Immune cells, including T cells, B cells, and osteoclasts, in conjunction with their associated cytokines, have been studied as primary molecular therapeutic targets for the management of rheumatoid arthritis (RA) patients. The increase in cytosolic Ca2+ levels through the activation of store-operated Ca2+ release–activated channels (CRACs) is involved in mediating a disparate array of cellular responses by these immune cells. This study was undertaken to investigate the feasibility and efficiency of the regulation of Ca2+ entry in the treatment of RA. To moderately suppress Ca2+ entry via CRACs, we gene silenced CRACM3, which was induced by systemic application of specific short hairpin RNAs (shRNAs) using a lentiviral-delivery system, in a murine model of collagen-induced arthritis (CIA). The inflammatory responses were determined by measuring the levels of a panel of cytokines and chemokines in the joints and serum. Ag-specific responses were evaluated by determining the cytokine profile of T cells stimulated with autoantigen. We also analyzed the ability of specific CRACM3-shRNA to regulate mature osteoclast function in CIA mice. The therapeutic effect of lentiviral-delivered CRACM3-shRNA was associated with gene silencing of CRACM3, along with the successful biodistribution of the virus. Extracellular Ca2+ influx in the splenocytes, thymocytes, and knee joint synovial cells was moderately suppressed. Inflammatory responses and autoimmune responses were reduced by CRACM3 gene silencing. A decrease in mature osteoclast activity also was observed in CRACM3-shRNA–treated CIA mice. These results indicate that regulation of Ca2+ entry through lentivirus-mediated CRACM3 gene silencing is beneficial in the treatment of RA.

To reach a treatment target of remission or low disease activity in rheumatoid arthritis (RA) patients, the management of RA relies primarily on the use of disease-modifying antirheumatic drugs (DMARDs). DMARDs consist of two major classes: synthetic chemical compounds and biological agents. Modern cytokine-targeted therapies with TNF inhibitors, IL-1 inhibitors, IL-6R–blocking mAbs, T cell costimulation inhibitors, and anti–B cell agents are recommended as biological originator DMARDs (boDMARDs) for patients in whom the treatment targets were not achieved using conventional synthetic DMARDs (1). In addition to the boDMARDs that target immunological targets, denosumab, which is a fully human mAb that inhibits the formation, function, and survival of osteoclasts by preventing the interaction of RANKL with RANK, also showed a potential protective ability against bone erosion in RA patients (2). The primary targets for both approved boDMARDs and boDMARDs still in clinical trials are immune cells (T cells, B cell, and osteoclasts) or their associated cytokines.

In recent years, one widespread and potentially important type of Ca2+ channel, the store-operated Ca2+ release–activated channel (CRAC), also known as ORAI, has been targeted in drug discovery for rheumatoid disease. Ca2+ entry through CRACs drives exocytosis, stimulates mitochondrial metabolism, activates gene expression, and promotes cell growth and proliferation in nonexcitable cells (3). The downregulation of CRACs resulted in a reduction of IFN-γ, IL-2, and IL-17 produced by T cells. Ca2+ signals via CRACs regulate several important T cell functions, especially cell tolerance. Reduced CRAC activity is linked to tolerance, whereas increased CRAC activity correlates with autoimmune diseases, such as inflammatory bowel disease (4). Impaired Ca2+ signals also were linked to defects in early development and IL-10 secretion in B cells. The negative selection of B cells, which occurs in the bone marrow to prevent the generation of self-reactive cells and subsequent autoimmunity, is regulated by the selective activation of ERK signaling triggered by CRAC-dependent Ca2+ signals (5). Although CRACs are not the only class of calcium channels expressed on osteoclasts, they are required for cell fusion, a late event in osteoclast differentiation (6). Because osteoclasts cannot function properly without multinucleation, selective CRAC inhibition may be useful in the management of hyperresorptive states in RA patients. These results raise the possibility that selective CRAC inhibitors could be of considerable clinical benefit. CRACs could be a single drug that simultaneously targets T cells, B cells, and osteoclasts, aimed at managing RA.

To demonstrate the therapeutic effects of the regulation of CRACs in RA, we partially blocked Ca2+ influx via CRACs by gene silencing CRACM3 using a systemically lentivirus-delivered short hairpin RNA (shRNA) in a murine model of collagen-induced arthritis (CIA). The present study was undertaken to investigate the feasibility and efficiency of partially regulating Ca2+ influx via CRACs by CRACM3 gene silencing for the treatment of RA.

