Lipopolysaccharide-induced M2 to M1 macrophage transformation for IL-12p70 production is blocked by Candida albicans mediated up-regulation of EBI3 expression

PLoS One. 2013 May 27;8(5):e63967. doi: 10.1371/journal.pone.0063967. Print 2013.


Macrophages are heterogeneous cell populations that are present in all tissues. Macrophages can be divided into classically activated inflammatory macrophages (M1) and alternatively activated anti-inflammatory macrophages (M2). It has been generally accepted that M1 macrophages are polarised in an inflammatory environment to produce pro-inflammatory cytokines, whilst M2 macrophages are involved in anti-inflammation and aid tissue repair in wound healing. Bacterial endotoxin (lipopolysaccharide; LPS) is a potent factor in infection, which induces M1 macrophages resulting in higher levels of iNOS, TNFα and IL-12p70 which dictate inflammatory T cell responses. M2 macrophages can be transformed into M1 macrophages following LPS stimulation to promote inflammation. Candida albicans is a commensal fungal microorganism, which has been suggested to induce immune tolerance; however, the mechanism of C. albicans-induced immune tolerance has not been investigated in detail. IL-35 is a recently identified anti-inflammatory cytokine which is a heterodimeric protein consisting of the Epstein-Barr virus-induced gene 3 (EBI3) and IL-12p35. IL-35 shares the protein subunit p35, with IL-12p70. IL-12p70 is the most potent cytokine to induce Th1 responses during inflammation. In this study, we demonstrate that heat-killed C. albicans (HKC) strongly suppressed LPS-induced IL-12p70 production in M2 macrophages. Candida albicans induced a high level of EBI3 expression in M2 macrophages, which served as a mechanism for IL-12p70 suppression by competitive binding of the common protein subunit (p35) of IL-35 and IL-12p70. To demonstrate that EBI3 expression had the ability to block IL-12p70 production intracellularly, a Chinese Hamster Ovary (CHO) cell line with biscistronic expression of IL-12p40 and p35 was constructed, followed by ectopic over-expression of EBI3. The over-expression of EBI3 in the IL-12p70 producing cell line effectively suppressed IL-12p70 production. IL-35 secretion was also detected in the cell line, with suppressed IL-12p70 production by immune-precipitation Western blotting. However, this secretion was not evident in M2 macrophages following stimulation by HKC. This can be explained by the constitutive expression of IL-35 receptors (gp130 and IL-12Rβ2) in M2 macrophages for cytokine consumption. Our results have indicated that C. albicans can suppress host inflammatory responses in mucosal skin by suppressing LPS-induced IL-12p70 production. Lower IL-12p70 production may avoid an unnecessary Th1 response in order to retain immune tolerance, which may be one of the mechanisms by which C. albicans achieves a successful commensal lifestyle without having a detrimental effect on the host's health.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bone Marrow Cells
  • CHO Cells
  • Candida albicans / immunology*
  • Cell Line
  • Cricetulus
  • Gene Expression
  • Gene Expression Regulation*
  • Interleukin-12 / biosynthesis*
  • Lipopolysaccharides / immunology*
  • Macrophages / immunology*
  • Macrophages / metabolism*
  • Macrophages / microbiology
  • Mice
  • Minor Histocompatibility Antigens
  • Phenotype
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Receptors, Cytokine / genetics*
  • Receptors, Cytokine / metabolism


  • Ebi3 protein, mouse
  • Lipopolysaccharides
  • Minor Histocompatibility Antigens
  • RNA, Messenger
  • Receptors, Cytokine
  • Interleukin-12

Grants and funding

This work was supported by Cardiff University research funding and funding for XFZ from China Scholarship Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.