IL-17A/IL17F: Core pro-inflammatory cytokines driving autoimmune skin and joint diseases

• Gene Information: The IL17A and IL17F genes are both located on human chromosome 6 (6p12.2) and are highly adjacent to each other. They are the most structurally similar core members of the IL-17 family (sharing approximately 50% structural homology).
• Protein Expression: IL-17A and IL-17F are predominantly produced by specialized CD4+ T cells (Th17 cells), as well as various innate immune cells (such as innate lymphoid cells [ILC3s] and γδ T cells) and neutrophils. In vivo, they primarily exist and function as secreted, disulfide-linked homodimers (IL-17A/A or IL-17F/F) or as a heterodimer (IL-17A/F).
• Signaling Pathway: Driven by the upstream IL-23/Th17 axis, IL-17A and IL-17F bind to the IL-17RA/IL-17RC receptor complex on target cells. This activates NF-κB and MAPK pathways, triggering strong local inflammation.
• Therapeutic Inhibition: Because IL-17A and IL-17F drive inflammation synergistically, simultaneously blocking both (e.g., using the dual-inhibitor Bimekizumab) is highly effective for treating autoimmune diseases like plaque psoriasis and psoriatic arthritis.

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/hIL17F 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.

IL17F

• The exons 2-3 of mouse Il17f gene that encode full-length protein were replaced by human IL17F exons 2-3 in B-hIL17A/hIL17F mice.
• The endogenous mouse promoter, 5′ UTR, and 3′ UTR regions are retained, allowing human IL17F expression to be driven by the native mouse Il17f promoter, while endogenous mouse Il17f transcription and translation are abolished.

• Mouse Il17a mRNA was detectable only in small intestine  of wild-type mice.
• Human IL17A mRNA was detectable in homozygous B-hIL17A/hIL17F mice but not in wild-type mice. 

Strain specific analysis of IL17A gene expression in wild type (WT) mice and B-hIL17A/hIL17F mice by RT-PCR. Small intestine RNA were isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hIL17A/hIL17F mice (H/H, H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human IL17A primers. Mouse Il17a mRNA were detectable in wild-type mice. Human IL17A mRNA was detectable in homozygous B-hIL17A/hIL17F mice but not in wild-type mice.

• Mouse Il17f mRNA were detectable in wild-type mice.
• Human IL17F mRNA was detectable in homozygous B-hIL17A/hIL17F mice but not in wild-type mice. 

Strain specific analysis of IL17A gene expression in wild type (WT) mice and B-hIL17A/hIL17F mice by RT-PCR. Kidney RNA were isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hIL17A/hIL17F mice (H/H, H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human IL17F primers. Mouse Il17f mRNA were detectable in wild-type mice. Human IL17F mRNA was detectable in homozygous B-hIL17A/hIL17F mice but not in wild-type mice.

• Mouse IL17A was detectable in WT mice. Human IL17A was detectable in homozygous B-hIL17A/hIL17F mice.

Strain specific IL17A expression analysis in homozygous B-hIL17A/hIL17F mice by ELISA. Serum were collected from WT mice and homozygous B-hIL17A/hIL17F mice (H/H, H/H) stimulated with anti-mCD3ε and anti-mCD28 antibody in vivo, and analyzed by ELISA with mouse IL-17A ELISA Kit (Biolegend, 432507) and human IL-17A ELISA Kit (Biolegend, 433917). Mouse IL17A was detectable in WT mice. Human IL17A was detectable in homozygous B-hIL17A/hIL17F mice (H/H). ND: not detectable.

• Mouse IL17F was detectable in WT mice. Human IL17F was exclusively detectable in homozygous B-hIL17A/hIL17F mice. 