The lentiviral vectors were produced by cotransfection of 293T kidney cells with the following three plasmids: (1) a specific vector plasmid encoding shRNAs targeting mouse CRACM3 (M3shRNA) (NM_198424) (TRC2-pLKO-puro): 5′-CCGGCACCAACGACTGCACAGATACCTCGAGGTATCTGTGCAGTCGTTGGTGTTTTTG-3′, mouse CRACM1 (M1shRNA) (NM_175423) (pLKO.1-puro): 5′-CCGGCACAACCTCAACTCGGTCAAACTCGAGTTTGACCGAGTTGAGGTTGTGTTTTTG-3′, or a negative control shRNA vector (ncshRNA) (Sigma-Aldrich, Tokyo, Japan); (2) the packaging plasmid psPAX2 (Addgene, Cambridge, MA); and (3) the envelope plasmid pMD2.G (Addgene). Viral particles of CRACM3-shRNA, CRACM1-shRNA, and ncshRNA were produced, and their titers were determined using a Lenti-X p24 Rapid Titer Kit (Clontech Laboratories, Mountain View, CA).

Male DBA/lJ mice (7–10 wk age) used for the induction of arthritis were bred and maintained under standard conditions. The experimental protocols were in accordance with the guidelines of the Animal Care Committee of Ehime University and were approved by the University Committee for Animal Research.

To induce arthritis, mice were injected s.c. with 200 μg bovine collagen type II (CII; Chondrex, Redmond, WA) emulsified in an equal volume of CFA containing heat-killed Mycobacterium tuberculosis H37 RA (4 mg/ml). Twenty-one days later, mice were boosted by the s.c. injection of 200 μg of CII emulsified in IFA. Mice injected s.c. with saline were used as negative controls.

Lentivirus expressing M3shRNA, M1shRNA, or ncshRNA was administered i.p. to CIA mice using the virus dose of 109 particles/7 d/mouse or 3 × 108 particles/2 d/mouse from day 21 after the first CII immunization. On day 35, the number of integrated provirus copies in tissues was determined using a Lenti-X Provirus Quantitation Kit (Clontech Laboratories). Genomic DNA was extracted from tissues of transfected CIA mice and then subjected to quantitative real-time PCR (qPCR) amplification with a dilution of a calibrated provirus control template. The final results are expressed in terms of provirus copies/cell.

The ankle circumferences and articular indexes were evaluated every 5 d, from days 20 to 50, under blinded conditions by two independent examiners. The articular indexes were scored from 0 to 4 as follows: 0 = no swelling, 1 = slight swelling and erythema, 2 = moderate swelling and edema, 3 = severe swelling and edema, and 4 = joint rigidity. Each limb was graded, and a total score for all four limbs was averaged, for a maximum possible score of 4 in each limb/animal. Mice also were assessed by magnetic resonance imaging (MRI) after the scoring for signs of arthritis. MRI imaging was performed using a MRmini SA110 scanner (DS Pharma Biomedical, Osaka, Japan). The animals were anesthetized using 1.5 vol% isoflurane vaporized in 100% medical oxygen. Imaging was performed using a three-dimensional T2-weighted flash sequence. The coronal and sagittal images were collected and reconstructed to obtain the volume of arthritic ankles and forepaws.

Single-cell suspensions (106 cell/ml) pooled from spleen, thymus, or knee joint synovial membrane were obtained at 35 d postimmunization. Cells were loaded with 1 μM fura 2–AM at a concentration of 1 × 106 cells/ml for 1 h at 37°C and then seeded into poly-d-lysine–coated 96-well plates. The assay was performed using a fluorometric imaging plate reader (FLIPR; FlexStation II; Molecular Devices Japan, Tokyo, Japan). The plates were equilibrated to room temperature for 15 min before the start of the assay and were read for a total of 700 s, including an initial 100-s reading window to measure the baseline fluorescence levels before the application of any compound with 0-mM Ca2+ bath solution. The plates were read for an additional 300 s after 0.5 μM thapsigargin was applied by FLIPR. Ca2+ (2 mM) was then applied to the wells, and the plates were read for an additional 300 s. Calcium signals were read using the 340/380-nm excitation and 510-nm emission set. The results are presented as the ratio of relative fluorescence units (RFU) (340 nm/380 nm) and initial rates of Ca2+ influx (in the first 15 s after Ca2+ addition). Because the final concentration of DMSO in the assay was 1%, vehicle controls of 1% DMSO were included in each assay plate.

