**6.3. OSM**

**6.2. INF-α/IFN-γ**

382 Drug Discovery

MAPK appears to be significant [47, 111, 112].

ing independent of IL-2.

The interferon protein family in conjunction with the interferon-regulated gene (IRG) path‐ way plays an important role in RA, SLE and other autoimmune diseases because the IRG pathway is a critical mediator of autoimmune-dependent inflammation [104-106]. INF-γ is known to be one of the strongest activators of JAK/STAT and Tyk2 resulting in IRG-mediat‐ ed responses [16, 107, 108]. INF-γ has also been shown to play a role in the epigenetic regu‐ lation of specific gene activation as evinced by the finding of an association of pJAK2 and IFN-γ receptor in the nucleus with histone H3 in IFN-γ-treated human amnionic (WISH; American Tissue Culture Collection; CCL 25) cells *in vitro* [109]. AG-490 a JAK2 inhibitor, also down-regulated STAT1 gene expression and AG-490 inhibited prolactin-induced IFN-γ, TNF-α, IL-1β and IL-12p40 synthesis in mouse peritoneal macrophages *in vitro* [110]. Of note, inhibition of JNK activity with the SMI, SP600125, also resulted in down-regulating IFN-γ and TNF-α indicating that both the JAK/STAT and MAPK pathways contributed to alterations in the expression of these cytokines. Although the importance of these results in providing a rationale for manipulating signal transduction pathways in human RA remains to be fully elucidated, the fact that the expression of several pro-inflammatory cytokines rel‐ evant to RA pathology are potentially controlled by cross-talk between JAK/STAT and

Three DNA-binding sites related to STAT protein-DNA binding have been recognized with‐ in the IFN-γ promoter. These DNA binding sites include an IL-12-mediated STAT4/DNA binding site, an IL-2-induced STAT5/DNA binding site and a CD2-mediated STAT/IFN-γ binding site [113]. Thus, CD2-mediated activation of human peripheral blood mononuclear cells was shown to result in STAT/DNA binding to a 3.6kb DNA motif within the IFN-γ pro‐ moter which occurred principally via STAT5A binding and less so by STAT5B, with both be‐

Induction of some of the IFN-regulatory factors (Irfs), including those gene responses brought about by activation of irf9 via IFN-α were found to be STAT protein-independent [114]. In ad‐ dition, results from other studies showed that Akt activity was also involved in key IFN-α, -γ gene responses [115]. Moreover, regulation of IFN-α, -γ-mediated responses required the di‐

In RA, the depressed level of IL-4 and IL-10 in mononuclear phagocytes is, in part, responsible for the imbalance in Th1/Th2 cytokines [3, 16]. The primary model employed to describe the re‐ lationship between IFN-γ and IL-4/IL-10 is dependent on several factors. This view was origi‐ nally proposed by Hamilton et al. [118] as follows; IL-4 was shown to markedly suppress the transcriptional activity of IFN-γ because the promoter sequence between IL-4 and IFN-γ were essentially identical. Proof of this came from the results of experiments that showed that IFN-γ/ STAT1 and IL-4/STAT6 both formed complexes at the same regulatory sequence, but whereas activated STAT1 promoted IFN-γ transcription, activated STAT6 did not. However, activated STAT6 was required to suppress the transcriptional up-regulation of IL-4. Thus, in the model, IL-4 appeared to be necessary to reduce IFN-γ gene expression (Table 1) and was related to a competition between activated STAT1 and activated STAT6 for binding to the IFN-γ promoter. In keeping with this model, the expression of IL-10 is also known to be suppressed by INF-γ

rect control of mTOR [116] beginning with the initiation of protein translation [117].

