**8.5. IL-3**

IL-3 is one of several major cytokines that drive the differentiation of cells of the hemato‐ poietic lineage. The interaction between IL-3 and its cognate receptor activates several sig‐ naling pathways, including, JAK/STAT, PI3K/Akt/mTOR and the Ras/Raf/MAPK pathways [199]. Downstream events that are regulated by IL-3 which are germane to RA and autoim‐ munity, in general, include the findings that depending on the conditions in the microenvir‐ onment, IL-3 can alter cell proliferation, survival or induce cell death by apoptosis [30].

IL-3 was identified as an activator of JAK2 and STAT5 [200] and the expression of the proapoptotic protein, c-myc. This finding provided the initial evidence that cell proliferation and apoptosis was regulated, in part, by activated STAT5. However, a subsequent study by Chaturvedi et al. [201] provided evidence to the contrary in that the interaction of IL-3 with its receptor activated STAT3 via the phosphorylation of tyr701. Moreover, the results of this study [201] also showed that myeloid cell proliferation was regulated by IL-3-activated Src kinase and not by IL-3-actiivated JAK3. This conclusion was based on the following results. Inhibition of c-Src kinase activation using a dominant-negative (dn) Src mutant also blocked STAT3 activation and, this in turn, inhibited proliferation of the 32Dcl3 myeloid cell line in response to IL-3. Moreover, expression of a dn-JAK2 mutation increased apoptosis in 32Dcl3 cells in the absence of IL-3 which also involved the concomitant down-regulation of ERK-2. Taken together these results indicated that Src kinase activation of STAT proteins regulated myeloid cell proliferation whereas JAKs controlled the activation of ERK-2 and associated anti-apoptotic signals [202].

and IFN-γ production suggesting that blockade of IL-7Rα in RA patients reduced the ex‐

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

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

393

With respect to the putative role of IL-7 in regulating certain aspects of cartilage responses in arthritis, Yammani et al. [213] reported that IL-7, IL-6 or IL-8 stimulated the production of the Ca2+-binding protein, S100A4, by cultured human articular chondrocytes. Importantly, IL-7 increased the synthesis of S100A4 to a greater extent than either IL-6 or IL-8 with IL-7 stimulated S100A4 resulting from JAK3/STAT3 activation. In that regard, pre-treating chon‐ drocytes with the experimental JAK3, inhibitor, WHI-P154, or with cyclohexamide blocked S100A4 synthesis which also inhibited the production of MMP-13. Because S100A4 has been implicated as significantly contributing to pannus-mediated destruction of cartilage in RA inflammation [214], blockade of IL-7R may be useful for down-regulating the expression of

The interaction between S100A4 and the tumor suppressor p53 protein was purported to be related to the role of S100A4 as a promoter of cancer metastasis [215]. IL-7 via S100A4 was also shown to induce the expression of MMP-13 as well as MMP-1, MMP-9 and S100A4 was also shown to be involved in the neoangiogenesis and aberrant cell proliferation of rheuma‐ toid synovium [216]. Importantly a selective inhibitor of MMP-13 reduced the level of carti‐ lage destruction in 2 of 3 animal models of RA, including the SCID-mouse co-implantation model and CIA, but not adjuvant arthritis. [217]. Thus, evidence has gradually accumulated to show that up-regulation of S100A4 via activation of STAT3 significantly alters the pro‐

**9. Experimental therapies that inhibit activated stat proteins: Is cytokine**

The results of a Phase 2B RA clinical trial have recently been published which showed that the JAK3-specific SMI, tofacitinib (CP690, 550) had clinical efficacy as measured by the ACR response criteria [218]. However, there has been less progress on developing novel strategies to directly inhibit activated STAT proteins or dampen STAT gene re‐ sponses. Noteworthy have been proof-of-principle studies that activated STAT proteins can be experimentally 'deactivated' which result in the inhibition of STAT/DNA binding. Thus, JNK-mediated phosphorylation of the STAT6 ser707 decreased the DNA binding ca‐ pacity of IL-4-stimulated STAT6 resulting in the inhibition of STAT6-responsive genes [219]. Using immunosuppressive STAT oligodeoxynucleotides (ODN) to inhibit activated STAT proteins have also been relatively successful. These ODN have been shown to in‐ terfere with the phosphorylation of STAT1 and STAT4 [220] and STAT1 and STAT3 [221]. Lastly, administration of a single dose of a STAT1 decoy ODN suppressed joint swelling and the histological appearance of acute and chronic adjuvant-induced experi‐ mental arthritis in the mouse [222]. Electrophoretic mobility shift analysis of the nuclear extracts from synoviocytes from the STAT1 decoy ODN-treated animals incubated with the STAT-1 decoy ODN inhibited STAT-1 binding to DNA. Of note, STAT-1 decoy ODN

S100A4 and MMP-13 with associated blunting of pannus invasion into cartilage.

pression of the STAT-responsive gene, IFN-γ.

gression of inflammatory arthritis.

**gene expression altered?**

The results of another study [203] showed that IL-3 played an important role in regulating SOCS3 and PIAS proteins [16, 20, 21] both of which are important in regulating cytokine sig‐ naling as well as the fine-tuning of the survival and/or cell death pathways for immune and non-immune cells in general. IL-3 plays a particularly critical role in regulating these events in mast cells [203], plasmacytoid dendritic cells [204], osteoclast-like cells, [205] and osteo‐ clasts [206, in particular. All of these cell types are involved in some aspect of RA pathology.

To further illustrate this point, Gupta et al. [206] showed that osteoclasts treated with IL-3 were diverted to the dendritic cell lineage which may also be related to the finding that that IL-3 dampened human osteoclast-mediated bone resorption. Most recently, Srivastava et al. [207] showed that IL-3 increased the number of functionally active Treg cells by stimulating the production of IL-2 by non-Treg cells the latter being dependent on the dose of IL-3. Of note, treating mice with CIA with IL-3 significantly reduced the severity of arthritis and also increased the frequency of Treg cells found in the thymus, lymph nodes and spleen. Al‐ though this study [207] did not directly measure the status of activated STAT proteins in the CIA mice treated with IL-3, these additional results showed that treatment of CIA with IL-3 decreased production of IL-6, IL-17A, TNF-α and IL-1 whilst increasing IFN-γ and IL-10 (Table 1).
