**8. The extended IL-10 cytokine superfamily**

In summary, cell-fate determination induced by the IL-12-mediated activation of STAT4, IL-4-mediated activation of STAT6, transforming growth factor-β (TGF-β), IL-6 plus TGF-β and IL-27 activation of STAT3 profoundly influence the balance of Th1 and Th2 cells, Th17 cells and Treg cell production, respectively [80, 146-149]. This conclusion must, however, be tempered by results of recent studies which also showed that formal interplays occurred be‐ tween IL-4-induced STAT6 phosphorylation, the GATA-binding protein-3 (GATA3) zinc-fin‐ ger transcription factor [150] and the Treg cell transcription factor, FoxP3 as well. Importantly, GATA3 was revealed as the key transcription factor in this complex interplay because GATA3 could 1) directly inhibit Th1 differentiation through its capacity to block upregulation of the IL-12β2 receptor; 2) inhibit the activity of STAT4; and 3) neutralize the ac‐ tivity of runt-related transcription factor 3 (runx3), via its capacity to induce protein-protein interactions [150]. Thus, by modulating the activities of IL-4/STAT6, GATA3/STAT4 and runx3 one could potentially alter the activity of pro-inflammatory and anti-inflammatory cy‐

IL-21 is a member of the Type I cytokine superfamily of cytokine receptors. In this group, the common γ cytokine receptor complex is the functional component for receptor-mediated signal transduction of IL-2, IL-4, IL-7, IL-9 and IL-15 [151-153]. Although IL-21 has strong structural ho‐ mology to IL-15, IL-21 interacts with a unique receptor, termed, IL-21Rα, which pairs with the γcommon cytokine receptor chain (i.e. CD132) to form the active IL-21 receptor complex [154].

IL-21-mediated events affect the functions of NK cells, T-cells and B-cells. Although devel‐ opment of Treg cells from the Th17 lineage is generally considered to require IL-6 because IL-6 reciprocally controls Th17 and Treg cell development through its ability to inhibit TGF-βinduced FoxP3 and by inducing RORγ, in fact, IL-21 can also induce RORγ and Th17 devel‐ opment in the absence of IL-6. However, evidence also showed that the number of Th17 cells, the recruitment of Th17 cells to inflamed tissues and the development of autoimmune encephalitis and myocarditis did not differ between IL-21R and IL-21 deficient mice com‐ pared to their wild-type counterparts [155, 156]. More importantly, IL-6 was the more potent inducer of Th17 differentiation compared to IL-21 thus calling into question, whether IL-21

Despite the emerging controversies regarding how important IL-21 is in T-cell development and immune responses, a therapeutic intervention designed to limit the responses of im‐ mune cells to IL-21 has long been considered for treating cancer and autoimmune diseases [157]. In addition, because the binding of IL-21R induces activation of several of the JAK iso‐ forms [153], it became apparent that it would be necessary to elucidate which cellular events were controlled by STAT proteins activated by phosphorylated JAKs in response to IL-21/ IL-21R. Attempting to address this point, Habib et al. [151] found that IL-21 induced prolif‐ eration of pro-B-lymphoid cells *in vitro* which was dependent on both γc and the γc-associ‐ ated JAK3 complex. However, a monoclonal antibody reactive only with γc was effective in limiting the proliferation of BaF3/IL21R α cells [151] indicating that neutralization of γc

tokines as well as overcome immune tolerance.

was even required for Th17 development.

alone could cause inhibition of JAK activation by IL-21/IL-21R.

**7.3. IL-21**

386 Drug Discovery

IL-19, IL-20, IL-22, IL-24 (melanoma differentiation-associated gene 7; mda-7), and IL-26 (AK155) are all structurally similar to IL-10 and these interleukins constitute members of the extended IL-10 cytokine superfamily [161-163]. Three additional members of the IL-10 cyto‐ kine superfamily have recently been added to this list, namely, IL-28A, IL-28B and IL-29 which now comprise the IFN-λ cytokine subfamily [164-166].

IL-19 and IL-20 are α-helical proteins. They have similar cysteine sites; their amino acid se‐ quences are approximately 30% identical. In the human genome, the genes encoding these IL-10 superfamily members are located in two clusters; one cluster comprises the genes for IL-10, IL-19, IL-20, and IL-24/mda-7 which are located on chromosome 1q31-32 [167]. IL-19 and IL-20 were predominately expressed in monocytes, as well as non-immune cells under inflam‐ matory conditions [168], whereas IL-22 and IL-26 was only produced by T-cells, especially Th1 cells and NK cells, whilst IL-24 synthesis was restricted to monocytes and T-cells [169].