Recent advances have assigned OSM, a member of the IL-6 protein superfamily an impor‐ tant role in the pathogenesis and progression of RA and OA [101]. In that regard, one of the more important experimental results involving OSM were reported by Hams et al. [122] who compared the inflammatory responses in wild-type mice to IL-6-deficient and mice de‐ ficient in the OSM receptor β (OSMRβ). They showed that the OSMRβ knockout mice showed enhanced trafficking of monocytes to sites of inflammation when these mice were compared to the wild-type or IL-6-knockout mice. However, the OSMRβ knockout mice did not demonstrate any differences in neutrophil or lymphocyte migration to inflamed tissue when compared to their wild-type or IL-6-deficient counterparts. These results suggested that the OSM/OSMRβ-pathway probably regulated chemokine production and chemokine function. Indeed this proved to be the case when the up-regulated chemokine in response to the activation of the OSMRβ-pathway was eventually identified as CCL5. CCL5 has been shown to be a critical chemokine for regulating the recruitment and retention of monocytes in inflamed RA synovial joints [3]. Although the evidence was indirect, these results sug‐ gested that a drug with the capacity to neutralize the interaction between IL-6 and IL6R in arthritic joints would not alter OSM/OSMRβ-mediated STAT activation [9]. This view was supported by the results from several previous studies which showed that 1) although the OSMR consisted of a heterodimer of the LIF receptor and gp130, the alternative form of OSMR, namely, OSMRβ, was activated only by OSM and not by LIF [123]; 2) OSM, but nei‐ ther IL-6 nor LIF induced tyrosine phosphorylation in the Shc adaptor protein p52 and p66 isoforms which in association with growth factor receptor-bound protein 2 (Grb2) were both recruited to OSMR, but not to gp130 [124]; and 3) at least in human or canine osteosarcoma cell lines, treatment with OSM phosphorylated JAK2/STAT3 and Src, each of which was shown to be involved in an OSM dose-dependent-mediated increase in expression of the MMP-2 gene (i.e. 72kDa gelatinase) and vascular endothelial growth factor (VEGF) gene [125]. Of note, the STAT3 SMI, LLL3, inhibited MMP-2 and VEGF gene expression indicat‐ ing that MMP-2 and VEGF were genes targeted by activated STAT3. Importantly, Clarkson et al. [126] showed that another one of the activated STAT-responsive genes in mammary epithelial cells was OSMR. This finding was critical for completing the circle which showed that activation of OSMRβ was central to the upstream activation of the OSM-mediated path‐ way as well as to the downstream increase in the expression of the OSMRβ gene, both events involving STAT proteins.

the combination therapy of infliximab and methotrexate reduced the level of IL-18 in serum whilst the level of the chemokine, CXCL12 was unaltered [135]. Moreover, synovial fluid from these RA patients had higher levels of IL-18 (as well as TNF-α and IL-15) prior to be‐ ginning combination therapy with infliximab and methotrexate compared to the level of these cytokines in a patient's sera. In addition, the level of IL-18/TNF-α in synovial fluid was strongly correlated with a patient's high Disease Activity Score-28 [136]. Thus, it may be in‐ formative going forward to assess the level of activated STAT3 and IL-18 in the synovial flu‐ id and sera of RA patients before and after treatment with TNF antagonists or other biological drugs that neutralize the activation of JAK/STAT and MAPK pathways to deter‐ mine the extent to which the level of activated STAT3, p38 kinase or JNK is correlated with

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

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

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IL-12 is made up of 2 disulfide-linked protein subunits, termed IL-12p35 and IL-12p40 linked in a heterodimer configuration [137, 138]. Whilst the IL-12p40 subunit has structural similarities with cytokine receptors, the IL-12p35 component is structurally similar to IL-6 and granulocyte-colony stimulating factor (G-CSF) [139]. Of note, if IL-12p35 and IL-12p40

IL-12 is synthesized by many cell types of the innate and adaptive immune systems, includ‐ ing, monocytes, macrophages, dendritic cells and neutrophils. IL-12 is a minor product of Bcells [140]. Although IL-12p35 is constitutively expressed at low levels by many of these cells, the expression of IL-12p40 is limited to those phagocytic cells that synthesize IL-12p70.

The connection between IL-12 and activation of the JAK/STAT pathway stems from the find‐ ing that IL-12 production was positively regulated by IFN-γ, the latter cytokine which is al‐ so induced by IL-12. Thus, IFN-γ regulates IL-12 gene expression and vice versa. By contrast, two of the anti-inflammatory cytokines, namely, IL-10 and IL-13 which also acti‐ vate JAK/STAT, suppressed IL-12 production [140] (Table 1). In addition, the type I interfer‐ on proteins, exemplified by IFN-β, which activates STAT1 [141] was shown to inhibit IL-12

The main immune functions of IL-12 involve the regulation of Th1 differentiation via the ac‐ tivation of STAT4 which induces the synthesis of the T-bet transcription factor [143]. T-bet

had been characterized as principally IFN-γ/STAT1-dependent, and IL-12/STAT4 independ‐ ent. In fact, expression of T-bet was shown to require activated STAT4 to achieve total IL-12 dependent Th1 cell-fate determination [143]. However, Yang et al. [144] showed that the effect of IL-12/STAT4 was more complex. Thus, IL-12 -induced activated STAT4 bound to a distant but highly conserved STAT-responsive T-bet enhancer region where it induced IFNγ-activated STAT1 independent T-bet gene expression in CD8+ cells. Importantly, IL-4-in‐ duced STAT6 activation regulates the development and effector functions, not of Th1 cells, but rather of Th2 cells in peripheral tissues such as skin, lung and gut [145]. However, Th2 cell produced in lymph nodes did not require IL-4-mediated activation of STAT6 [145].

suppressor T-cell development which

are produced by the same cell, the bioactive heterodimer is termed, IL-12p70 [140].

IL-18 gene expression by synovium and cartilage *ex vivo*.

**7.2. IL-12**

gene expression in mice [142].

was shown to regulate IFN-γ expression and CD8+
