The immunomodulatory effects of TNF-α inhibitors on human Th17 cells via RORγt histone acetylation

Abstract

The presence of interleukin (IL)-17-related cytokines correlates with rheumatoid arthritis (RA) pathogenesis. Epigenetic modifications, including histone acetylation, regulate gene expression in RA pathogenesis. Tumour necrosis factor-alpha (TNF-α) inhibitors such as etanercept and adalimumab, represent a breakthrough in RA treatment. We aimed to investigate the effects of etanercept and adalimumab on human Th17-polarized cells and the possible intracellular regulators of these effects, including the Th17-specific transcription factors signal transducer, activator of transcription 3 (STAT3), retinoid-related orphan receptor γ-T (RORγt) and epigenetic modification. Human CD4+ T cells from healthy subjects and patients with RA were pretreated with TNF-α inhibitors and then being polarized into IL-17-producing cells. The Th17-related cytokine levels in the culture supernatants were determined with an enzyme-linked immunosorbent assay. Intracellular signalling was investigated by western blot, real-time RT-PCR, and chromatin immunoprecipitation. Th17-polarized cells from patients with RA produced more IL-17A, IL-17F and IL-22 than those from healthy subjects. Etanercept and adalimumab suppressed IL-17A, IL-17F and IL-22 levels in Th17-polarized cells from healthy subjects and patients with RA. Western blot analysis revealed that etanercept and adalimumab decreased mitogen-activated protein kinase-phospho-p38, nuclear factor-κB-phospho-p65, phospho-STAT3 and RORγt levels. Etanercept and adalimumab decreased histone (H)3 and H4 acetylation in the RORγt gene promotor region by decreasing the recruitment of the acetyltransferases p300, CBP and PCAF. The present study broadens our knowledge of the mechanisms underlying the immunomodulatory effects of TNF-α inhibitors in rheumatoid arthritis treatment.

Keywords: RORγt; TNF-α; Th17; histone acetylation; rheumatoid arthritis.

Figure 1. Etanercept and adalimumab suppress IL-17A and IL-17F production in human Th17-polarized T cells

Human naïve CD4⁺ T cells polarized towards the Th17 phenotype in the presence of rhIL-2, rhIL-1β, rhIL-23, rhTGF- β and rhIL-6 with anti-hCD3, anti-hCD28, anti-hIL-4, and anti-hINF-γ, and then were cultured for 3 days or 5 days. Control naïve T cells were cultured only in the presence of anti-hCD3, anti-hCD28 and rhIL-2. The supernatants were collected for (A) IL-17A and (B) IL-17F detection by ELISA. The results represent the means ± standard deviations of three independent experiments. Pretreatment with etanercept (0.1 and 1 μg/ml) or adalimumab (1 and 10 μg/ml) suppressed (C) IL-17A and (D) IL-17F production in human CD4⁺ T cells from a healthy individual after 5 days of Th17-polarized conditions. The results represent the means ± standard deviations of three experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 between the Th17-polarized conditions with and without TNF-α inhibitor pretreatment. (E) The viability of human CD4⁺ T cells pretreated with or without etanercept (0.1 and 1 μg/ml) or adalimumab (1 and 10 μg/ml) was determined after 5 days of Th17 polarization using the WST-1 assay and expressed as a percentage of the control. The results represent the means ± standard deviations of 5 individual experiments.

Figure 2. The effects of etanercept and adalimumab on IL-17A, IL-17F and IL-22 production in Th17-polarized cells from patients with RA

The levels of Th17-related cytokines, including (A) IL-17A, (B) IL-17F and (C) IL-22, in the supernatants of Th17-polarized cells from four healthy donors (HD) and six patients with RA that were pretreated in vitro with or without etanercept (1 μg/ml) or adalimumab (1 or 10 μg/ml) were determined by ELISA. Horizontal bars indicate the median. *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 3. The suppressive effects of etanercept and adalimumab on IL-17A, IL-17F and IL-22 expression in human Th17-polarized cells through MAPK pathways

(A) Human Th17-polarized cells were induced from purified CD4⁺ T cells from healthy subjects. Pretreatment with SB203580 (a p38 inhibitor, 10⁻⁶–10⁻⁵ M), SP600125 (a JNK inhibitor, 10^-5 M) or PD98059 (an ERK inhibitor, 10⁻⁵ M) significantly suppressed IL-17A expression in Th17-polarized cells. (B) SB203580 (10^-6 M) and SP600125 (10⁻⁵ M) significantly suppressed IL-17F expression in human Th17-polarized cells. (C) Pretreatment with SB203580 (10⁻⁵ M), SP600125 (10⁻⁶–10⁻⁵ M) and PD98059 (10⁻⁶–10⁻⁵ M) could significantly suppress IL-22 expression in Th17-polarized cells. ^#P < 0.05 for the comparison of Th17-polarized conditions with and without MAPK inhibitor pretreatment. (D) Etanercept (0.1 and 1 μg/ml) and (E) adalimumab (1 and 10 μg/ml) decreased pp38 levels in human Th17-polarized cells. (F) Etanercept (0.1 and 1 μg/ml) but not (G) adalimumab decreased pERK levels in human Th17-polarized cells. For western blot analysis, the standard deviations of the optical density data were calculated for three independent experiments, and one experiment representative of the set of three is shown. *P < 0.05 for the comparison of Th17-polarized conditions with and without etanercept or adalimumab pretreatment.

