IL6/IL6R: A key cytokine signaling axis in inflammation and its therapeutic intervention.

•  Gene Information: Interleukin 6 (IL6) is a protein coding gene located on chromosome 7p15.3, encoding a pleiotropic cytokine that serves as a core mediator of the acute phase response. Its cognate receptor alpha subunit, IL6R (CD126), is encoded by a gene situated on chromosome 1q21.3.
• Protein Expression: IL6 is transiently produced and secreted primarily by activated macrophages, endothelial cells, and fibroblasts in response to stimulation. The receptor, IL6R, exists in two main forms: a transmembrane protein (mIL6R) constitutively expressed on hepatocytes and specific leukocyte subsets, and a functional soluble isoform (sIL6R) generated via alternative splicing or proteolytic shedding.
• Signaling Pathway: IL6 exerts its biological effects by binding to either mIL6R (classic signaling) or sIL6R (trans signaling). Both pathways require the subsequent recruitment of the shared transmembrane signal transducer gp130 (IL6ST), forming a hexameric signaling complex that activates the downstream intracellular JAK STAT3 cascade.
• Therapeutic Inhibition: By blocking the interaction within this cytokine axis, therapeutic monoclonal antibodies such as tocilizumab (targeting IL6R) or siltuximab (targeting IL6) effectively halt hyperactive downstream inflammatory cascades, delivering proven clinical benefits in treating rheumatoid arthritis, systemic juvenile idiopathic arthritis, and induced cytokine release syndrome.

IL6

• The exons 1 5 of mouse Il6 gene that encode whole protein is replaced by human counterparts in B hIL6/hIL6R mice(C).
• The promoter, 5’UTR and 3’UTR region of the mouse Il6 are also replaced by human counterparts.

IL6R

• The CDS of human IL6R gene was inserted after the start codon ATG of mouse Il6ra gene in B hIL6/hIL6R mice(C).
• Expression of the human IL6R protein is driven by the native mouse Il6r promoter, while endogenous mouse Il6r transcription and translation are disrupted.

Strain specific IL6 expression analysis in wild-type BALB/cCrSlcNifdc mice and homozygous B-hIL6/IL6R mice (C) by ELISA. Serum was collected from wild type BALB/cCrSlcNifdc mice (female, n=3), homozygous B hIL6/IL6R mice (C) (female, n=3), the production of IL6 in serum were assessed after 3 h of stimulation with LPS (20 μg/200uL, i.p.) in vivo. Expression level of mouse and human IL6 were analyzed by ELISA (ELISA MAX™ Deluxe Set Mouse IL 6 (431304); ELISA MAX™ Deluxe Set Human IL 6 (430508)). Mouse IL6 was only detectable in wild type BALB/cCrSlcNifdc mice. Human IL6 was only detectable in homozygous B hIL6/IL6R mice (C). Values are expressed as mean ± SEM.

Strain specific IL6R expression analysis in wild-type BALB/cCrSlcNifdc mice and homozygous B-hIL6/IL6R mice(C) by flow cytometry. Splenocytes were collected from wild type BALB/cCrSlcNifdc mice and homozygous B hIL6/IL6R mice(C), and analyzed by flow cytometry with species specific anti mIL 6Rα Antibody  (PE anti mouse CD126 (IL 6Rα chain) Antibody, biolegend, 115805) and anti hIL 6Rα Antibody  (PE anti human CD126 (IL 6Rα) Antibody, biolegend, 352803). Mouse IL6R was detectable in T cells of wild type mice. Human IL6R was detectable in T cells of homozygous B hIL6/IL6R mice(C).

Strain specific soluble IL6R expression analysis in wild-type BALB/cCrSlcNifdc mice and homozygous B-hIL6/IL6R mice(C) by ELISA. Serum was collected from wild type BALB/cCrSlcNifdc mice (female, n=3), homozygous B hIL6/IL6R mice(C) (female, n=3). Expression level of soluble human IL6R were analyzed by ELISA (Human IL6R ELISA Kit (DR600)). Human IL6R was detectable in homozygous B hIL6/IL6R mice(C). Values are expressed as mean ± SEM.

Human IL6, mouse IFNγ, and mouse CRP expression analysis in homozygous humanized B-hIL6/hIL6R mice (C) by ELISA. Mice received LPS injection (i.p.) 2 h following PBS or tocilizumab analog (i.p., in house) treatment. Serum was collected from homozygous B hIL6/hIL6R mice (C) twenty four hours after LPS injection. Expression level of human IL6, mouse IFNγ, and mouse CRP were analyzed by ELISA. Pre treatment of tocilizumab analog significantly decreased human IL6, mouse IFNγ, and mouse CRP expression at twenty four hours after LPS injection, indicating a successful inhibition of the downstream activation caused by hIL6R. Values are expressed as mean ± SEM. Significance was determined by one way ANOVA test. ***p < 0.001, p < 0.01, *p < 0.05.