The histological examination of the joints was performed after routine fixation, decalcification, and paraffin embedding. Tissue sections from hind paws were stained with H&E. The slides were evaluated for synovial hypertrophy, formation, and cartilage/subchondral bone destruction by two blinded observers. Each joint was scored from 0 to 3, and two joints were analyzed for each animal.

The presence of CRACM3 in the joints of CIA mice was assessed after lentivirus-mediated M3shRNA treatment. The sections were incubated with a rabbit polyclonal anti-CRACM3 Ab (ProSci, Poway, CA), and HRP-conjugated anti-rabbit IgG was used as the secondary Ab. Sections were examined by microscopy, and the captured image data were analyzed with image analysis software (NIH Image 1.62). Representative brown-orange field images (1 = 0.237 mm2) that showed CRACM3+ cells were segmented from background objects and scored. The total areas were segmented using gray-scale image. The expression of CRACM3 was divided by the area of the tissue sections to determine the number of CRACM3+ cells/mm2.

For tartrate-resistant acid phosphatase (TRAP) staining, sections were stained using a TRAP/ALP Staining Kit (Wako, Osaka, Japan), according to the manufacturer’s instructions, and counterstained with hematoxylin. A representative red field that showed a TRAP+ area was segmented and divided by the area of the tissue section.

Sera from CIA mice were collected at the peak of disease (day 35 postimmunization), and anti-CII IgG, IgG1, and IgG2 levels were measured by ELISA, as previously described (7). The protein extract from joints was prepared by homogenization of joints (50 mg tissue/ml) in 50 mM Tris HCl (pH 7.4) with 0.5 mM DTT and a protease-inhibitor mixture (10 μg/ml). Cytokines and chemokine levels in the serum and joint protein extract were detected by sandwich ELISA using a Q-plex array (Quansys Biosciences, Logan, UT), according to the manufacturer’s instructions. The lower limits of detection are 87.50 pg/ml for IL-1β, 1.33 pg/ml for IL-2, 2.34 pg/ml for IL-3, 4.30 pg/ml for IL-4, 18.75 pg/ml for IL-5, 1.72 pg/ml for IL-6, 3.52 pg/ml for IL-10, 15.63 pg/ml for IL-17, 28.13 pg/ml for INF-γ, 2.81 pg/ml for TNF-α, and 12.50 pg/ml for RANTES.

Single-cell suspensions pooled from spleens were obtained 35 d postimmunization. For cytokine determination, cells were stimulated in complete RPMI 1640 with heat-inactivated CII, in a dose-dependent manner, for 48 h. Cytokine levels were determined using a Q-plex array, as described above.

The methods for purification of differentiated osteoclasts from collagen films were adapted from a previous protocol (8, 9). Briefly, bone marrow cells, which include osteoclast precursors, were plated in culture plates coated with a type I collagen film. Cells were fed with fresh α-MEM, recombinant mouse (rm)M-CSF (20 ng/ml), and RANK (150 ng/ml) every other day. After 6 d in culture, the plates were washed with PBS, and the collagen film was digested with a solution of 0.2% collagenase A. Harvested cells used for intracellular Ca2+ influx assay were plated on chamber slides or bone slices and fed with rmM-CSF (20 ng/ml) and RANK (150 ng/ml) in α-MEM. After 48 h of incubation, the cells that were plated on the chamber slides were stained for TRAP using a TRAP/ALP staining kit, and the bone slices were subjected to Mayer’s hematoxylin staining and scanning electron microscopy.

The RANKL levels in sera and joint protein extracts from CIA mice were determined using a sandwich ELISA. Briefly, 96-well plates were coated using a specific mAb for mouse RANKL (GeneTex, Irvine, CA). After blocking, standards and samples were pipetted into the wells and incubated at room temperature for 2 h. The wells were washed, and a goat polyclonal biotinylated anti-mouse RANKL Ab (R&D Systems, Minneapolis, MN) was added. HRP-conjugated streptavidin was pipetted into the wells, and a 3,3′,5,5′-tetramethylbenzidine substrate solution was added for color development. The intensity of the color was measured at 450 nm.

All experiments were designed in a completely randomized multifactorial format with 3–25 mice/group. The results are expressed as mean ± SEM. Repeated-measures ANOVA, followed by the Scheffé F-test, was performed to evaluate the statistical significance of differences with regard to survival rate, arthritis score, swelling rate, peak Ca2+ influx, and autoimmune response. Other data were analyzed using the two-sample t test assuming unequal variance. The p values < 0.05 were considered significant.

