Inhibition of BET Proteins Regulates Fcγ Receptor Function and Reduces Inflammation in Rheumatoid Arthritis

Abstract

Overactivation of immune responses is a hallmark of autoimmune disease pathogenesis. This includes the heightened production of inflammatory cytokines such as Tumor Necrosis Factor α (TNFα), and the secretion of autoantibodies such as isotypes of rheumatoid factor (RF) and anticitrullinated protein antibody (ACPA). Fcγ receptors (FcγR) expressed on the surface of myeloid cells bind Immunoglobulin G (IgG) immune complexes. Recognition of autoantigen-antibody complexes by FcγR induces an inflammatory phenotype that results in tissue damage and further escalation of the inflammatory response. Bromodomain and extra-terminal protein (BET) inhibition is associated with reduced immune responses, making the BET family a potential therapeutic target for autoimmune diseases such as rheumatoid arthritis (RA). In this paper, we examined the BET inhibitor PLX51107 and its effect on regulating FcγR expression and function in RA. PLX51107 significantly downregulated expression of FcγRIIa, FcγRIIb, FcγRIIIa, and the common γ-chain, FcϵR1-γ, in both healthy donor and RA patient monocytes. Consistent with this, PLX51107 treatment attenuated signaling events downstream of FcγR activation. This was accompanied by a significant decrease in phagocytosis and TNFα production. Finally, in a collagen-induced arthritis model, PLX51107-treatment reduced FcγR expression in vivo accompanied by a significant reduction in footpad swelling. These results suggest that BET inhibition is a novel therapeutic approach that requires further exploration as a treatment for patients with RA.

Keywords: BET inhibition; FcγR; monocytes; rheumatoid arthritis.

Figure 1

BET inhibition reduces FcγR transcript in human monocytes. Healthy donor CD14+ monocytes were cultured for 24 h with PLX51107 (50, 100, 200, 250 nM) or with vehicle control (DMSO). Transcript expression was analyzed by real-time PCR for (A) FcγRIIa, (B) FcγRIIb, (C) FcγRIIIa, (D) FcεRIγ, and (E) FcγRIa. (n = 8). * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001 respectively.

Figure 2

BET inhibition reduces FcγR protein expression in human monocytes. Healthy-donor CD14⁺ monocytes were cultured for 24 h with PLX51107 (50, 100, 200, 250 nM) or with vehicle control (DMSO) for 24 h. Whole cell lysates were then obtained, and protein expression was measured by immunoblotting (IB). Expression of (A) FcγRIIa, (B) FcγRIIb, (C) common Fc γ chain (FcεRIγ), and (D) FcγRIa were analyzed. Top panels show a representative western blot and densitometry quantifications are shown in the bottom panels. Calreticulin or GAPDH were used as loading controls (n = 5, n = 3 for FcγRIa). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 3

BET inhibition reduces FcγR surface expression in healthy human monocytes. Healthy-donor CD14⁺ monocytes were cultured for 24 h with PLX51107 (50, 100, 200, 250 nM) or with vehicle control (DMSO) for 24 h. Then surface expression of (A) FcγRIIa, (B) FcγRIIIa, (C) total FcγRs and, and (D) FcγRIa was quantified by flow cytometry. Top panels show mean fluorescence intensities (n = 4), while bottom panels show representative histograms. * p ≤ 0.5, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 respectively.

Figure 4

BET inhibition downregulates FcγR-mediated signaling. Healthy-donor CD14⁺ monocytes were treated with PLX51107 (250 nM) or vehicle control (DMSO) for 24 h and then subsequently activated with heat-aggregated IgG for 7 min. Whole cell lysates were obtained, and immunoblotting was done to detect activation of the (A) NK-κB, (B) MEK1/2 and (C) PI3K pathways with phospho-specific antibodies. GAPDH was used as loading controls. Representative western blots are shown (n = 3).

Figure 5

BET inhibition attenuates monocyte FcγR function. Healthy-donor PBMs were treated with PLX51107 for 24 h and then analyzed for antibody-mediated functions. (A) Treated CD14⁺ monocytes were incubated with IgG-opsonized sheep RBCs (SRBCs). Phagocytic index was measured using microscopy and defined as the total number of ingested RBCs per 100 CD14⁺ monocytes (n = 5). (B) PLX51107 treated CD14⁺ monocytes were incubated on ice with IgG opsonized SRBCs. Rosetting index was measured using microscopy and defined as total number of surface embedded RBCs per 100 CD14⁺ monocytes (n = 4). (C) After BET inhibition, PBMs were stimulated with heat-aggregated IgG for additional 24 h; supernatants were collected and TNFα levels were measured by ELISA (n = 5). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 respectively.

Figure 6

BET inhibition regulates FcγR expression and function in monocytes from rheumatoid arthritis patients. CD14⁺ monocytes from RA patients were treated with PLX51107 (50 nM, 250 nM) or vehicle control (DMSO) for 48 h and mRNA expression of (A) FcγRIIa, (B) FcγRIIb, (C) FcγIIIa, (D) FcεRIγ, and (E) FcγRIa, was analyzed by real-time PCR (n = 6). To analyze antibody-mediated functions, 24-h, PLX51107-treated RA patient CD14⁺ monocytes were (F) incubated at 37 °C with IgG opsonized sheep RBCs (SRBCs). Phagocytic index was measured using microscopy and defined as number of ingested RBCs per 100 monocytes (n = 4). (G) Similarly, PLX51107-treated RA patient CD14⁺ monocytes were stimulated with plate-bound IgG for another 24 h; supernatants were collected and TNFα levels were measured by ELISA; TNFα levels in control (DMSO) or PLX51107 conditions are shown for 6 different patients, represented as individual values (left) and a graph (right). * p ≤ 0.05, ** p ≤ 0.01 respectively.

Figure 7

BET inhibition reduces footpad inflammation and FcγR expression in vivo. DBA mice were injected with collagen type II as indicated in the methods to induce rheumatoid arthritis. After 2 weeks from the last collagen injection, mice were treated with vehicle or PLX51107 (iBET) at 10mg/kg three times a week for 5 weeks. During the treatment, (A) footpad swelling was measured every week, showing the average between left and right front foot (n = 10 per group). Mice were then sacrificed 2 weeks after the last treatment, spleens obtained and used to detect expression of FcγRs in (B) monocytes, and (D) macrophages, as well as the percentage of (C) monocytes and macrophages. Graphs show the mean fluorescence intensities, as well as representative histograms below. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 respectively.