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. 2015 Nov;104(6):733-42.
doi: 10.1002/bip.22692.

Inhibition of cell adhesion and immune responses in the mouse model of collagen-induced arthritis with a peptidomimetic that blocks CD2-CD58 interface interactions

Ameya S Gokhale  1 , Rushikesh Sable  1 , Jason D Walker  2 , Leslie McLaughlin  2 , Konstantin G Kousoulas  2 , Seetharama D Jois  1
Affiliations

Inhibition of cell adhesion and immune responses in the mouse model of collagen-induced arthritis with a peptidomimetic that blocks CD2-CD58 interface interactions

Ameya S Gokhale et al. Biopolymers. 2015 Nov.

Abstract

CD2 and CD58 are two important costimulatory molecules involved in generating the signal II required for normal immune signaling. However, this interaction can be targeted to be of benefit in cases of abnormal immune signaling seen in autoimmune diseases. Our objective in this study was to design a peptidomimetic (compound 7) based on a β-strand structure of the adhesion domain of CD2 protein to inhibit CD2-CD58 protein-protein interaction and its effect on immunomodulation in the collagen-induced arthritis (CIA) model. The ability of compound 7 to bind to CD58 protein was assessed using flow cytometry. The effect of compound 7 on modulating the immune response was evaluated in an autoimmune disease using CIA in mice. The stability of compound 7 was evaluated in mouse serum using mass spectrometry. Antibody (Ab) binding inhibition studies suggested that compound 7 binds to CD58 protein. Compound 7 was successful in modulating immune responses when administered in the CIA mouse model along with reducing anti-collagen Ab levels and decreasing the level of interferon gamma (IFN-γ) relative to control treatments. Compound 7 was found to be nonimmunogenic and stable in mouse serum up to 48 h. Results suggest that compound 7 can serve as a lead compound for immunomodulation, and could be a therapeutic agent for the autoimmune disease rheumatoid arthritis (RA).

Keywords: costimulatory molecules; immunomodulation; peptidomimetic; protein−protein interaction; rheumatoid arthritis.