To determine the potential therapeutic application of lentiviral shRNAs targeting CRACMs, a single administration/wk of a high dose (109) of lentiviral particles or the repeated administration of low doses (3 × 108) of lentiviral particles were used in the experimental CIA mouse model. Low survival rates (40%) were observed in CIA mice treated with a combination of CRACM1-shRNA–expressing lentivirus (Lenti-M1shRNA) and CRACM3-shRNA–expressing lentivirus (Lenti-M3shRNA) (Fig. 1a). The survival rates were 100% for Lenti-M3shRNA–treated CIA mice (109 particles/wk), 90% for Lenti-M3shRNA–treated mice (3 × 108 particles/2 d), and 100% for untreated CIA mice. We then determined the biodistribution of the inoculated vector. After systemic delivery of lentiviral particles, the number of integrated provirus copies in individual tissues was determined (Table I). Although more provirus copies were observed in the tissues from Lenti-M3shRNA–treated mice using the dose of 3 × 108 particles/2 d, considering the lower survival rate (Fig. 1a) and splenomegaly (data not shown) in the mice subjected to repeated administrations of low doses (3 × 108) of lentiviral particles, the administration of Lenti-M3shRNA at a dose of 109 particles/wk was chosen for further in vivo assessments.

FIGURE 1.

shRNA-mediated inhibition of CRACM3 expression in mice. (a) A comparison of mouse survival rates in saline-injected control mice, untreated CIA mice, Lenti-ncshRNA–treated CIA mice, Lenti-M3shRNA–treated CIA mice (H) (109 particles/week), Lenti-M3shRNA–treated mice (L) (3 × 108 particles/2 d), and Lenti-M3shRNA+Lenti-M1shRNA–treated mice. The number of surviving mice was counted every day following the final Ag boost. *p < 0.05, ANOVA. (b) PCR amplification of reverse-transcription products produced a band reflecting the predicted shRNA-mediated mRNA suppression. The expression of CRACM3 mRNA or actin mRNA was analyzed in Lenti-NcshRNA–treated mice (upper two panels). The expression of CRACM3 mRNA or actin mRNA was analyzed in Lenti-M3shRNA–treated mice (lower two panels). (c) Calibrated CRACM3 mRNA expression in Lenti-shRNAs–treated mice. *p < 0.05, **p < 0.001, versus Lenti-NcshRNA–treated mice (n = 5), two-sample t test assuming unequal variance. (d) The joints of CIA mice obtained after Lenti-M3shRNA treatment were examined for the presence of CRACM3 by immunological staining. The sections were incubated with a rabbit polyclonal anti-CRACM3 Ab and HRP-conjugated anti-rabbit IgG (original magnification ×200). (e) CRACM3 expression in the joints of normal control mice, Lenti-ncshRNA–treated CIA mice, and Lenti-M3shRNA–treated CIA mice. Captured image data were analyzed with image analysis software (NIH Image 1.62). A representative brown-orange field image (1 = 0.237 mm2) showing CRACM3+ cells was segmented from background objects and scored. The total areas were segmented using the gray-scale image. The expression of CRACM3 was divided by the area of the tissue sections to determine the expression of CRACM3/mm2. *p < 0.05, **p < 0.001, two-sample t test assuming unequal variance (n = 3–5). The results are mean ± SEM. (f) Ca2+ influx peaks in splenocytes, thymocytes, and synovial cells obtained from normal control mice, Lenti-M3shRNA–treated mice, Lenti-ncshRNA–treated CIA mice, and M3shRNA-treated CIA mice. Single-cell suspensions (106 cell/ml) were obtained 35 d postimmunization. Cells isolated from mice were loaded with 1 μM fura 2–AM, and the assay was performed using a fluorometric imaging plate reader. The assay was read for a total of 700 s, including an initial 100-s reading window to measure the baseline fluorescence levels before the application of any compound. The plates were read for an additional 300 s after 0.5 μM thapsigargin was applied by FLIPR. Ca2+ (2 mM) was then applied to the wells, and the plates were read for an additional 300 s. Calcium signals were read using the 340/380-nm excitation and 510-nm emission set. The results are presented as the peak ratio of RFU (340 nm/380 nm). (g) Ca2+ influx initial rates (in the first 15 s after Ca2+ addition) in splenocytes, thymocytes, and synovial cells obtained from normal control mice, Lenti-M3shRNA–treated mice, Lenti-ncshRNA–treated CIA mice, and Lenti-M3shRNA–treated CIA mice. Results are mean ± SEM. *p < 0.05, two-sample t test assuming unequal variance (n = 3–5). a.u., area unit.

