Bim-Bcl-2 homology 3 mimetic therapy is effective at suppressing inflammatory arthritis through the activation of myeloid cell apoptosis

Abstract

Objective: Rheumatoid arthritis (RA) is a destructive autoimmune disease characterized by an increased inflammation in the joint. Therapies that activate the apoptotic cascade may have potential for use in RA; however, few therapeutic agents fit this category. The purpose of this study was to examine the potential of Bim, an agent that mimics the action of Bcl-2 homology 3 (BH3) domain-only proteins that have shown success in preclinical studies of cancer, in the treatment of autoimmune disease.

Methods: Synovial tissues from RA and osteoarthritis patients were analyzed for the expression of Bim and CD68 using immunohistochemistry. Macrophages from Bim(-/-) mice were examined for their response to lipopolysaccharide (LPS) using flow cytometry, real-time polymerase chain reaction analysis, enzyme-linked immunosorbent assay, and immunoblotting. Bim(-/-) mice were stimulated with thioglycollate or LPS and examined for macrophage activation and cytokine production. Experimental arthritis was induced using the K/BxN serum-transfer model. A mimetic peptide corresponding to the BH3 domain of Bim (TAT-BH3) was administered as a prophylactic agent and as a therapeutic agent. Edema of the ankles and histopathologic analysis of ankle tissue sections were used to determine the severity of arthritis, its cellular composition, and the degree of apoptosis.

Results: The expression of Bim was reduced in RA synovial tissue as compared with controls, particularly in macrophages. Bim(-/-) macrophages displayed elevated expression of markers of inflammation and secreted more interleukin-1beta following stimulation with LPS or thioglycollate. TAT-BH3 ameliorated arthritis development, reduced the number of myeloid cells in the joint, and enhanced apoptosis without inducing cytotoxicity.

Conclusion: These data demonstrate that BH3 mimetic therapy may have significant potential for the treatment of RA.

Figure 1. Bim expression in RA and OA synovial tissue

(A) Bim expression is decreased in RA compared to OA synovial tissue. Representative photomicrographs of adjacent RA (n=8) and OA (n=8) synovial tissue sections stained for Bim (brown) or CD68 (brown) and counterstained with hematoxylin (blue). (B) Quantitative analysis of expression of Bim in RA and OA synovial tissue. RA and OA synovial tissue adjacent sections stained for Bim and CD68 were scored by a pathologist blinded to the study.

Figure 2. Deficiency for Bim leads to increased activation of macrophages

(A) Increased LPS-induced expression of activation markers on Bim−/− BMDMs. Wt and Bim−/− macrophages stimulated with LPS were examined by flow cytometry. Data are representative of four independent experiments. (B) Thioglycollate elicited Bim−/− peritoneal macrophages exhibit enhanced expression of markers of activation. Wt (n=4/time point) and Bim−/− (n=4/time point) peritoneal macrophages were isolated prior and following thioglycollate injection and analyzed by flow cytometry.

Figure 3. Increased synthesis of IL-1β in Bim−/− macrophages

(A) The mRNA levels of IL-1β are increased in Bim−/− BMDMs following LPS stimulation. Macrophages stimulated with LPS were examined over time for IL-1β and GAPDH by real time PCR. Data are representative of two independent experiments. (B) Bim−/− macrophages secrete increased IL-1β production following stimulation with LPS. LPS-treated macrophages were examined for IL-1β secretion using ELISA. * indicates p<0.05. (C) Bim−/− mice have increased in vivo production of IL-1β following LPS injection. Serum from untreated (Wt, n=21: Bim−/−, n=15) or LPS injected (Wt, n=12: Bim−/−, n=7) mice were examined for IL-1β levels using a Luminex based assay.

Figure 4. TAT-BH3 peptide is effective as a (A) prophylactic or as a (B) therapeutic

For prophylactic (saline, n=24; TAT-inactive BH3, n=16; or TAT-BH3 peptide, n=32) and therapeutic study (saline, n=36; TAT-inactive BH3, n=18; or TAT-BH3 peptide, n=22). (C) Representative photomicrographs of ankle sections from the prophylactic study. C= cartilage, B= bone, P=pannus, SL= synovial lining. (D, E) Histological scores of ankle sections from the prophylactic (saline, n=8);TAT-inactive BH3, n=16; TAT-BH3 peptide, n=18 and therapeutic study (saline, n=20; TAT-inactive BH3, n=18;TAT-BH3, n=22). * =p<0.05 as compared to saline; †=p<0.05 as compared to TAT-inactive BH3.

Figure 5. TAT-BH3 peptide induces apoptosis in myeloid cells

Peritoneal cells from mice treated with the prophylactic regimen were harvested at 6 hours post K/BxN serum injection, stained with annexin V-APC, anti-F4/80 antibody, and anti-Gr-1 antibody, and analyzed by flow cytometry. Shown are the cells gated for total population, FITC-TAT-BH3, or annexin V-APC.

Figure 6. Fewer myeloid cells are recruited to joints of TAT-BH3 peptide-treated arthritic mice

(A) Representative photomicrographs of macrophages in ankle sections from the prophylactic study. C=cartilage, B=bone, P=pannus, SL=synovial lining. Arrows represent F4/80-positive cells in the untreated mouse. (B, C) TAT-BH3 peptide-treated mice have decreased numbers of macrophages and PMNs. * =p<0.05 as compared to saline; †=p<0.05 as compared to TAT-inactive BH3. (D) Representative photomicrographs of TUNEL and Hoechst stained ankle sections from therapeutic study.