Lower level of IL‑35 and its reduced inhibition in Th17 cells in patients with bone marrow mononuclear cells Coombs test‑positive hemocytopenia

Abstract

Interleukin (IL)-35 is the latest member of IL-12 family, which plays an important role in other autoimmune diseases. Bone marrow mononuclear cells Coombs test‑positive hemocytopenia, also termed immunorelated hemocytopenia (IRH) is a type of autoimmune-associated diseases. The present study investigated the relationship of IL‑35 in patients with IRH. A total of 43 patients with IRH and 19 normal controls were enrolled in the current study. Serum levels of IL‑35 and IL‑17 in peripheral blood were evaluated by ELISA. Regulatory T cells (Tregs) level was detected by flow cytometry and IL‑35 subunits mRNA in Treg was determined using reverse transcription‑quantitative polymerase chain reaction: Epstein‑Barr virus induced 3 (EBI3) and IL‑12α chain p35. Effect of IL‑35 on T helper 17 cells (Th17) cells was determined by mix‑culture of IL‑35 with CD4+ T lymphocytes. Serum level of IL‑35 was decreased in untreated patients with IRH compared with remission patients (P<0.01) and was significantly associated with clinical indexes. Frequency of IL‑35 produced Tregs was lower and IL‑35 subunits mRNA in CD4+CD25+ Tregs were decreased in patients with IRH compared with health controls (P<0.01). Serum level of IL‑17 was increased in patients with IRH (P<0.01) and there was a negative correlation between IL‑35 and IL‑17 (r=‑0.553; P<0.01). The production of Th17 cells and IL‑17A mRNA expression were reduced (P<0.05) after mix‑culture of CD4+ T lymphocytes with IL‑35 compared with mix‑culture of CD4+ T lymphocytes without IL‑35. In conclusion, the present study revealed that IL‑35 may be a monitoring indicator of IRH occurrence and progression. IL‑35 level was lower and the inhibition on Th17 cells was reduced in the patients with IRH.

Figure 1.

(A) Serum level of IL-35 was from untreated IRH patients (n=18), remission patients (n=25) and normal controls (n=19). (B) Serum level IL-35 was from IRH patients (n=18), remission patients with positive cell membrane antibodies (remission⁺, n=11) and negative cell membrane antibodies (remission⁻, n=14) according to the cell membrane antibody results. Relative mRNA expression of (C) EBI3 and (D) p35 in isolated CD4⁺CD25⁺ Tregs from untreated patients (n=18), remission patients (n=21) and normal controls (n=19) detected by reverse transcription-quantitative polymerase chain reaction. Level of Tregs (CD4⁺CD25⁺Foxp3⁺) in peripheral blood in untreated patients (n=18), remission patients (n=21) of IRH and normal controls (n=19). (E) Level of Tregs in IRH patients detected by flow cytometry. (F) Levels of CD4⁺ T cells in lymphocyte population (G) Level of Tregs in normal control detected by flow cytometry. (H) Level of Tregs in CD4⁺ T lymphocytes. (I) Level of Tregs in lymphocytes. Data were expressed as median and range. *P<0.05, **P<0.01. IL-35, interleukin-35; IRH, immune-related hemocytopenia; EBI3, Epstein-Barr virus induced 3; Tregs, regulatory T cells; FOXP3, forkhead box P3.

Figure 2.

(A) Level of Th17 cells in patients with IRH detected by flow cytometry. Level of Tregs in (B) normal control and the percentage of (C) Th17 cells in CD4⁺ T lymphocytes. (D) Correlation between the level of Th17 cell and concentration of IL-35 in patients with IRH. Expression of PB serum level of IL-17 determined by ELISA. (E) Serum level of IL-17 of untreated IRH patients (n=18), remission patients (n=25) and normal controls (n=19). Data were expressed as the median and range. (F) Correlation between IRH patient PB serum level of IL-17 and IL-35. *P<0.05, **P<0.01. Th17, T helper 17; IRH, immune-related hemocytopenia; Tregs, regulatory T cells; IL, interleukin; PB, peripheral blood.

Figure 3.

(A) Levels of Th17 cells in the 3 groups; (B) level of Th17 cells in CD4⁺ T cells population. (C) mRNA expression levels of gp130, IL-12R-β2, RORγt, and IL-17a obtained using reverse transcription-quantitative polymerase chain reaction. *P<0.05. group (a), blank control; group (b), anti-CD3 MAbs, anti-CD28 MAbs, TGF-β, IL-6; group (c), anti-CD3 MAbs, anti-CD28 MAbs, TGF-β, IL-6, IL-35. Th17, T helper 17; IL, interleukin; ROR, RAR-related orphan receptor; TGF, transforming growth factor.