FIGURE 1.

shRNA-mediated inhibition of CRACM3 expression in mice. (a) A comparison of mouse survival rates in saline-injected control mice, untreated CIA mice, Lenti-ncshRNA–treated CIA mice, Lenti-M3shRNA–treated CIA mice (H) (109 particles/week), Lenti-M3shRNA–treated mice (L) (3 × 108 particles/2 d), and Lenti-M3shRNA+Lenti-M1shRNA–treated mice. The number of surviving mice was counted every day following the final Ag boost. *p < 0.05, ANOVA. (b) PCR amplification of reverse-transcription products produced a band reflecting the predicted shRNA-mediated mRNA suppression. The expression of CRACM3 mRNA or actin mRNA was analyzed in Lenti-NcshRNA–treated mice (upper two panels). The expression of CRACM3 mRNA or actin mRNA was analyzed in Lenti-M3shRNA–treated mice (lower two panels). (c) Calibrated CRACM3 mRNA expression in Lenti-shRNAs–treated mice. *p < 0.05, **p < 0.001, versus Lenti-NcshRNA–treated mice (n = 5), two-sample t test assuming unequal variance. (d) The joints of CIA mice obtained after Lenti-M3shRNA treatment were examined for the presence of CRACM3 by immunological staining. The sections were incubated with a rabbit polyclonal anti-CRACM3 Ab and HRP-conjugated anti-rabbit IgG (original magnification ×200). (e) CRACM3 expression in the joints of normal control mice, Lenti-ncshRNA–treated CIA mice, and Lenti-M3shRNA–treated CIA mice. Captured image data were analyzed with image analysis software (NIH Image 1.62). A representative brown-orange field image (1 = 0.237 mm2) showing CRACM3+ cells was segmented from background objects and scored. The total areas were segmented using the gray-scale image. The expression of CRACM3 was divided by the area of the tissue sections to determine the expression of CRACM3/mm2. *p < 0.05, **p < 0.001, two-sample t test assuming unequal variance (n = 3–5). The results are mean ± SEM. (f) Ca2+ influx peaks in splenocytes, thymocytes, and synovial cells obtained from normal control mice, Lenti-M3shRNA–treated mice, Lenti-ncshRNA–treated CIA mice, and M3shRNA-treated CIA mice. Single-cell suspensions (106 cell/ml) were obtained 35 d postimmunization. Cells isolated from mice were loaded with 1 μM fura 2–AM, and the assay was performed using a fluorometric imaging plate reader. The assay was read for a total of 700 s, including an initial 100-s reading window to measure the baseline fluorescence levels before the application of any compound. The plates were read for an additional 300 s after 0.5 μM thapsigargin was applied by FLIPR. Ca2+ (2 mM) was then applied to the wells, and the plates were read for an additional 300 s. Calcium signals were read using the 340/380-nm excitation and 510-nm emission set. The results are presented as the peak ratio of RFU (340 nm/380 nm). (g) Ca2+ influx initial rates (in the first 15 s after Ca2+ addition) in splenocytes, thymocytes, and synovial cells obtained from normal control mice, Lenti-M3shRNA–treated mice, Lenti-ncshRNA–treated CIA mice, and Lenti-M3shRNA–treated CIA mice. Results are mean ± SEM. *p < 0.05, two-sample t test assuming unequal variance (n = 3–5). a.u., area unit.

