Regulation of inflammatory responses in tumor necrosis factor-activated and rheumatoid arthritis synovial macrophages by JAK inhibitors

Abstract

Objective: JAK inhibitors have been developed as antiinflammatory and immunosuppressive agents and are currently undergoing testing in clinical trials. The JAK inhibitors CP-690,550 (tofacitinib) and INCB018424 (ruxolitinib) have demonstrated clinical efficacy in rheumatoid arthritis (RA). However, the mechanisms that mediate the beneficial actions of these compounds are not known. The purpose of this study was to examine the effects of both JAK inhibitors on inflammatory and tumor necrosis factor (TNF) responses in human macrophages.

Methods: In vitro studies were performed using peripheral blood macrophages derived from healthy donors and treated with TNF and using synovial fluid macrophages derived from patients with RA. Levels of activated STAT proteins and other transcription factors were detected by Western blotting, and gene expression was measured by real-time polymerase chain reaction analysis. The in vivo effects of JAK inhibitors were evaluated in the K/BxN serum-transfer model of arthritis.

Results: JAK inhibitors suppressed the activation and expression of STAT-1 and downstream inflammatory target genes in TNF-stimulated and RA synovial macrophages. In addition, JAK inhibitors decreased nuclear localization of NF-κB subunits in TNF-stimulated and RA synovial macrophages. CP-690,550 significantly decreased the expression of interleukin-6 in synovial macrophages. JAK inhibitors augmented nuclear levels of NF-ATc1 and cJun, followed by increased formation of osteoclast-like cells. CP-690,550 strongly suppressed K/BxN serum-transfer arthritis, which is dependent on macrophages, but not lymphocytes.

Conclusion: Our findings demonstrate that JAK inhibitors suppress macrophage activation and attenuate TNF responses and further suggest that suppression of cytokine/chemokine production and innate immunity contribute to the therapeutic efficacy of JAK inhibitors.

Figure 1

Dose-dependent effect of JAK inhibitors on STAT signaling in human macrophages. Human MΦs were stimulated with IFNα (A) or IFNγ (B) or left untreated in the presence of increasing concentrations of JAK inhibitors CP-690,550 or INCB018424 for 15 min and nuclear extracts were analyzed by immunoblotting for expression and tyrosine phosphorylation of STAT proteins. Data are representative of two independent experiments.

Figure 2

JAK inhibitors downregulate TNF-induced STAT1 activation and expression of STAT1-dependent genes. (A) Human MΦs were stimulated with TNF in the presence of increasing concentrations of CP-690,550 or INCB018424 for 6 h and expression of STAT1 dependent genes was analyzed by qPCR. Data are representative of two independent experiments. (B and C) MΦs were stimulated with TNF for indicated times in the presence or absence of JAK inhibitors and mRNA expression of STAT1-dependent chemokines (B) and interferon response genes (C) was measured by qPCR. Target gene expression is presented relative to expression in untreated cells 1h after TNF simulation (set as 1). Results of six (CP) and five (INCB) independent experiments (mean ± SD) are shown. *, p<0.05; **, p<0.01; ***, p<0.001 (repeated-measures two-way ANOVA with Bonferroni post hoc test for multiple comparisons). (D) Human MΦs were stimulated with TNF (+) or left untreated (−) in the presence (+) or absence (−) of CP-690,550 or INCB018424 for indicated times and tyrosine phosphorylated- and total STAT1 in nuclear (upper panels) and cytoplasmic (lower panels) extracts was analyzed by immunoblotting. Data are representative of five independent experiments.

Figure 3

Jak inhibitors augment TNF-induced expression of NFATc1 and cJun, and increase formation of osteoclast-like cells. Human macrophages were stimulated with TNF (+) or left untreated (−) in the presence (+) or absence (−) of CP-690,550 or INCB018424. Nuclear extracts were analyzed for expression of NFATc1 (A) and cJun (B, left). Data are representative of 5 independent experiments. (B) Right, the intensity of cJun bands at 24 hours was quantified by densitometry. Results of five independent experiments (mean ± SEM) are shown. (C) Quantification of TNF-induced TRAP+ multinuclear cells. Cells were fixed and stained for TRAP activity after 8–12 days of culture. Each symbol indicates an independent experiment, n=11; small horizontal lines, mean; **, p<0.01 (one-way ANOVA with Dunn post hoc test). (D) Quantification of TNF-induced resorption. Results of four independent experiments (mean ± SEM) are shown; *, p<0.05 (one-way ANOVA with Dunn post hoc test).

Figure 4

Effect of JAK inhibition on the expression of inflammatory cytokines and NF-κB signaling. Human MΦs were stimulated with TNF (+) or left untreated (−) in the presence (+) or absence (−) of CP-690,550 or INCB018424 for the indicated times. Data are representative of six (CP) and five (INCB) independent experiments. (A) and (B, upper panel) TNF, IL-6 and IL-1β mRNA levels were measured by qPCR. Gene expression is presented relative to expression in untreated cells 1h after TNF simulation (set as 1). (B) Lower panel, effects of CP-690,550 and INCB018424 on IL-1β mRNA expression 48 h after TNF stimulation. Each symbol indicates an independent experiment; n=5; *, p<0.05 (Wilcoxon matched-pairs signed rank test). (C) Nuclear extracts were analyzed by immunoblotting for expression of NF-κB subunits (upper panel). Data are representative of three (p-p65) and five (RelB and p52) independent experiments. The intensity of RelB and p52 bands was quantified by densitometry (lower panel). Results of five independent experiments (mean ± SEM) are shown; *, p<0.05 (one-way ANOVA with Dunn post hoc test).

Figure 5

Effects of JAK inhibitors on synovial macrophages isolated from patients with RA. CD14+ cells were isolated from synovial fluids of patients with RA and were cultured for 24 h with (+) or without (−) CP-690,550 or INCB018424. (A) Gene expression in JAK inhibitor-treated cells shown as percent of expression in untreated cells (set at 100%, shown as dotted line). Results of seven (CP) and six (INCB) independent experiments are shown (mean ± SEM). *, p<0.05 (Wilcoxon matched-pairs signed rank test). (B) Synovial MΦs isolated from patients with RA (lanes 3 –7) were treated as in (A) and nuclear extracts were analyzed by immunobloting for expression of transcription factors. Human donor MΦs (HD) cultured with TNF (+) for 24 hours (lane 2) or 72 hours (lane 8) served as a positive control, whereas cells cultured without TNF (−) for same time (lanes 1 and 9) represent a negative control.

Figure 6

CP-690,550 attenuates K/BxN serum induced arthritis. Arthritis was induced in C57BL/6 mice as described in Methods. (A) Time course of arthritis development with or without CP-690,550 treatment. Data represents means ± SEM from 5 mice in each group. *, p<0.05; **, p<0.01; ****, p<0.0001 (repeated-measures two-way ANOVA with Bonferroni post test for multiple comparisons). (B) Representative histology of ankle joints. Animals were sacrificed on day 9 after first serum injection, hind legs were fixed and decalcified, and ankle joint sections were stained with hematoxylin, fast green and safranin O. Original magnification, 40 ×. Bigger arrows, synovium; asterisks, bone; smaller arrows, cartilage.