IL23: A key inflammation cytokine in the inflammatory response driven by Th17 cells

• Gene Information:  IL-23 is a heterodimeric cytokine composed of p19 and p40 subunits, and is encoded by interleukin-23 subunit alpha (IL23A) and  interleukin-12 subunit beta (IL12B) respectively. IL23A is located on chromosome 12q13.3, IL12B is located on chromosome 5q33.3.
• Protein Expression: Mature, biologically active IL-23 protein is secreted only when a cell expresses both the p19 and p40 subunits and assembles them correctly. IL-23 is mainly produced by activated antigen-presenting cells, such as macrophages and dendritic cells, as well as damaged keratinocytes or intestinal epithelial cells.
• Signaling Pathway: IL23 binds to the IL23R/IL12RB1 receptor complex to trigger JAK-STAT, p38 MAPK, and NF-κB signaling. This initiates the transcription of downstream pro-inflammatory factors, such as IL-17A, IL-17F, IL-22 and RORγt, thereby promoting the differentiation and maintenance of pathogenic Th17 cells and exacerbating chronic tissue inflammation.
• Therapeutic Inhibition: Blocking the interaction between IL23 and IL23 receptor can reduce the severity of autoimmune diseases, such as the IBD model and psoriasis.

α4β7: A central pathway for directing lymphocyte migration to the gut and other mucosal sites during inflammation

• Gene Information:  Integrin α4β7 is composed of a 150 kD (α4 or CD49d) and a 130 kD (β7) heterodimer, also known as CD49d/β7 or LPAM-1. ITGA4 is located on chromosome 2q31.3, ITGB7 is located on chromosome 12q13.13.  Integrin α4β7 binds its ligand MAdCAM-1, and plays an important role in lymphocytes adhesion.
• Protein Expression: The α4β7 integrin is a heterodimer, composed of an α4 subunit and a β7 subunit. α4β7 is mainly expressed in leukocytes, such as T cells, B cells, NK cells, macrophages, dendritic cells, monocytes, and others.
• Signaling Pathway: Integrin α4β7 interacts with the cell surface adhesion molecules MAdCAM-1 which is normally expressed by the vascular endothelium of the gastrointestinal tract, which is a central pathway for directing lymphocyte migration to the gut and other mucosal sites during inflammation.
• Therapeutic Inhibition: Blocking the binding of α4β7 to MAdCAM-1, such as with Vedolizumab, inhibits lymphocyte homing and infiltration into inflammatory tissues such as the intestine.

IL23A

• The exons 1-4 of mouse Il23a gene that encode the whole molecule (ATG to STOP codon) were replaced by human counterparts in B-hIL23A/hIL12B/hα4β7 mice.
• The promoter, 5’UTR and 3’UTR region of the mouse gene were retained. The human IL23A expression was driven by endogenous mouse Il23a promoter, while mouse Il23a gene transcription and translation will be disrupted.

IL12B

• The exons 2-8 of mouse Il12b gene that encode the whole molecule (ATG to STOP codon), including 3’UTR were replaced by human counterparts in B-hIL23A/hIL12B/hα4β7 mice.
• The promoter and 5’UTR region of the mouse gene were retained. The human IL12B expression was driven by endogenous mouse Il12b promoter, while mouse Il12b gene transcription and translation will be disrupted.

ITGA4

• The exons 2-27 of mouse Itga4 gene that encode the extracellular domain were replaced by human counterparts in B-hIL23A/hIL12B/hα4β7 mice.
• The promoter, 5’UTR and 3’UTR region of the mouse gene were retained. The human ITGA4 expression was driven by endogenous mouse Itga4 promoter, while mouse Itga4 gene transcription and translation will be disrupted.

ITGB7

• The exons 2-14 of mouse Itgb7 gene that encode the extracellular domain were replaced by human counterparts in B-hIL23A/hIL12B/hα4β7 mice.
• The promoter, 5’UTR and 3’UTR region of the mouse gene were retained. The human ITGB7 expression was driven by endogenous mouse Itgb7 promoter, while mouse Itgb7 gene transcription and translation will be disrupted.

Note: B-hIL23A/hIL12B/hα4β7 mice were obtained by mating B-hIL23A/hIL12B mice (120553) and B-hα4β7 mice (111913).

• Human IL23 was exclusively detectable in homozygous B-hIL23A/hIL12B/hα4β7 mice but not wild-type C57BL/6JNifdc mice.

Mouse IL23 and human IL23 expression analysis in B-hIL23A/hIL12B/hα4β7 mice by ELISA. BMDCs were produced by culturing the bone marrow from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hIL23A/hIL12B/hα4β7 mice (H/H;H/H;H/H;H/H), which were stimulated with LPS in vitro. After stimulation, the supernatants were collected and the levels of mouse and human IL23 were analyzed by ELISA (R&D, M2300; R&D, D2300B). Mouse IL23 was only detectable in wild-type C57BL/6JNifdc mice. Human IL23 was exclusively detectable in homozygous B-hIL23A/hIL12B/hα4β7 mice. Values are expressed as mean ± SEM. ND: not detectable. 

• Human ITGA4 and ITGB7 were exclusively detectable in T cells of homozygous B-hIL23A/hIL12B/hα4β7 mice but not wild-type C57BL/6JNifdc mice.