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Table I.
Tissue distribution of systemic delivered lentiviral particles as provirus quantification using qPCR
TissueSingle i.p. (Provirus Copies/Cell)Consecutive i.p. (Provirus Copies/Cell)
Blood 1.01 ± 0.12 1.32 ± 0.45 
Lung 1.11 ± 0.35 1.64 ± 0.54 
Liver 0.88 ± 1.21 1.75 ± 4.5 
Kidney 0.87 ± 0.15 1.5 ± 0.31 
Spleen 1.25 ± 0.01 1.53 ± 0.23 
Muscle 0.62 ± 3.21 3.85 ± 6.11 
Bone marrow 3.73 ± 2.45 12.21 ± 7.46 
Heart 0.45 ± 0.21 1.12 ± 1.03 
Intestine 0.95 ± 0.1 1.86 ± 0.32 
Lymph node 1.2 ± 0.4 2.87 ± 0.65 
TissueSingle i.p. (Provirus Copies/Cell)Consecutive i.p. (Provirus Copies/Cell)
Blood 1.01 ± 0.12 1.32 ± 0.45 
Lung 1.11 ± 0.35 1.64 ± 0.54 
Liver 0.88 ± 1.21 1.75 ± 4.5 
Kidney 0.87 ± 0.15 1.5 ± 0.31 
Spleen 1.25 ± 0.01 1.53 ± 0.23 
Muscle 0.62 ± 3.21 3.85 ± 6.11 
Bone marrow 3.73 ± 2.45 12.21 ± 7.46 
Heart 0.45 ± 0.21 1.12 ± 1.03 
Intestine 0.95 ± 0.1 1.86 ± 0.32 
Lymph node 1.2 ± 0.4 2.87 ± 0.65 

Based on the experiments mentioned above, CRACM3 levels were significantly decreased at the mRNA level in peripheral blood, lung, liver, spleen, muscle, bone marrow, and lymph nodes, but not in kidney and heart, in Lenti-M3shRNA–treated mice (Fig. 1b, 1c). CRACM3 protein expression in the knee joints of Lenti-M3shRNA–treated mice was decreased to 6.1% (p < 0.001) of the levels observed in mice treated with ncshRNA-expressing lentivirus (Lenti-ncshRNA) (Fig. 1d, 1e). Together, these data confirm the silencing of CRACM3 expression by lentivirus-mediated shRNA transfection in mice.

Single-cell suspensions pooled from spleen, thymus, and knee joint synovial membrane were obtained and loaded with fura 2–AM, and a Ca2+ assay was performed (Fig. 1f, 1g). A slight increase (∼10–15%) in the RFU340/380 was detected in this study after the administration of thapsigargin into the 0-mM Ca2+ bath solution, indicating that the intracellular Ca2+ stores were depleted (Supplemental Fig. 1). Next, a sustained increase in the Ca2+ influx, as reflected by RFU340/380, was observed in thapsigargin-stimulated cells upon the addition of 2 mM Ca2+ to the bath solution. No significant differences in the RFU340/380 among these groups were detected after the addition of thapsigargin to the 0-mM Ca2+ bath solution.

According to the maximum RFU340/380 values for each group, treatment of CIA mice with Lenti-M3shRNA reduced the cell Ca2+ influx peaks in splenocytes, thymocytes, and synovial cells to 54.54 (p < 0.05), 54.15 (p < 0.05), and 47.49% (p < 0.05), respectively, of the peaks in the cells from Lenti-ncshRNA–treated CIA mice (Fig. 1f). The initial rates of Ca2+ influx of cells from Lenti-M3shRNA–treated CIA mice also decreased to 69.64 (p < 0.05), 71.50 (p < 0.05), and 68.23% (p < 0.05), respectively, of the rates in the cells from Lenti-ncshRNA–treated CIA mice (Fig. 1g). Both the peaks and the initial rates of Ca2+ influx of cells from CIA mice were higher in splenocytes, thymocytes, and synovial cells (peak: 1.45-fold [p < 0.05], 3.08-fold [p < 0.05], and 2.77-fold [p < 0.05], respectively; influx rate: 1.28-fold, 1.46-fold (p < 0.05), and 1.29-fold, respectively) compared with those from nonsensitized mice. Although splenocytes, thymocytes, and synovial cells isolated from saline- and Lenti-M3shRNA–treated nonsensitized mice presented lower Ca2+ influx peaks and initial rates compared with the same cell populations isolated from nonsensitized mice not treated with shRNA, these differences were not significant.

These results indicate that the Ca2+ influx in tissue-derived cells was partially inhibited by lentivirus-mediated CRACM3 silencing.

To examine the efficiency of Lenti-M3shRNA treatment, we first evaluated the clinical signs of arthritis in our experimental CIA mouse model. Lenti-M3shRNA–treated CIA mice showed a marked decrease in arthritis severity compared with Lenti-ncshRNA–treated CIA mice. The swelling rates of paws, which were quantified using three-dimensional MRI analysis, were 33.42%, and the arthritic clinical score was 55.94% lower in the Lenti-M3shRNA–treated group compared with the control Lenti-ncshRNA–treated group (Fig. 2a–c).

FIGURE 2.

