CP-690,550 is currently being studied in a variety of autoimmune diseases. In this study, we show that the inhibitor blocks signaling by JAK3-dependent γc cytokine receptors, as well as by other cytokine receptors that signal through JAK1. Accordingly, we found that CP-690,550 interfered with Th1 and Th2 differentiation, and also impaired the production of inflammatory Th17 cells generated in response to IL-1β, IL-6 and IL-23. In contrast, the JAK inhibitor enhanced production of IL-17A in cells cultured with IL-6 and TGF-β1. These effects were associated with amelioration of murine arthritis, which correlated with reduced expression of STAT1-dependent genes. Furthermore, CP-690,550 also blocked cytokine production in a sepsis model suggesting that the mechanism of action of this drug involves blocking the action of cytokines during innate and adaptive responses.
Despite its advanced stage of clinical development, the mode of action by which CP-690,550 exerts efficacy in RA and other autoimmune settings remains unresolved (15
). In contrast to its activity against isolated kinases, CP-690,550 demonstrates functional specificity for JAK1 and JAK3 over other JAK family members in cells, although the basis of this apparent discrepancy has not been determined (18
). Since many of the cytokines involved in RA and other autoimmune diseases signal through receptors associated with JAKs, the question arises as to how the effects of CP-690,550 relate to the apparent efficacy of the drug in the setting of autoimmune disease. A central component of the pathophysiology of RA and psoriasis is the action of autoreactive T cells and the inflammatory cytokines that act upon them (61
). As was expected, CP-690,550 potently inhibited γc-cytokine signaling pathways in the current studies by targeting JAK1 and JAK3 in T cells. Similar results have been observed in JAK1 and JAK3 deficient cells (2
) and with JAK1-selective inhibitors (unpublished results) suggesting that blockade of either or both of these kinases can modulate γc-cytokine receptor signals. A recent study has also demonstrated that a selective JAK3 inhibitor, WYE-151650, is effective in collagen-induced arthritis (24
Neither the clinical efficacy of CP-690,550 nor the potential efficacy of other JAK inhibitors is likely to be explained by inhibition of γc-cytokine receptor signaling alone. By such a mechanism, the differentiation of naive T cells to Th2 effector cells would be inhibited, but Th2 cells are likely not relevant to the pathogenesis of CIA in mice or RA and psoriasis in humans (41
). Surprisingly, CP-690,550 also prevented Th1 differentiation. While previous observations have indicated that cellular JAK3-deficiency or inhibition of JAK3 can suppress Th1 differentiation (64
), our data suggest a different mechanism since CP-690,550 suppressed expression of the Th1-associated transcription factor T-bet. Th1 differentiation is driven by IL-12 and IFN-γ and by the activation of STAT1 and T-bet (42
). Our results indicate that CP-690,550 has only a modest effect on IL-12-induced STAT4 activation while profoundly inhibiting STAT1 activation in T cells induced either by IL-12 or IFN-γ. Indeed, the inhibition of IFN-γ signaling alone could likely account for the observed Th1 suppression as demonstrated by the effect of anti-IFN-γ neutralizing antibodies. The consequences of CP-690,550 treatment on Th1 differentiation and STAT1 signaling could also explain efficacy of the inhibitor in a mouse Graft-versus-Host Disease (GVHD) model, where Th1 responses were limited by CP-690,550 without affecting cell proliferation (66
While blocking Th1 responses can be highly efficient in GVHD and transplant rejection, this mechanism alone would likely be less successful in autoimmune diseases in which Th17 cells also play a major role. Thus, using inhibitors that target not only JAK3 but also JAK1 or JAK2 and subsequently affect the differentiation of Th1 as well as Th17 cells could be of benefit in autoimmune settings. The generation of Th17 cells is regulated by multiple factors. While IL-6 and TGF-β1 can efficiently induce IL-17 production, IL-6 together with IL-23 and IL-1β, in the absence of TGFβ-1, can also induce IL-17 in naïve Th cells (50
). Indeed, we have shown recently that Th17 cells generated in the absence of TGF-β are more pathogenic in vivo
than those generated in the presence of this cytokine (51
). Moreover, we have found that the balance between STAT3 and STAT5 activation can have opposing regulatory effects on IL-17 expression (68
). Results from the present studies demonstrate that CP-690,550, most likely by inhibiting STAT5, increases IL-17 expression when Th17 cells are generated with TGF-β and IL-6. In contrast, in the absence of TGF-β-signaling CP-690,550 blocked IL-17 expression.
