Modulation of innate and adaptive immune responses by tofacitinib (CP-690,550)

Abstract

Inhibitors of the JAK family of nonreceptor tyrosine kinases have demonstrated clinical efficacy in rheumatoid arthritis and other inflammatory disorders; however, the precise mechanisms by which JAK inhibition improves inflammatory immune responses remain unclear. In this study, we examined the mode of action of tofacitinib (CP-690,550) on JAK/STAT signaling pathways involved in adaptive and innate immune responses. To determine the extent of inhibition of specific JAK/STAT-dependent pathways, we analyzed cytokine stimulation of mouse and human T cells in vitro. We also investigated the consequences of CP-690,550 treatment on Th cell differentiation of naive murine CD4(+) T cells. CP-690,550 inhibited IL-4-dependent Th2 cell differentiation and interestingly also interfered with Th17 cell differentiation. Expression of IL-23 receptor and the Th17 cytokines IL-17A, IL-17F, and IL-22 were blocked when naive Th cells were stimulated with IL-6 and IL-23. In contrast, IL-17A production was enhanced when Th17 cells were differentiated in the presence of TGF-β. Moreover, CP-690,550 also prevented the activation of STAT1, induction of T-bet, and subsequent generation of Th1 cells. In a model of established arthritis, CP-690,550 rapidly improved disease by inhibiting the production of inflammatory mediators and suppressing STAT1-dependent genes in joint tissue. Furthermore, efficacy in this disease model correlated with the inhibition of both JAK1 and JAK3 signaling pathways. CP-690,550 also modulated innate responses to LPS in vivo through a mechanism likely involving the inhibition of STAT1 signaling. Thus, CP-690,550 may improve autoimmune diseases and prevent transplant rejection by suppressing the differentiation of pathogenic Th1 and Th17 cells as well as innate immune cell signaling.

Trial registration: ClinicalTrials.gov NCT00615199.

Figures

Figure 1

CP-690,550 inhibits γc-chain cytokine signaling in T cells. Equal numbers of mouse CD4+ T cells were pre-incubated with the indicated concentrations of CP-690,550 before stimulation with or without IL-2 for 15 minutes (ns, no cytokine stimulation). Activation of STAT5 and AKT were determined in cell lysates by immunoblotting with phospho-specific antibodies (A). Mouse CD4+ T cells were pre-incubated with or without 0.3 μM CP-690,550 and stimulated with IL-21 for the indicated time periods (ns, no cytokine stimulation). Activation of STAT3 and STAT1 was determined as in A (B). Human whole blood was pre-incubated with or without CP-690,550 before stimulation with the indicated cytokines for 15 minutes. The extent of specific STAT phosphorylation within the CD3+ T cell population was assessed by intracellular flow cytometry using phospho-specific antibodies. Results are representative of 8 separate experiments (C).

Figure 2

Inhibition of JAK-mediated IL-6, IFN-γ and IL-12 signaling by CP-690,550. Mouse CD4+ T cells (A) or human whole blood CD3+ T cells (B) were pre-incubated with the indicated concentrations of CP-690,550 and stimulated with IL-6 for 15 minutes (ns, no cytokine stimulation). Activation of STAT3 or STAT1 was determined by phospho-specific antibodies and immunoblotting (A) or flow cytometry (B). Cytokine-induced STAT activation following JAK1 or JAK3 siRNA transfection of human CD4+ T cells was assessed by flow cytometry and represents the percentage of STAT phosphorylation compared to control siRNA-transfected cells (C). Data represents the mean ± SEM from 3 separate experiments. Mouse CD4+ T cells were pre-incubated with CP-690,550 at the indicated concentrations before stimulation with IFN-γ for 30 minutes (D) or IL-12 for 15 minutes (E). STAT activation was determined as in A (ns, no cytokine stimulation).

Figure 3

CP-690,550 inhibits Th2 and Th1 differentiation. Naïve mouse CD4+ T cells were stimulated with anti-CD3/anti-CD28 antibodies in the presence or absence of the indicated concentrations of CP-690,550, using either Th2 polarizing conditions (IL-4 and anti-IFN-γ) or Th1 polarizing conditions (IL-12 and anti-IL-4). On day 3, the expression of lineage-associated transcription factors GATA3 and T-bet was determined by quantitative RT-PCR. Results were normalized using β-actin transcripts and represent relative expression ± SEM, *P<0.001, (A). After 3 days in culture, cells were activated with PMA/ionomycin. Th cell differentiation and proliferation were assessed by intracellular cytokine staining and CFSE dilution. Th2 cell polarization was evaluated by IL-13 expression (B), and Th1 cell polarization by IFN-γ expression (C). Data are representative of 3 separate experiments. Naïve CD4+ T cells from wild type (WT) or STAT1-deficient (STAT1 KO) mice were activated with IL-12 and anti-IL-4 for 3 days in the presence or absence of either CP-690,550 or anti-IFN-γ neutralizing antibodies. Intracellular staining of PMA/ionomycin activated cells shows IFN-γ and T-bet expression (D). Data are representative of 3-4 separate experiments with similar results.