Application of Lenti-M3shRNA reduces the severity of CIA. To induce CIA, mice were injected s.c. with 200 μg of bovine CII emulsified in an equal volume of CFA containing heat-killed M. tuberculosis H37 RA (4 mg/ml). Twenty-one days later, mice were boosted by s.c. injection of 200 μg of CII emulsified in IFA. (a) MRI of arthritic ankles and forepaws. Imaging was performed using a three-dimensional T2-weighted flash sequence. (b) Swelling rates of arthritic ankles and forepaws. Coronal and sagittal images were collected every 5 d and reconstructed to obtain the volume of arthritic ankles and forepaws. The ratio of increased volume after Ag sensitization/volume in the presensitization period was calculated. Results are mean ± SEM. *p < 0.05 compared with saline-sensitized control mice, ƚp < 0.05, versus CIA mice, ANOVA, followed by the Scheffé test (n = 10–25). (c) Clinical severity of arthritis was scored every 5 d from experimental days 20 to 50. Results are mean ± SEM. *p < 0.05, versus saline-injected control mice, ƚp < 0.05, versus CIA mice, ANOVA, followed by the Scheffé test (n = 10–25). (d) Histological analysis of paw joints of Lenti-ncshRNA– and Lenti-M3shRNA–treated CIA mice using H&E staining (original magnification ×100). (e) Histological analysis quantitated by scoring for inflammation, bone erosion, and cartilage damage. Results are mean ± SEM. *p < 0.05, versus Lenti-ncshRNA–treated mice, two-sample t test assuming unequal variance (n = 3).

FIGURE 2.

Application of Lenti-M3shRNA reduces the severity of CIA. To induce CIA, mice were injected s.c. with 200 μg of bovine CII emulsified in an equal volume of CFA containing heat-killed M. tuberculosis H37 RA (4 mg/ml). Twenty-one days later, mice were boosted by s.c. injection of 200 μg of CII emulsified in IFA. (a) MRI of arthritic ankles and forepaws. Imaging was performed using a three-dimensional T2-weighted flash sequence. (b) Swelling rates of arthritic ankles and forepaws. Coronal and sagittal images were collected every 5 d and reconstructed to obtain the volume of arthritic ankles and forepaws. The ratio of increased volume after Ag sensitization/volume in the presensitization period was calculated. Results are mean ± SEM. *p < 0.05 compared with saline-sensitized control mice, ƚp < 0.05, versus CIA mice, ANOVA, followed by the Scheffé test (n = 10–25). (c) Clinical severity of arthritis was scored every 5 d from experimental days 20 to 50. Results are mean ± SEM. *p < 0.05, versus saline-injected control mice, ƚp < 0.05, versus CIA mice, ANOVA, followed by the Scheffé test (n = 10–25). (d) Histological analysis of paw joints of Lenti-ncshRNA– and Lenti-M3shRNA–treated CIA mice using H&E staining (original magnification ×100). (e) Histological analysis quantitated by scoring for inflammation, bone erosion, and cartilage damage. Results are mean ± SEM. *p < 0.05, versus Lenti-ncshRNA–treated mice, two-sample t test assuming unequal variance (n = 3).

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General histological analysis using H&E staining revealed a significant decrease in cellular infiltration into the joint cavity and periarticular soft tissue, pannus formation, cartilage destruction, and bone erosion in the knee joints of Lenti-M3shRNA–treated CIA mice compared with the control Lenti-ncshRNA–treated group. Quantification analysis indicated that Lenti-M3shRNA treatment reduced inflammation by 37.86%, bone erosion by 40%, and cartilage damage by 26.15% (Fig. 2e). Taken together, these results indicated that gene silencing of CRACM3 decreased arthritis severity in the CIA murine model.

To investigate the mechanisms underlying the decrease in severity of CIA with Lenti-M3shRNA treatment, we evaluated the released inflammatory mediators in joint protein extraction and serum. The profile of joint protein extraction indicates that gene silencing of CRACM3 significantly reduced the protein expression of inflammatory cytokines, including TNF-α, INF-γ, IL-2, IL-6, IL-12, and IL-17, and some chemokines, such as RANTES, in the local joints of CIA mice (Fig. 3a). The release of the suppressive cytokine IL-10 was increased after Lenti-M3shRNA treatment. Decreased levels of TNF-α, INF-γ, IL-6, and IL-12 also were observed in the sera of Lenti-M3shRNA–treated CIA mice compared with control Lenti-ncshRNA–treated mice (Fig. 3b). These data suggest that the broad anti-inflammatory effect of Lenti-M3shRNA in joints was accompanied by the downregulation of the systemic inflammatory response in CIA mice.