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Figures

FIGURE 1
FIGURE 1
Schematic diagram showing the importance of adhesion molecules CD2-CD58 in immune response and inhibition of protein-protein interactions of CD2-CD58 by compound 7 and modulation of immune response.
FIGURE 2
FIGURE 2
Structure of compound 7.
FIGURE 3
FIGURE 3
Competitive binding of compound 7 with FITC-labeled antibody to Caco-2 cells expressing CD58 protein. Antibody binding to Caco-2 cells in the presence of compound 7 at different concentrations is shown. 1 × 106 cells/100 μL were redistributed into different wells of 96-well plates. After 24 h, attached cells were incubated with FITC-CD58Ab (pre-diluted, 20 μL) and/or compound 7 for 1 h. The plate was washed 3 times after incubation and fluorescence was read using a microplate reader at excitation λ 485 nm and emission λ 528 nm. Fluorescence from the blank was subtracted for representation. Data are from triplicate experiments.
FIGURE 4
FIGURE 4
Competitive binding of compound 7 with FITC-labeled antibody to Caco-2 cells expressing CD58 monitored by flow cytometry (BD FACS Calibur). Cells were distributed in 7 different vials and added with 50 μL of FITC-labeled anti-CD58 as positive control, 50 μL of FITC-CD58Ab along with 100 μL of compound 7 at concentrations of 10, 50, 100 and 200 μM and controls. The shift in number of cells with or without FITC-CD58Ab was observed. Histograms show Caco-2 cells without peptide or antibody (more than 80% of the unstained cells are observed), cells with FITC-AbCD58 (there was a shift of cell population to the right and nearly 75% of cells were stained), and cells with compound 7 and FITC-CD58Ab at compound 7 concentrations of 10, 50, 100, and 200 μM, respectively. Note that in the presence of compound 7 more Caco-2 cells without antibody labeling (cell population shifted to left compared to cells + FITC antibody) were observed, suggesting the inhibition of FITC-CD58Ab to Caco-2 cells. A vertical dotted line was drawn for comparison of shifts of cells.
FIGURE 5
FIGURE 5
Suppression of collagen-induced arthritis by compound 7. Treatment of mice by compound 7 shows suppression of collagen-induced arthritis (statistically significant) from days 40–46 at concentrations of 0.5 mg/kg and 1 mg/kg when compared to control peptide and vehicle. Scoring (0–4) was done according to the published procedure as described in the text for all the four limbs with a maximum score of 16. For plotting, the average score from 8 mice was used.
FIGURE 6
FIGURE 6
Histopathological analysis of samples from hind limbs of normal, arthritic, and treated DBA/1 mice. Sections of paw (interphalangeal joints) and hind limb (tarsal joints) from 12 mice from different groups were examined. Representative sections from the tarsal joints of three mice are shown above. (A) Normal tarsocrural joint (tibia and talus). Articular cartilaginous surfaces of both bones are intact and smooth (arrow heads) and the joint space is clear (*); H&E staining, 200×. (B) Arthritic proximal intertarsal joint (talus and calcaneus) with grade 4 arthritis. Abundant pannus with thick articular cartilage in some areas (#) and cartilage ulceration in others (thick arrows); periosteal bone proliferation (not shown); moderate inflammatory infiltrates consisting of histiocytes, plasma cells, and neutrophils. H&E staining, 100×. (C) Proximal intertarsal joint (central and fourth tarsal bones) of mouse treated with compound 7 (0.5mg/kg). Severe pannus with moderate multifocal articular cartilage erosion and ulceration (thick arrows); bone lysis and destruction (thin arrows) with periosteal proliferation; histiocytic and neutrophilic infiltration. H&E staining; 100×
FIGURE 7
FIGURE 7
A) Relative amounts of collagen II antibody in terms of absorbance was measured in different treatment groups. Serum was collected from blood samples of mice that developed arthritis and treatment groups as well as controls. These samples were added in triplicate into each well (50 μL). Immulon II plates were coated with bovine type II collagen and incubated. After washing, bound anti-collagen Abs were determined using HRP-labeled goat anti-mouse IgG Readings were in triplicate, and the experiment was repeated twice. The figure clearly indicates the difference between collagen II Ab levels in the arthritis group those in groups treated with compound 7. Control peptide groups also showed higher collagen II Ab levels than the compound 7-treated groups. B) Levels of IFN-γ were measured in different groups of DBA/1 mice using BD biosciences bead array method and flow cytometry. The compound 7-treated group at 1 mg/kg showed significant reduction in levels of IFN-γ compared to the arthritic group. C) Cells from spleens of mice primed and challenged with compound 7 for antigenicity. DBA-1 female mice were immunized with 100 μg of native type II collagen emulsified with CFA 12 days prior to the actual day of assay. Antigen-presenting cells were generated from naïve mice without type II collagen injection. Cells were isolated from the immunized and control mice. After harvesting the cells, compound 7 was added at different concentrations. Plates were incubated for 45 min in a CO2 incubator at 37 °C. This was followed by the final step of reading the fluorescence using a celltiter-glo (CTG) assay (Promega, Inc.) with a plate reader. Con A was used as positive control (p<0.05).
FIGURE 8
FIGURE 8
Stability of compound 7 in serum by MALDI-TOF. Y-axis represents amount of intact compound 7 remaining. 200 μL of stock solutions of compound 7 was treated with 1 mL mouse serum. At different time points ranging from 0 min to 48 h, 100 μL of the above sample was withdrawn and treated with 500 μL of chilled acetonitrile for compound 7 extraction. These samples were analyzed by TOF-MS. The spectra were acquired after calibration of the instrument with a peptide standard. A minimum of 500 laser shots per sample was used to generate each mass spectrum. Leuprolide was used as an internal standard against which the sample peaks were ing 0.05% Tweenive intensity is plotted with respect to time, and data are from triplicate experiments.
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