IL-35 recombinant protein reverses inflammatory bowel disease and psoriasis through regulation of inflammatory cytokines and immune cells

Abstract

Interleukin-35 (IL-35), a member of the IL-12 family, functions as a new anti-inflammatory factor involved in arthritis, psoriasis, inflammatory bowel disease (IBD) and other immune diseases. Although IL-35 can significantly prevent the development of inflammation in many diseases, there have been no early studies accounting for the role of IL-35 recombinant protein in IBD and psoriasis. In this study, we assessed the therapeutic potential of IL-35 recombinant protein in three well-known mouse models: the dextransulfate sodium (DSS)-induced colitis mouse model, the keratin14 (K14)-vascular endothelial growth factor A (VEGF-A)-transgenic (Tg) psoriasis mouse model and the imiquimod (IMQ)-induced psoriasis mouse model. Our results indicated that IL-35 recombinant protein can slow down the pathologic process in DSS-induced acute colitis mouse model by decreasing the infiltrations of macrophages, CD4⁺ T and CD8⁺ T cells and by promoting the infiltration of Treg cells. Further analysis demonstrated that IL-35 recombinant protein may regulate inflammation through promoting the secretion of IL-10 and inhibiting the expression of pro-inflammatory cytokines such as IL-6, TNF-α and IL-17 in acute colitis model. In addition, lower dose of IL-35 recombinant protein could achieve long-term treatment effects as TNF-α monoclonal antibody did in the psoriasis mouse. In summary, the remarkable therapeutic effects of IL-35 recombinant protein in acute colitis and psoriasis mouse models indicated that IL-35 recombinant protein had a variety of anti-inflammatory effects and was expected to become an effective candidate drug for the treatment of inflammatory diseases.

Keywords: IL-35 recombinant protein; immunoregulation; inflammatory bowel disease; psoriasis.

Figure 1

IL‐35 recombinant protein effectively relieves pathological features in DSS‐induced acute colitis mouse model. (A) The DSS‐induced process and dosage regimen of IL‐35 recombinant protein. (B) Mice body weight of different groups was measured everyday (n = 8 per group). (C) Colon bleeding score was assessed as the combined score of faecal consistency and blood in the stool throughout the DSS treatment and recovery period (scored as 0–4). (D and E) Representative photographs of mouse colon in different groups (D) and the length of the colon were measured (E). (F) H&E staining of mice colon sections. Scale bar, 100 μm. Data are shown as means ± S.D. Statistical significance was assessed by unpaired, two‐tailed Student's t‐test (***P < 0.001, *P < 0.05).

Figure 2

IL‐35 recombinant protein regulates the infiltration of macrophages in colonic lesions. (A) Left: Flow cytometry analysed the proportion of macrophages (CD11b⁺F4/80⁺) in control and IL‐35 group (n = 8 per group). Right: quantitative analysis the proportion of macrophages in different groups. (B) Left: immunofluorescence analysis of macrophages in colon sections. F4/80 was detected in red and DAPI (for nuclei staining) was blue. Right: quantitative analysis of F4/80⁺ cells. (C) Flow cytometry analysed the proportion of M1 macrophages in control and IL‐35 group (Left) and corresponding statistical data were showed (Right). Scale bar, 50 μm. Data are shown as means ± S.D. Statistical significance was assessed by unpaired, two‐tailed Student's t‐test (**P < 0.01, *P < 0.05).

Figure 3

IL‐35 recombinant protein regulates the infiltration of T cells in colonic lesions. (A) Left: flow cytometry analysed the proportion of Treg cells (CD25⁺Foxp3⁺) in control and IL‐35 group (n = 8 per group). Right: quantitative analysis the proportion of Treg cells in different groups. (B) Left: immunofluorescence analysis of CD4⁺ T cells in colon sections. CD4 was detected in red and DAPI (for nuclei staining) was blue. Right: quantitative analysis of CD4⁺ T cells. (C) Left: immunofluorescence analysis of CD8⁺ T cells in colon sections. CD8 was detected in red and DAPI (for nuclei staining) was blue. Right: quantitative analysis of CD8⁺ T cells. Scale bar, 50 μm. Data are shown as means ± S.D. Statistical significance was assessed by unpaired, two‐tailed Student's t‐test (**P < 0.01, *P < 0.05).

Figure 4

IL‐35 recombinant protein regulates inflammatory cytokines in acute colitis mouse model. (A) Quantitative RT‐PCR analysis of IL‐4, IL‐6, IL‐10, IL‐17, IFN‐γ and TNF‐α messenger RNA (mRNA) expression in colorectal tissues (normalized to β‐actin). (B) Determination of the IL‐4, IL‐6, IL‐10, IL‐17, IFN‐γ and TNF‐α concentration in serum by ELISA. (C) Determination of the IL‐4, IL‐6, IL‐10, IL‐17, IFN‐γ and TNF‐α concentration in colorectal tissues lysate by ELISA. Data are shown as means ± S.D. Statistical significance was assessed by unpaired, two‐tailed Student's t‐test (***P < 0.001, **P < 0.01, *P < 0.05).

Figure 5

IL‐35 recombinant protein can improve pathological phenotype in K14‐VEGF‐A‐tg psoriasis model. (A) Upper: mice (n = 8 per group) psoriatic symptoms were assessed via macroscopic observation at day 30 (indicated as 30 days) after the treatment period. Middle: cumulative scores (erythema + scaling + thickness) were calculated and depicted. Lower: H&E staining of ear skin at 30 days. (B) Upper: mice psoriatic symptoms were assessed via macroscopic observation at day 60 (indicated as 60 days) after the treatment period. Middle: cumulative scores (erythema + scaling + thickness) were calculated and depicted. Lower: H&E staining of ear skin at 60 days. (C) Representative photographs of mice psoriatic symptoms in different groups treated with PBS, IL‐35 recombinant protein or IBI303. Scale bar, 100 μm. Data are shown as means ± S.D. Statistical significance was assessed by unpaired, two‐tailed Student's t‐test (***P < 0.001, *P < 0.05).

Figure 6

IL‐35 recombinant protein can slow down the pathological process in the IMQ‐induced psoriasis mouse model. (A) Representative photographs of the skin tissue in IMQ‐induced psoriasis mouse with or without IL‐35 recombinant protein treatment. (B) H&E‐stained skin sections from IMQ‐treated mice showed increased epidermal thickening in control group compared with IL‐35 group. Scale bar, 100 μm.