Strain specific ITGA4 and ITGB7 expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hIL23A/hIL12B/hα4β7 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hIL23A/hIL12B/hα4β7 mice (H/H;H/H;H/H;H/H). Protein expression was analyzed with anti-mouse ITGA4 antibody (Biolegend, 103705), anti-mouse ITGB7 antibody (Biolegend, 120607), anti-human ITGA4 antibody (Biolegend, 304307) anti-human ITGB7 antibody (Invitrogen, MA5-23541) by flow cytometry. Mouse ITGA4 and ITGB7 were only detectable in T cells of wild-type C57BL/6JNifdc mice. Human ITGA4 and ITGB7 were exclusively detectable in T cells of homozygous B-hIL23A/hIL12B/hα4β7 mice.

• Human ITGA4 and ITGB7 were exclusively detectable in B cells of homozygous B-hIL23A/hIL12B/hα4β7 mice but not wild-type C57BL/6JNifdc mice.

Strain specific ITGA4 and ITGB7 expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hIL23A/hIL12B/hα4β7 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hIL23A/hIL12B/hα4β7 mice (H/H;H/H;H/H;H/H). Protein expression was analyzed with anti-mouse ITGA4 antibody (Biolegend, 103705), anti-mouse ITGB7 antibody (Biolegend, 120607), anti-human ITGA4 antibody (Biolegend, 304307) anti-human ITGB7 antibody (Invitrogen, MA5-23541) by flow cytometry. Mouse ITGA4 and ITGB7 were only detectable in B cells of wild-type C57BL/6JNifdc mice. Human ITGA4 and ITGB7 were exclusively detectable in B cells of homozygous B-hIL23A/hIL12B/hα4β7 mice.

• Human ITGA4 and ITGB7 were exclusively detectable in DCs of homozygous B-hIL23A/hIL12B/hα4β7 mice but not wild-type C57BL/6JNifdc mice.

Strain specific ITGA4 and ITGB7 expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hIL23A/hIL12B/hα4β7 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hIL23A/hIL12B/hα4β7 mice (H/H;H/H;H/H;H/H). Protein expression was analyzed with anti-mouse ITGA4 antibody (Biolegend, 103705), anti-mouse ITGB7 antibody (Biolegend, 120607), anti-human ITGA4 antibody (Biolegend, 304307) anti-human ITGB7 antibody (Invitrogen, MA5-23541) by flow cytometry. Mouse ITGA4 and ITGB7 were only detectable in DCs of wild-type C57BL/6JNifdc mice. Human ITGA4 and ITGB7 were exclusively detectable in DCs of homozygous B-hIL23A/hIL12B/hα4β7 mice.

• Human ITGA4 and ITGB7 were exclusively detectable in monocytes of homozygous B-hIL23A/hIL12B/hα4β7 mice but not wild-type C57BL/6JNifdc mice.

Strain specific ITGA4 and ITGB7 expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hIL23A/hIL12B/hα4β7 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hIL23A/hIL12B/hα4β7 mice (H/H;H/H;H/H;H/H). Protein expression was analyzed with anti-mouse ITGA4 antibody (Biolegend, 103705), anti-mouse ITGB7 antibody (Biolegend, 120607), anti-human ITGA4 antibody (Biolegend, 304307) anti-human ITGB7 antibody (Invitrogen, MA5-23541) by flow cytometry. Mouse ITGA4 and ITGB7 were only detectable in monocytes of wild-type C57BL/6JNifdc mice. Human ITGA4 and ITGB7 were exclusively detectable in monocytes of homozygous B-hIL23A/hIL12B/hα4β7 mice.

• Human ITGA4 and ITGB7 were exclusively detectable in macrophages of homozygous B-hIL23A/hIL12B/hα4β7 mice but not wild-type C57BL/6JNifdc mice.

Strain specific ITGA4 and ITGB7 expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hIL23A/hIL12B/hα4β7 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hIL23A/hIL12B/hα4β7 mice (H/H;H/H;H/H;H/H). Protein expression was analyzed with anti-mouse ITGA4 antibody (Biolegend, 103705), anti-mouse ITGB7 antibody (Biolegend, 120607), anti-human ITGA4 antibody (Biolegend, 304307) anti-human ITGB7 antibody (Invitrogen, MA5-23541) by flow cytometry. Mouse ITGA4 and ITGB7 were only detectable in macrophages of wild-type C57BL/6JNifdc mice. Human ITGA4 and ITGB7 were exclusively detectable in macrophages of homozygous B-hIL23A/hIL12B/hα4β7 mice.

• The synergistic stimulation of TL1A and IL23 could promote the production of downstream cytokines in wild-type C57BL/6 mice and homozygous B-hIL23A/hIL12B/hα4β7 mice.
• Both mIL23 and hIL23 could bind to mouse IL23 receptor.

Ex vivo functional analysis in B-hIL23A/hIL12B/hα4β7 mice. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hIL23A/hIL12B/hα4β7 mice (H/H;H/H;H/H;H/H), then the production of mouse IFN-γ, mouse IL17A, and mouse IL22 in supernatants were assessed after 72 h of incubation with mTL1A (300 ng/mL), mIL23 (10 ng/mL), and hIL23 (10 ng/mL) in vitro.