**6.2. INF-α/IFN-γ**

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

[119]. Thus, it was shown that when transfected RPMI 8226.1 B-cells were incubated with IFNγ followed by lipopolysaccharide (LPS), IFN-γ reduced LPS-induced IL-10 promoter activity which was independent of the irf, but dependent on an activated STAT-motif. Further analyses indicated that IFN-γ down-regulated IL-10 gene expression via displacement of the trans-acti‐

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

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

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Experimental strategies could be designed to increase the mononuclear cell expression of IL-4/IL-10 by manipulating the ultra-sensitive INF-γ promoter region with various activated STAT protein types. Another strategy for potentially improving the level of IL-10 in RA would involve manipulating natural Treg cells in a cell-based therapy mode because Treg cells are a rich source of IL-10 [120, 121]. However, as pointed out by Nandakumar et al. [121] one must be mindful that the antigen specificity of natural Treg cells must be carefully regu‐ lated to protect against the development of self-reactive effector T-cells or for that matter,

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

vated STAT3 by STAT1 induced by IFN-γ.

Treg cells with inappropriate antigen specificity.

**6.3. OSM**

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‐ ing independent of IL-2.

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

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 [119]. Thus, it was shown that when transfected RPMI 8226.1 B-cells were incubated with IFNγ followed by lipopolysaccharide (LPS), IFN-γ reduced LPS-induced IL-10 promoter activity which was independent of the irf, but dependent on an activated STAT-motif. Further analyses indicated that IFN-γ down-regulated IL-10 gene expression via displacement of the trans-acti‐ vated STAT3 by STAT1 induced by IFN-γ.

Experimental strategies could be designed to increase the mononuclear cell expression of IL-4/IL-10 by manipulating the ultra-sensitive INF-γ promoter region with various activated STAT protein types. Another strategy for potentially improving the level of IL-10 in RA would involve manipulating natural Treg cells in a cell-based therapy mode because Treg cells are a rich source of IL-10 [120, 121]. However, as pointed out by Nandakumar et al. [121] one must be mindful that the antigen specificity of natural Treg cells must be carefully regu‐ lated to protect against the development of self-reactive effector T-cells or for that matter, Treg cells with inappropriate antigen specificity.
