NCBI: National Center of Biotechnology Information; IDs: Identifications; EST: Expressed sequence tag.

A. Data presentation format (The data presented in X-, Y-axis, and tissue order of ARHGDIA and Idha are applied to all the human and mouse genes examined respectively). As an example, the gene expression profiles of human housekeeping gene Rho GDP dissociation inhibitor (GDI) alpha (ARHGDIA) in the eighteen tissues are presented, with the tissue names and position numbers shown on the X-axis. The gene expression data were normalized by the β-actin (Hs. 520640) expression data from the same tissue, which are presented on the left Y-axis. The expression ratios among tissues were generated by normalizing the arbitrary units of the gene in the tissues with the median level of the arbitrary units of the gene in all the tissues which are presented on the right Y-axis. In order to define confidence intervals for statistically higher expression levels of given genes, we calculated the confidence intervals of tissue expression for three housekeeping genes [the mean X+2×standard deviations (SD) = 2.83] including ARHGDIA (Hs. 159161), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Hs. 544577), and ribosomal protein S27a (RPS27A, Hs. 311640). The expression variations of given genes in tissues, when they were larger than 2.83-fold, were defined as the high expression levels with statistical significance (the right Y-axis). To define confidence intervals for statistically higher expression levels of given genes in 14 mouse tissues, we calculated the confidence intervals of tissue expression [the mean X+2×standard deviations (SD) = 3.1] for three mouse house keeping genes including Lactate dehydrogenase A (Ldha, Mm. 29324), non-POU-domain-containing octamer binding protein (Nono, Mm. 280069), and ribosomal protein L32 (Rpl32, Mm. 104368). The expression variations of given genes in tissues, when they were larger than 3.1-fold, were defined as the high expression levels with statistical significance (the right Y-axis). B. The expression profiles of suppressive cytokines in human (Left 2 columns, with cytokine members designated with capital letters) and mouse (right 2 columns, with cytokine members designated with lowercase letters) tissues. C. The expression profiles of IL-35 related subunits in human and mouse tissue.

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.

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.

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.