level of boney erosions and an improvement in the quality of subchondral bone. Moreover, treatment of rats with CIA with 1BB1 reduced the expression of TNF-α, IL-1β, IL-6 and Re‐ ceptor Activator of Nuclear Factor Kappa-B Ligand (RANKL) genes in synovial tissue and also lowered IL-6 levels in serum. Synovial fibroblasts isolated from rats with CIA respond‐ ed to treatment with IL-19 in a similar fashion seen with synovial tissue *in situ* where in‐

Suppression of Pro-Inflammatory Cytokines via Targeting of STAT-Responsive Genes

http://dx.doi.org/10.5772/52506

389

There is now compelling evidence that IL-19-mediated activation of STAT3 was associated with the development and progression of inflammatory arthritis which was characterized by the elevated expression of many of the pro-inflammatory cytokines pertinent to human RA joint destruction. These data also showed that the rat CIA model could be further ex‐ ploited to determine the extent to which specific dampening or up-regulation of STAT-re‐ sponsive cytokine genes would ameliorate inflammatory responses associated with CIA.

IL-20 interacts with IL-20R1/IL-20R2 to activate the JAK/STAT pathway [166] and IL-20 has been implicated in the pathogenesis of autoimmune diseases [178]. However, IL-20R2 sig‐ naling was shown to blunt mouse CD4 and CD8 T-cell responses to antigen *in vitro* and *in vivo* [179]. Thus, it remains to be determined the extent to which IL-20 promotes or sup‐

In the CIA model in the rat, treatment with an anti-IL-20 antibody 7E, either alone, or in combination with the TNF blocker, etanercept was compared to etanercept alone for their capacity to 1) ameliorate cartilage damage; 2) stabilize bone mineral density; and 3) alter cy‐ tokine production [180]. In addition, the effect of antibody 7E on expression of various genes implicated in the progression of CIA was evaluated on rat synovial fibroblasts *in vitro.* Treat‐ ment with 7E or etanercept or the combination of 7E and etanercept significantly reduced the severity of arthritis as measured by rat hind paw thickness and swelling. These treat‐ ments also prevented cartilage degradation and bone loss whilst reducing the level of syno‐ vial tissue IL-20, IL-1β, IL-6, RANKL and MMPs. Of note, IL-20 induced the expression of TNF-α in synovial fibroblasts isolated from rats with CIA. Moreover, IL-20 induced RANKL production in synovial fibroblasts, osteoblasts and Th17 cells. In another study, antibody 7E was shown to inhibit mouse osteoclast differentiation induced by macrophage-CSF and RANKL [181]. These results [181] coupled with results from the CIA model [180] indicated that IL-20 was likely to have promoted the increased bone loss in CIA by promoting osteo‐

clast differentiation and the activity of osteoclast-mediated bone resorption.

Correlative human studies of IL-20-mediated responses in RA are just emerging. However, the results have differed somewhat from those seen in the CIA model. Thus, Kragstrup et al. [182] showed that plasma IL-20 levels were increased in RA compared to OA patients with the elevated level of IL-20 primarily localized to mononuclear cells and neutrophils. Stimu‐ lating mononuclear cells isolated from RA synovium with recombinant IL-20 resulted in the increased secretion of the chemoattractant CCL2/MCP-1. However, at variance with find‐ ings in the CIA model, recombinant IL-20 did not alter the expression of TNF-α or IL-6 by

creased synthesis of TNF-α, IL-1β, IL-6 and RANKL was detected.

**8.2. IL-20**

presses immune-mediated inflammation.

mononuclear cells *in vitro*.

Both IL-20 and IL-24 bind to the IL-20R complex which is made up of the cytokine receptor family 2-8/IL-20Rα (IL-20R1) [170], although it was previously shown that IL-19/IL-19 recep‐ tor binding was similar to IL-20/IL-24 receptor binding [170]. IL-19 was also shown to inter‐ act with a DIRS1-like element which is composed of tyrosine recombinase-encoding transposons/IL-20Rβ (IL-20R2) [170-172].

In all cases, the binding of IL-19, IL-20 or IL-24 to these receptors caused activation of STAT3 and activation of a minimal promoter region containing those sequences identified as STATbinding sites. Importantly, absent either of the R1 proteins in the two types of receptor com‐ plexes, IL-20R1/IL-20R2 and IL-22R1/IL-22R2 reduced the affinity of IL-19 or IL-24 for these receptors. Furthermore, IL-20R2, and not IL-20R1, was identified as the high affinity recep‐ tor chain for these cytokines [173].

The functional significance of the IL-10-related cytokines, IL-19, IL-20, IL-21, IL-22 and IL-24 in terms of the pathophysiology of RA and other autoimmune diseases is systematically be‐ ing elucidated. In most cases, the role played by these cytokines has been inferred from measurements in sera of RA patients before and/or after medical therapy.