Strain specific IL17F expression analysis in homozygous B-hIL17A/hIL17F mice by ELISA. Naïve CD4+ T cells were sorted from splenocytes of wild type (WT) mice and homozygous B-hIL17A/hIL17F mice (H/H, H/H), and induced into Th17 cells. Th17 cells were stimulated by PMA and lonomycin. The Th17 cells culture supernatants were collected and analyzed by ELISA with with mouse IL-17F ELISA Kit (Biolegend, 436107) and human IL-17F ELISA Kit (Biolegend, 435107). Mouse IL17F was detectable in WT mice. Human IL17F was exclusively detectable in homozygous B-hIL17A/hIL17F mice (H/H, H/H). ND: not detectable.

• Percent of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hIL17A/hIL17F mice was similar to those in the C57BL/6 mice.
• Humanization of IL17A/IL17F does not affect normal immune cell development or splenic distribution.

Analysis of leukocyte subpopulations by FACS. Splenocytes and lymph nodes were isolated from female C57BL/6 and B-hIL17A/hIL17F mice(n=3, 7-week-old). Flow cytometry analysis of the splenocytes and lymph nodes was performed to assess leukocyte subpopulations. Percent of T, B, NK, DC, granulocyte, monocyte, and macrophage cells in heterozygous B-hIL17A/hIL17F mice was similar to those in the C57BL/6 mice, demonstrating that introduction of B-hIL17A/hIL17F in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in spleen.

• Percent of CD8+ T cells, CD4+ T cells and Treg cells in homozygous B-hIL17A/hIL17F mice was similar to those in the C57BL/6 mice
• Humanization of IL17A/IL17F does not change the overall development, differentiation or distribution of these T cell subtypes.

Analysis of spleen T cell subpopulations by FACS. Splenocytes and lymph nodes were isolated from female C57BL/6 and B-hIL17A/hIL17F mice (n=3, 7-week-old).  Flow cytometry analysis of the splenocytes and lymph nodes was performed to assess leukocyte subpopulations. Percent of CD8+ T cells, CD4+ T cells, and Treg cells in homozygous B-hIL17A/hIL17F mice was similar to those in the C57BL/6 mice, demonstrating that introduction of B-hIL17A/hIL17F mice in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in lymph node. Values are expressed as mean ± SEM.

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

Complete blood count (CBC) of B-hIL17A/hIL17F  mice. Values are expressed as mean ± SD.

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

Blood biochemical parameters of B-hIL17A/hIL17F  mice are shown. Values are expressed as mean ± SD.

Experimental schedule for induction of IMQ-induced psoriasis model in B-hIL17A/hIL17F 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 IMQ-induced psoriasis model. Control mice were treated similarly with Vaseline cream. Severity of skin inflammation was daily scored and back skin was collected at the endpoint. IMQ: imiquimod.

• After treatment of bimekizumab-analog, the phenotypical presentation level and erythema and scaling score of the back were much lower than the control in homozygous B-hIL17A/hIL17F mice.

IMQ-induced psoriasis model in B-hIL17A/hIL17F mice. Mice (female, 10 week-old, n=5) were scored daily for up to 6 days for body weight and clinical signs of IMQ-induced psoriasis model following treatment with imiquimod (IMQ) cream. Mice in each group were treated with different dose of bimekizumab-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 bimekizumab-analog on IMQ-induced psoriasis model in B-hIL17A/hIL17F mice were dose dependent, confirming that B-hIL17A/hIL17F mice provide a powerful model for in vivo evaluation of anti-human IL17A/IL17F antibodies.

Dose dependent effects of antibody on keratinocyte proliferation and inflammatory cell infiltration in IMQ-induced psoriasis model in B-hIL17A/hIL17F mice. Back skin was collected at the endpoint and stained with Hematoxylin and eosin (H&E). (A) H&E staining of the back skin of mice. (B) Epidermal thickness of the mice. (C) Histological changes were scored on a scale from 0 to 11. Results indicated that bimekizumab-analog (in house) significantly reduced psoriasis-like skin lesions in B-hIL17A/hIL17F mice, confirming that B-hIL17A/hIL17F mice is a powerful model for in vivo evaluation of anti-human IL17A and IL17F antibody.