While the regulation of IL-17A and IL-17F expression are more complex (68
), the expression of IL-23R and IL-22 are strictly dependent on STAT3 activation (51
). We show in these studies that CP-690,550 interferes with IL-23 action by blocking upregulation of its receptor and subsequent IL-17 induction. Moreover, CP-690,550 inhibited IL-23R expression under either Th17 condition. Similarly, the JAK inhibitor abrogated STAT3-mediated IL-22 and IL-21 expression in Th17 cells, and also inhibited RORγt and T-bet expression. Thus, CP-690,550 potently suppresses the generation of pathogenic Th17 cells with an IL-23/STAT3-signature. Inhibitory effects on Th17-associated cytokines have also been suggested for the JAK1/JAK2 inhibitor INCB028050 (25
This mode of action of CP-690,550 may be of interest in a number of autoimmune diseases where interfering with IL-23 signaling attenuates disease (41
). Thus, it may very well be that a clinically important action of CP-690,550 is to block the combined actions of IL-23.
On the other hand, IL-6 has wide-ranging biological activities in various target cells. In addition to promoting Th17 differentiation, it regulates immune responses, the acute phase reaction, hematopoiesis, and bone metabolism (73
). IL-6-deficient mice are protected from experimental autoimmune diseases such as CIA (34
). Additionally, elevated serum IL-6 levels have been observed in patients with inflammatory diseases such as RA and Crohn's disease, and tocilizumab, a humanized anti-IL-6R antibody that blocks IL-6 signaling, has shown clinical efficacy in these indications, ameliorating inflammation and normalizing acute phase protein levels (75
). Our data indicate that CP-690,550 interferes with production of IL-6 and also blocks IL-6 signaling, which could be explained by effects of the inhibitor on JAK1 and/or JAK2. Thus, an additional mechanism underlying CP-690,550 efficacy in RA is likely mediated through effects on IL-6.
We were surprised by the rapid effects of CP-690,550 on established disease in the mouse CIA model. Indeed, effects of the inhibitor were observable within hours of initiating treatment. Despite the inhibitory consequences of CP-690,550 on Th cell differentiation, it seemed unlikely that this could induce such rapid effects in vivo
. Rather, the rapid suppression of inflammatory responses suggested that blockade of innate immune mechanisms might represent part of the salutatory effects of JAK inhibition. This led us to examine the efficacy of the JAK inhibitor in the sepsis model. Importantly, we found that CP-690,550 had no direct effect on TLR4 signaling in vitro
, as we did not observe inhibition of LPS induced TNF or IL-6 production from human PBMC (data not shown). Rather, the suppression of acute TNF responses in vivo
following LPS administration is more consistent with inhibition of IFN-γ signaling by blockade of JAK1, since both STAT1-deficient and IFN-γR-deficient mice are resistant to LPS-induced endotoxemic shock (58
,). In contrast, IFN-γ priming of macrophages has been shown to enhance both LPS-stimulated TNF production in vivo
) and STAT1 expression (77
), and it has been suggested that IFN-γ activation of STAT1 may alter signaling pathways downstream of anti-inflammatory cytokines such as IL-10 or TGF-β, leading to antagonism of their suppressive function (78
). If this were the case, CP-690,550 suppression of STAT1-responsive genes could override the effect of priming. IL-10 responses to LPS are enhanced in mice made deficient for IFN-α/β/γ or STAT1 (60
), suggesting that STAT1 is a negative regulator of IL-10 gene expression. Our observations were consistent with this hypothesis, as we observed enhanced IL-10 levels in LPS treated mice given the JAK inhibitor. Another possible contribution to CP-690,550 suppression of LPS responses in vivo
could involve blockade of IL-15 signaling since both IL-15 deficiency and anti-IL-15 neutralizing antibody have been shown to suppress LPS-induced endotoxemia in vivo
). While there is no doubt that IL-15 signaling is potently inhibited by CP-690,550, this mechanism can not completely explain the results from the current study since blockade of IL-15 signaling would not be expected to affect IL-10 in this model.
The simultaneous control of signaling pathways involved in innate and adaptive immune responses by CP-690,550 may explain why this JAK inhibitor has produced rapid clinical improvement in RA patients who have previously failed other disease-modifying anti-rheumatic drug therapies or TNF antagonists (22
). Based on the present data, it appears that the efficacy of CP-690,550 is likely based on its ability to block multiple cytokines and break the cycle of inflammation. Clearly, it will be important to try to understand which critical cytokines are blocked in humans undergoing JAK inhibitor treatment and the extent to which signaling is abrogated. As such, our findings have implications for the possible utility of CP-690,550 in a wide variety of inflammatory disorders.