Figure 4. Etanercept and adalimumab downregulate NFκB and STAT3 expression in human Th17-polarized cells

(A) Th17-polarized cells were induced from purified CD4⁺ T cells from healthy subjects. Etanercept at 1 μg/ml and (B) adalimumab at 1 and 10 μg/ml decreased pp65 levels in human Th17-polarized cells. (C) Etanercept at 0.1 and 1 μg/ml and (D) adalimumab at 10 μg/ml decreased pSTAT3 levels in human Th17-polarized cells. For western blot analysis, the standard deviations of the optical density data were calculated for three independent experiments, and one experiment representative of the set of three is shown. *P < 0.05 for the comparison of Th17-polarized conditions with or without etanercept or adalimumab pretreatment.

Figure 5. Etanercept and adalimumab downregulate RORγt expression in human Th17-polarized T cells via histone acetylation

Western blot analysis revealed that (A) etanercept (0.1 and 1 μg/ml) and (B) adalimumab (1 and 10 μg/ml) suppressed RORγt expression 24 h after Th17 polarization. (C) Real-time quantitative PCR was used to evaluate RORγt mRNA expression in CD4⁺ T cells after the cells were cultured under Th17-polarized conditions for 12 h with or without pretreatment with TNF-α inhibitors for 2 h. The results represent the means ± standard deviations of three independent experiments. (D) Pretreatment with anacardic acid (an acetyltransferase inhibitor) suppressed RORγt expression 24 h after Th17 polarization. For western blot analysis, the standard deviations of the optical density data were calculated from three independent experiments, and one experiment representative of the set of three is shown. *P < 0.05, **P < 0.01 and ***P < 0.001 between Th17-polarized conditions with and without TNF-α inhibitor pretreatment. ChIP analysis revealed that histone (E) H3 and (F) H4 acetylation was decreased by etanercept (1 μg/mL) and adalimumab (10 μg/mL) 6 h after Th17-polarization. The standard deviations of the ChIP data were calculated from four independent experiments. ^#P < 0.05.

Figure 6. The effects of etanercept and adalimumab on NFκB-associated acetyltransferases in Th17-polarized cells

Etanercept (1 μg/mL) decreased the recruitment of the NFκB-associated acetyltransferases (A) p300, (B) CBP and (C) PCAF in Th17-polarized cells induced from purified CD4⁺ T cells (naïve T cells). Adalimumab (10 μg/mL) decreased the recruitment of the NFκB-associated acetyltransferases (D) p300, (E) CBP and (F) PCAF in Th17-polarized cells. The standard deviations of the ChIP data were calculated from four independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 7. The in vitro effects of etanercept and adalimumab on Th17-related cytokine production by Th17-polarized cells from an RA patient undergoing Enbrel™ treatment

Th17-polarized cells induced from CD4⁺ T cells that were collected from an RA patient 2 h before (at the trough level) and 48 h after (at the peak level) of Enbrel™ injection. The in vitro effects of etanercept (1 μg/mL) and adalimumab (1 and 10 μg/mL) on IL-17A, IL-17F and IL-22 production in Th17-polarized cells were determined by ELISA. P-values indicate comparison of the cytokine levels before and after Enbrel™ injection. (A) IL-17A production by Th17-polarized cells isolated from the RA patient 2 h before and 48 h after Enbrel™ injection was significantly suppressed by in vitro treatment with adalimumab (1 and 10 μg/mL) but not etanercept (1 μg/mL). (B) IL-17F and (C) IL-22 production was significantly suppressed by in vitro treatment with etanercept (1 μg/mL) and adalimumab (1 μg/mL) when cells were isolated from the RA patient 2 h before but not 48 h after Enbrel™ injection. In vitro pretreatment with adalimumab (10 μg/mL) significantly suppressed IL-17F and IL-22 production both before and after Enbrel™ injection. The bars represent the means ± standard deviations from three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 between the Th17-polarized cells with and without in vitro etanercept or adalimumab pretreatment.

Figure 8. Schematic of the proposed intracellular mechanisms underlying TNF-α inhibitor regulation in Th17 cells polarized from CD4+ T cells