Figure 4

Modulation of Th17 differentiation by CP-690,550. Sorted naïve CD4+ T cells were activated for 3 days with anti-CD3/anti-CD28 antibodies in the presence of IL-6 in combination with either TGF-β1, IL-23 or TGF-β1 and IL-23. CP-690,550 was added to the cultures at the indicated concentrations. Expression of IL-17A and IL-2 was determined by intracellular cytokine staining after stimulation with PMA/ionomycin. The effect of CP-690,550 on IL-17A expression was also studied in conditions, where TGF-β1 signaling was blocked by neutralizing antibodies. Representative flow cytometry plots (A) and summarized data of 3-4 separate experiments are shown (B). Expression of Rorc, Ahr and Il23r in cells activated as in A was determined by quantitative RT-PCR. Results were normalized using β-actin transcripts and represent fold increase of expression (mean ± SEM) compared to Th0 conditions (C).

Figure 5

CP-690,550 inhibits the differentiation of IL-23-induced Th17 cells and associated cytokines. Sorted naïve CD4+ T cells were activated in serum-free media in the absence or presence of CP-690,550 with anti-CD3/anti-CD28 antibodies and the combination of IL-6, IL-1β and either TGF-β1 or IL-23. After 4 days of culture cells were restimulated with PMA/ionomycin and the expression of IL-17A, IL-17F and IL-22 was assessed by flow cytometry (A). Expression of Il21 (B) and Tbx21 (C) was determined by quantitative RT-PCR. Results were normalized using β-actin transcripts and represent relative expression (mean ± SEM, B, *P<0.001, C, *P<0.01).

Figure 6

Rapid amelioration of established arthritis and inflammation by CP-690,550. Mice with fully developed CIA were orally administrated vehicle of CP-690,550 at 50 mg/kg b.i.d. beginning on day 48 post immunization (arrow). Data represent the mean ± SEM severity score from 8 mice in each group (A). Plasma concentrations of SAA, G-CSF, IL-6, CXCL1 (KC), CCL2 (MCP-1) and CXCL10 (IP-10) were measured 4 h after dosing on day 48 (B). Relative expression of STAT1-induced genes in paw tissue 4 h after dosing on day 48 (C). Usp18, ubiquitin specific peptidase 18; Isg15, interferon-stimulated gene 15; Oas1a, 2′-5′ oligoadenylate synthase 1A; Mx1, myxovirus resistance protein 1. Quantitative RT-PCR results were normalized to cyclophilin and represent mean ± SEM relative expression from non-diseased animals (normal), as well as vehicle and CP-690,550 treated mice with CIA (C). Cellular infiltration of inflamed paw tissue in mice treated as in A was assessed by histology and IHC at the indicated time points (D). Data represents the mean ± SEM score for all 4 paws from 8 mice per treatment group. For all panels, *P<0.01 and **P<0.001. Representative histology (H&E) and F4/80 or CD3 IHC of paw tissue sections collected 7 days after onset of treatment with vehicle or CP-690,550 (E).

Figure 7

CP-690,550 blocks innate JAK signaling in vivo. Mice were administered single doses of CP-690,550, whole blood was collected after 1 hour (∼Cmax) and stimulated ex vivo with the indicated cytokines. STAT phosphorylation expressed as percent of control cells (from non-treated mice) is plotted against plasma drug exposure in the same sample (A). Each data point represents an individual mouse from 1 of 4 separate experiments. EC50 corresponded to 470 nM, 273 nM and 6656 nM for JAK1/JAK2, JAK1/JAK3 and JAK2 inhibition, respectively. JAK inhibition following chronic dosing in mice induced with CIA (B). Data respresent the mean ± SEM JAK inhibition or mean AUC for efficacy from 1 of 2 separate studies involving 5 mice at each dose. Plasma cytokine levels were assessed 1, 2 and 6 hours after a 10 μg i.p. administration of LPS to DBA/1J mice pre-treated for 1 h with or without 5 mg/kg CP-690,550 (C). Time courses of TNF, IL-6, IL-12p70, IL-10, IFN-γ and IL-1β production in plasma are shown. Data represent plasma cytokine concentrations (mean ± SEM) from 1 of 2 separate studies with similar results involving 8 mice at each dose.