blocking IL-17A or its interaction with its receptors dramatically reduces inflammatory responses. Therapeutic monoclonal antibodies targeting this axis are highly effective and for autoimmune conditions such as plaque psoriasis, psoriatic arthritis, and ankylosing spondylitis.

IL17A

• The exons 1-3 of mouse Il17a gene that encode the extracellular domain were replaced by human IL17A exons 1-3 in B-hIL17A mice.
• The endogenous mouse promoter, 5′ UTR, and 3′ UTR regions are retained, allowing human IL17A expression to be driven by the native mouse Il17a promoter, while endogenous mouse Il17a transcription and translation are abolished.

• Mouse Il17a mRNA was detectable only in splenocytes of wild-type mice.
• Human IL17A mRNA was detectable only in homozygous B-hIL17A mice but not in wild-type mice.

Strain specific analysis of IL17A mRNA expression in C57BL/6 mice and homozygous B-hIL17A mice by RT-PCR.  Spleen RNA was isolated from C57BL/6 mice (+/+) and homozygous B-hIL17A mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human IL17A primers. Mouse Il17a mRNA was only detectable in wild-type mice. Human IL17A mRNA was exclusively detectable in homozygous B-hIL17A mice but not in wild-type mice.

• Mouse IL17A was detectable in wild-type mice. Human IL17A was detectable in homozygous B-hIL17A mice but not in wild-type mice.

Strain specific IL17A expression analysis in wild-type mice and B-hIL17A mice by ELISA. Serum was collected from C57BL/6 mice (+/+) and homozygous B-hIL17A mice (H/H) stimulated with anti-CD3ε (7.5 μg/mice, 2 hours, i.p.) and anti-CD28 (4 μg/mice, 2 hours, i.p.) in vivo, and analyzed by ELISA with species-specific IL17A ELISA kit. Mouse IL17A was detectable in WT mice. Human IL17A was detectable in homozygous B-hIL17A mice  but not in WT mice.

• Percent of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hIL17A mice were similar to those in the C57BL/6 mice.
• Humanization of IL17A does not affect normal immune cell development or distribution.

Analysis of leukocyte subpopulations by FACS. Splenocytes and peripheral blood were isolated from female C57BL/6 and B-hIL17A mice (n=3, 6 week-old). Flow cytometry analysis of the spleen and blood was performed to assess leukocyte subpopulations. Percent of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hIL17A mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hIL17A in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types. Values are expressed as mean ± SEM.

• Percent of CD8+ T cells, CD4+ T cells and Treg cells in homozygous B-hIL17A mice were similar to those in the C57BL/6 mice.
• Humanization of IL17A  does not change the overall development, differentiation or distribution of these T cell subtypes.

Analysis of spleen T cell subpopulations by FACS. Splenocytes and peripheral blood were isolated from female C57BL/6 and B-hIL17A mice (n=3, 6 week-old). Flow cytometry analysis of the spleen and blood was performed to assess leukocyte subpopulations. Percent of CD8+ T cells, CD4+ T cells and Treg cells in homozygous B-hIL17A mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hIL17A in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes. Values are expressed as mean ± SEM.

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

Complete blood count (CBC) of B-hIL17A mice. Values are expressed as mean ± SD.

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

Blood biochemical parameters of B-hIL17A mice are shown. Values are expressed as mean ± SD.

• After treatment of anti-hIL17A antibody, the phenotypical presentation level and erythema and scaling score of the back were much lower than the control in homozygous B-hIL17A mice.

IMQ-induced psoriasis model in B-hIL17A mice. Mice (female, 8 week-old, n=6) were scored daily for up to 5 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-analog (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.

• Therapeutic effects of secukinumab-analog (commercial drug) on IMQ-induced psoriasis model 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.

Dose dependent effects of antibodies on keratinocyte proliferation and inflammatory cell infiltration in IMQ-induced psoriasis model 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-analog (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.

Experimental schedule for Experimental Autoimmune Encephalomyelitis (EAE) in B-hIL17A mice. 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).

• B-hIL17A mice can be successfully used in the 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). MOG: myelin-oligodendrocyte glycoprotein; PTX: pertussis toxin. F: female; M: male.

• Infiltration of inflammatory cells in the MOG group was significantly increased, and the myelin protein was greatly reduced in B-hIL17A mice 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.

• 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 IFNγ+ T cells.

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 IFNγ. 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 IFNγ+ T cells.

• 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 IFNγ+ T cells.

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 IFNγ. 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 IFNγ+ T cells.

Experimental schedule for Experimental Autoimmune Encephalomyelitis (EAE) in B-hIL17A mice. 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). Anti-hIL17A antibody was applied at day 12 (i.p. QW×6).

• After treatment of anti-hIL17A antibody, the clinical score level was much lower than the control in homozygous 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.

Experimental schedule for IMQ-induced psoriasis model 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 6 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.

• After treatment of anti-hIL17A antibody, the phenotypical presentation level and erythema and scaling score of the back were much lower than the control in homozygous B-hIL17A mice.

IMQ-induced psoriasis model in B-hIL17A mice. 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-analog 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.

• Therapeutic effects of ixekizumab-analog on IMQ-induced psoriasis model 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.

Dose dependent effects of antibody on keratinocyte proliferation and inflammatory cell infiltration in IMQ-induced psoriasis model 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 on a scale from 0 to 11. (C) Epidermal thickness of the mice. Results indicated that therapeutic effects of ixekizumab-analog (in house) 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.

Experimental schedule for IMQ-induced psoriasis model in B-hIL17A mice. Mice at 8 week-old of age received a daily topical of commercially available IMQ cream on the shaved back for 6 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.