Since 2007, a new anti-inflammatory/immunosuppressive cytokine, IL-35, has been defined 
. Formed by heterodimerization of Epstein-Barr virus-induced gene 3 (EBI3) protein with the IL-12 p35 subunit (IL-12A) 
, IL-35 inhibits inflammation in various autoimmunity models such as experimental colitis 
, collagen-induced autoimmune arthritis 
, autoimmune demyelination in central nervous system 
, and type 2 T helper cell (Th2)-mediated allergic asthma 
. Although secretion of IL-35 has only been confirmed in Tregs 
, the contributions of IL-35 generated from tissues and cells other than T cells under non-stimulated conditions remains poorly identified. In an effort to bridge this knowledge gap we used a new experimental database mining and statistical analysis technique to determine the tissue expression of anti-inflammatory cytokines and receptors including novel cytokine IL-35 and made the following findings: 1)
anti-inflammatory cytokines IL-35 and IL-10 are not constitutively expressed in most tissues whereas TGF-βs have higher expression levels in non-stimulated tissues; 2)
IL-35 receptor subunit IL-6ST, IL-10RA, IL-10RB, TGF-βR1, and TGF-βR2 receptor complex are constitutively expressed in cardiovascular and other tissues; 3)
NF-κB transcription factor has higher binding frequencies in the promoter region of IL-35 subunits; 4)
alternative promoter and alternative splicing regulate the structures and expressions of IL-35 and other anti-inflammatory cytokines and receptors; 5)
higher expression of IL-35 could be induced with higher hypomethylation status. The higher binding frequencies of NF-κB-transcription factor in IL-35 promoters, alternative promoters, along with higher expression of IL-35 induced by hypomethylation all suggest that the expression of IL-35 is inducible via several mechanisms. To support our results, we also presented the reported experimental evidence that IL-35 is indeed induced by stimulations of various proinflammatory cytokines and bacterial endotoxin LPS in vascular endothelial cells, smooth muscle cells and monocytes; 6)
the expressions of IL-35 and IL-10 could be regulated by AU-rich element-mediated mRNA degradation mechanism; and 7)
the two subunits of IL-35 are subjected to regulation by different microRNAs. The last two structural evidences indicate that IL-35 can be degraded quickly by 3′UTR-mediated mechanisms, which correlates with the non-constitutive expression of IL-35 in tissues.
It is worth pointing out that the data retrieved from the expression sequence tag (EST) database analyzed in this study is more precise than that detected with traditional approaches including Northern blot analysis and PCR analysis due to the un-biased cDNA cloning and DNA sequencing procedures of EST database deposits 
. Thus, the expression patterns of IL-35, other anti-inflammatory cytokines, and receptors are experimentally-based and precise.
Transcription factors (TFs) are master genes which control the expression of other genes. It is well-accepted that multiple binding sites for a given TF within a promoter will increase the likelihood of actual binding 
. The most physiologically relevant TFs will bind to the putative core promoter region (1,500 base pairs upstream of the transcription start site) to fulfill their functions 
. Our strategy to identify TF binding profiles and transcriptional signaling is an important advance in merging bioinformatics and experimental science. This study, together with our previous database mining work 
, utilized novel database mining techniques to identify disease-related signaling pathways. Our research method is featured as; (1)
intensively grounded in the literature, (3)
panoramic and integrative for gene and TF regulation, (4)
based on the NCBI experimental databases, (5)
inclusive of well-characterized TFs in the searchable database TESS; (6)
statistically rigorous analysis of available public databases, and (7)
Alternative promoters play an important role in gene transcription in response to tissue/cell-specific and stimulation-specific transcription signaling 
. One of the best examples of multiple promoter usage is fibroblast growth factor-1 (FGF1) transcription, which is controlled by at least four distinct promoters in a tissue-specific manner. The 1.A and 1.B promoters of FGF1 are constitutively active in their respective cell types. In contrast, different biological response modifiers, including serum and transforming growth factor-β, can induce the 1.C and 1.D promoters of FGF1 
. Of note, our results showed that most anti-inflammatory cytokines and cytokine receptors have more than one promoter, suggesting the capacity of these genes to respond to tissue-/cell-specific and stimulation-specific transcription signaling 
. Although endothelial cells and vascular smooth muscle cells also generate various inflammation-regulating cytokines 
, the lack of a cell type-specific gene expression database prevents the analysis of databases in cell-specific manner. When the detailed sequences become available, it will be possible to compare the transcription factor binding profiles in the alternative promoters of the same genes.
To summarize our results, we propose a new system of categorizing anti-inflammatory cytokines () based on the following three criteria including (1) constitutive or non-constitutive expression in tissues, (2) non-responsiveness or responsiveness to proinflammatory stimuli, and (3) acceleration or no acceleration of autoimmune and inflammation by cytokine gene deficiency. We categorize anti-inflammatory cytokines into two groups: first, the house-keeping cytokines are defined with constitutive expression in tissues, non-responsiveness to proinflammatory stimuli, and acceleration of autoimmune and inflammation in the absence of the genes, such as TGF-βs; second, the definitions of responsive cytokines include cytokines with non-constitutive expression in tissues, responsiveness to proinflammatory stimuli and no acceleration of autoimmune and inflammation in the absence of the genes, including IL-35. IL-35 is not constitutively expressed in most tissues similar to IL-10. Instead it is upregulated, by hypomethylation and other proinflammatory signals, in human tissues and most mouse tissues except mouse blood, bone marrow, liver, and thymus. In addition, the expression and structure of IL-35 are under various regulations including NF-κB transcription factors, alternative promoter, alternative splicing, and mRNA degradation via AU-rich-dependent and microRNA-dependent mechanisms. These mechanisms underlie the upregulation and quick degradation of IL-35. Our new working model and new system in categorizing anti-inflammatory cytokines provide important insight into the following two important issues: first, how anti-inflammatory cytokines share their duties: the house-keeping cytokines, such as TGF-βs, inhibit the initiation of inflammation whereas the responsive cytokines including novel cytokine IL-35 suppress full-blown inflammation; and second how these two groups of anti-inflammatory cytokines orchestrate their roles in suppressing inflammation in different stages in various tissues and systems. Our findings are significant for future design of novel anti-inflammatory/immunosuppressive therapies.
A new working model of responsive anti-inflammatory cytokine and housekeeping cytokine.