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.
• 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.
• Expression of the human IL6R protein is driven by the native mouse Il6r promoter, while endogenous mouse Il6r transcription and translation are disrupted.

• Human IL6 and IL6R mRNA were exclusively detectable in B-hIL6/hIL6R mice.

Strain specific analysis of IL6 and IL6R gene expression in B-hIL6/hIL6R mice by RT-PCR. Mouse Il6 and Il6r mRNA were detectable in spleen of wild-type mice (+/+). Human IL6 was detectable only in homozygous B-hIL6/hIL6R mice (H/H; H/H) after stimulated with LPS for 3h. Human IL6R mRNA was detectable in homozygous B-hIL6/hIL6R mice, but not in wild-type mice with or without LPS stimulation.

• Mouse IL6 was detected exclusively in wild-type C57BL/6 mice.
• Human IL6 was detected in homozygous B-hIL6/hIL6R mice, but not in wild-type mice.

Strain specific IL6 expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hIL6/hIL6R mice by ELISA. Serum was collected from wild-type C57BL/6JNifdc  mice (+/+) (female, n=3, 8-week-old), homozygous B-hIL6/hIL6R mice(H/H ; H/H) (female, n=3, 8-week-old), the production of IL6 in serum were assessed after 3h of stimulation with LPS (20 μg/200uL, i.p.) in vivo. Expression level of human IL6 were analyzed by ELISA (Human IL-6 ELISA MAX TM Deluxe Set (430508)). Human IL6 was only detectable in homozygous homozygous B-hIL6/hIL6R mice (n=3). Values are expressed as mean ± SEM.

• Human IL6R was exclusively expressed on T cells in B-hIL6/hIL6R mice.

Strain specific IL6R expression analysis in wild-type C57BL/6 mice, homozygous B-hIL6/hIL6R mice by flow cytometry. Splenocytes were collected wild-type C57BL/6 mice(+/+) and homozygous B-hIL6/hIL6R mice (H/H;H/H). The protein expression was analyzed with anti-mouse CD126 (IL-6Rα chain) Antibody (Biolegend, 115805) and anti-human CD126 (IL-6Rα) Antibody (Biolegend, 352803) by flow cytometry. Mouse IL6R was detectable in T cells of C57BL/6 mice. Human IL6R was detectable in T cells of homozygous B-hIL6/hIL6R mice.

• Human IL6R was detected in brain, colon, kidney, liver, lung, spleen, and thymus, but not in heart, of homozygous B-hIL6/hIL6R mice.

Representative human IL6R expression in different tissues of B-hIL6/IL6R mice by IHC. Tissues were collected from homozygous B-hIL6/hIL6R mice and stained with antibodies for human IL6R (A-I) or anti-IgG antibodies (J). Human tonsils as positive control (I); Mouse lung stained with anti-IgG antibodies as a negative control (J); As shown in the figure, human IL6R was detected in brain, colon, kidney, liver, lung, spleen and thymus, but not in heart. Original magnification ×200. Abbreviations: IHC, immunohistochemistry.

• Soluble human IL6R was exclusively expressed in B-hIL6/hIL6R mice.

Strain specific IL6R expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hIL6/IL6R mice by ELISA. Plasma (EDTA) was collected from wild-type C57BL/6 mice (female, 6-week-old, n=3) and homozygous B-hIL6/IL6R mice (female, 6-week-old, n=3). Expression level of human IL6R were analyzed by ELISA (human IL6R ELISA kit: R&D, DR600). Human IL6R was exclusively detectable in homozygous B-hIL6/IL6R mice. Values are expressed as mean ± SEM.

• Soluble human IL6R was exclusively expressed in B-hIL6/hIL6R mice.

Soluble human IL6R expression analysis in homozygous B-hIL6/hIL6R mice by ELISA. Liver and pancreas were isolated from wild-type C57BL/6 mice (female, 7-week-old, n=3) and homozygous B-hIL6/hIL6R mice (female, 7-week-old, n=3), and analyzed by ELISA with species-specific anti-human IL6R ELISA kit (human IL6R ELISA kit: R&D, DR600). Soluble human IL6R was detectable in liver (A) and pancreas (B) of homozygous B-hIL6/hIL6R mice but not in wild-type mice.

• The percentages of T cells, B cells, NK cells, DCs, neutrophils, monocytes, and macrophages in homozygous B-hIL6/hIL6R mice were similar to those in C57BL/6 mice.
• Humanization of IL6 and IL6R does not affect normal immune cell development or splenic distribution.

Analysis of leukocyte subpopulations by flow cytometry in spleen and lymph node. Splenocytes and lymph nodes were isolated from female C57BL/6 and B-hIL6/hIL6R mice (female, 9-week-old, n = 3). Single live cells were gated on the CD45⁺ population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

• The proportions of CD4⁺ T cells, CD8⁺ T cells, and Tregs in homozygous B-hIL6/hIL6R mice were comparable to those in C57BL/6 mice.
• Humanization of  IL6 and IL6R does not affect normal T cell development, differentiation, or splenic distribution.

Analysis of T-cell subpopulations by flow cytometry in spleen and lymph node. Splenocytes and lymph nodes were isolated from female C57BL/6 and B-hIL6/hIL6R mice (female, 9-week-old, n = 3). Single live cells were gated on the TCRβ⁺ T-cell population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

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

Complete blood count (CBC) of B-hIL6/hIL6R mice. Values are expressed as mean ± SD.

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

Blood biochemical parameters of B-hIL6/hIL6R mice are shown. Values are expressed as mean ± SD.

Efficacy of anti-human IL6 antibodies in B-hIL6/hIL6R mice with collagen induced arthritis (CIA) model. Arthritis was induced by the subcutaneous injection of CII emulsion into B-hIL6/hIL6R mice on Day 0 and Day 21 (male, n=6 in each group). The development of arthritis was monitored and the arthritis score was evaluated every day. The mice were divided into groups at day 32 (Clinical score ≧ 1). The treatment group was intraperitoneally injected with different doses of anti-human IL6 antibody sirukumab analog (in house).

• Sirukumab analog significantly reduced clinical score compared with modelling group.

Efficacy of anti-human IL6 antibodies in B-hIL6/hIL6R mice with collagen induced arthritis (CIA) model. Mice in each group were treated with sirukumab analog (in house). Body weight change(A) and clinical score (B) were evaluated during treatment twice a week. There was no significant change in body weight, while total clinical score increased in the groups except control during days 21 to 28. It indicated that the arthritis mouse model had been constructed successfully. Clinical scores decreased in the two groups treated with sirukumab analog and dose-dependent. The results indicated the B-hIL6/hIL6R mice provide a powerful preclinical model for in vivo evaluation of anti-human IL6 antibodies.

• Sirukumab analog decreased pathological score compared with modelling group.

Efficacy of anti-human IL6 antibody in B-hIL6/hIL6R mice with collagen induced arthritis (CIA) model. Histopathological examination was performed on the joints of the extremities at endpoint. (A) Hematoxylin and eosin (H&E) staining. (B) Score of arthritis histology. G1 showed no significant abnormal changes. G2 showed bone structure damage(d), articular cavity or periarticular space disappeared (e), and pannus were observed (a), suggesting that the CIA model was successfully established. Compared with the G2 group, the low-dose group (G3) showed slight inflammatory cell infiltration (b) and pannus (a) and synovial hyperplasia (c). However, in the high dose group (G4), there was only partial pannus, the arthrosis disappeared and the articular cavity was obvious.