IL-35 Is a Novel Responsive Anti-inflammatory Cytokine — A New System of Categorizing Anti-inflammatory Cytokines

It remains unknown whether newly identified anti-inflammatory/immunosuppressive cytokine interleukin-35 (IL-35) is different from other anti-inflammatory cytokines such as IL-10 and transforming growth factor (TGF)-β in terms of inhibition of inflammation initiation and suppression of full-blown inflammation. Using experimental database mining and statistical analysis methods we developed, we examined the tissue expression profiles and regulatory mechanisms of IL-35 in comparison to other anti-inflammatory cytokines. Our results suggest that in contrast to TGF-β, IL-35 is not constitutively expressed in human tissues but it is inducible in response to inflammatory stimuli. We also provide structural evidence that AU-rich element (ARE) binding proteins and microRNAs target IL-35 subunit transcripts, by which IL-35 may achieve non-constitutive expression status. Furthermore, we propose a new system to categorize anti-inflammatory cytokines into two groups: (1) the house-keeping cytokines, such as TGF-β, inhibit the initiation of inflammation whereas (2) the responsive cytokines including IL-35 suppress inflammation in full-blown stage. Our in-depth analyses of molecular events that regulate the production of IL-35 as well as the new categorization system of anti-inflammatory cytokines are important for the design of new strategies of immune therapies

Differential response of IL-35 subunits, IL-10, and TGF-β1 in response to proinflammatory cytokine IL-1β.

<p>Differential response of IL-35 subunits, IL-10, and TGF-β1 in response to proinflammatory cytokine IL-1β.</p

A new working model of responsive anti-inflammatory cytokine and housekeeping cytokine.

<p>Homeostatic tissues express “house-keeping” anti-inflammatory cytokines TGF-β1, TGF-β2, TGF-β3 to prevent it from initiation of inflammation. When tissues get inflamed, proinflammatory factors may stimulate tissues to express “responsive” anti-inflammatory cytokines such as IL-35 by specific transcription factors to counteract inflammation response. Furthermore, ARE binding proteins and MicroRNAs are responsible of the quick degradation of IL-35 mRNA afterwards, by which IL-35 achieve non-constitutive expression status in tissues again.</p

3′-untranslated region in mRNA of anti-inflammatory cytokines in human contain signals for RNA protein binding.

<p>3′-untranslated region in mRNA of anti-inflammatory cytokines in human contain signals for RNA protein binding.</p

Positions of AUUA sequences in 3′-UTR of IL-12A mRNA are indicated.

<p>Positions of AUUA sequences in 3′-UTR of IL-12A mRNA are indicated.</p

Higher hypomethylation status is positively associated with higher expression of IL-35 gene.

<p>A. Concentrations of SAM and SAH in mouse tissues were previously examined by Ueland et al. B. Correlation of suppressive cytokines and TGF-β receptors with SAM/SAH ratios in mouse tissues. C. Schematic presentation of how IL-35 may be regulated by methylation status. S-Adenosylhomocysteine (SAH) and S-Adenosylmethionine (SAM) are intermediate metabolites of homocysteine-methionine metabolism cycle. SAH is a potent inhibitor of cellular methylation. High SAM/SAH ratio is associated with hypermethylation of DNA and no IL12A/Ebi3 expression. Low SAM/SAH ratio is associated with hypomethylation of DNA and Ebi3 can be expressed.</p

IL-35 subunit mRNAs have been shown to be induced by stimulation in various human cell types.

<p>IL-35 subunit mRNAs have been shown to be induced by stimulation in various human cell types.</p

Transcription factor binding frequencies in the promoter region of IL-35.

<p>The promoter sequences (1500 base pair upstream of the transcription start site) of 3 housekeeping genes (ACTB, GAPDH, ARHGDIA), IL12A, EBI3, and TGFB1 were retrieved from the NIH/NCBI Entrez Gene database, and were analyzed using TESS to determine the frequencies of 10 Transcription factors (TFs). The binding frequencies of the 10 TFs in the promoter region of each gene were counted. Confidence interval was set by using the mean+2×standard deviation (SD) of the TF binding frequencies in the promoter of 3 housekeeping genes. The binding frequency which is higher than the uppermost confidence interval (p<0.05) is considered significant.</p

Alternative promoter and alternative splicing regulate the expression and structures of anti-inflammatory cytokines.

<p>Alternative promoter and alternative splicing regulate the expression and structures of anti-inflammatory cytokines.</p

The two-tier expression status of IL-35 and related cytokines is identified.

<p>The two-tier expression status of IL-35 and related cytokines is identified.</p