FIGURE 3.

Administration of Lenti-M3shRNA inhibits the inflammatory response in CIA. The cytokine profiles in joint protein extracts (a) and serum (b) were determined on day 35 postimmunization using a Q-plex array. Results are mean ± SEM. *p < 0.05, versus Lenti-ncshRNA–treated mice, two-sample t test assuming unequal variance (n = 5). (c) The number of collagen-type–specific cytokine-producing T cells. Single-cell suspensions pooled from spleens were obtained 35 d postimmunization. Cells were stimulated with heat-inactivated CII in a dose-dependent manner for 48 h for cytokine determination. Cytokine levels were determined using a Q-plex array. Results are mean ± SEM. *p < 0.05, ANOVA, followed by the Scheffé test (n = 3). (d) CII-specific IgG, IgG1, and IgG2a levels in serum collected on day 35 from Lenti-ncshRNA– and Lenti-M3shRNA–treated CIA mice were measured by ELISA. Results are mean ± SEM. *p < 0.05, versus Lenti-ncshRNA–treated mice, two-sample t test assuming unequal variance (n = 8–12).

FIGURE 3.

Administration of Lenti-M3shRNA inhibits the inflammatory response in CIA. The cytokine profiles in joint protein extracts (a) and serum (b) were determined on day 35 postimmunization using a Q-plex array. Results are mean ± SEM. *p < 0.05, versus Lenti-ncshRNA–treated mice, two-sample t test assuming unequal variance (n = 5). (c) The number of collagen-type–specific cytokine-producing T cells. Single-cell suspensions pooled from spleens were obtained 35 d postimmunization. Cells were stimulated with heat-inactivated CII in a dose-dependent manner for 48 h for cytokine determination. Cytokine levels were determined using a Q-plex array. Results are mean ± SEM. *p < 0.05, ANOVA, followed by the Scheffé test (n = 3). (d) CII-specific IgG, IgG1, and IgG2a levels in serum collected on day 35 from Lenti-ncshRNA– and Lenti-M3shRNA–treated CIA mice were measured by ELISA. Results are mean ± SEM. *p < 0.05, versus Lenti-ncshRNA–treated mice, two-sample t test assuming unequal variance (n = 8–12).

Close modal

The suppression of Lenti-M3shRNA treatment also was Ag specific. High CII-specific cytokine production of Th1-associated cytokines (TNF-α and INF-γ) and Th17-associated cytokines (IL-17) was observed in the splenocytes derived from Lenti-M3shRNA–treated mice. Additionally, a decrease in CII-specific IL-10 was detected in Lenti-M3shRNA–treated CIA mice (Fig. 3c).

Lenti-M3shRNA treatment reduced total circulating Ag-specific Abs targeting CII, especially IgG2 anti-CII Abs (Fig. 3d), which are generally reflective of Th1 activity. There was no significant difference in the level of specific IgG1 expression between Lenti-M3shRNA–treated mice and Lenti-ncshRNA–treated mice.

These data indicate that Lenti-M3shRNA treatment reduces the inflammatory response and the Ag-specific response, particularly the Th1 response, in the joints and periphery of CIA mice.

We next investigated whether gene silencing of CRACM3 was able to regulate the resorptive activity of the mature osteoclast. Bone marrow cells from CIA mice, which were treated with Lenti-M3shRNA or Lenti-ncshRNA, were plated on collagen films in the presence of M-CSF and RANKL for 6 d, at which time they became mature and developed the ability to resorb bone. The cells were detached from the plate with collagenase and then cultured with M-CSF and RANKL on top of bone slices.

Based on the RFU340/380 values, although slight decreases in Ca2+ influx peaks and initial rates were observed in the detached osteoclasts from Lenti-M3shRNA–treated CIA mice compared with Lenti-ncshRNA–treated CIA mice, the differences were not significant (peak: 93.52% [p = 0.76]; influx rate: 72.6% [p = 0.08]) (Supplemental Fig. 2).

The total number of TRAP+ cells in CIA mice was significantly increased compared with saline-sensitized mice (Fig. 4a–c, 4m). M3shRNA treatment resulted in a reduced number of TRAP+ cells (71.42%) compared with Lenti-ncshRNA–treated CIA mice, but this trend was not statistically significant. According to the results of pit-formation assays, the bone-resorptive capacity of primary mature osteoclasts obtained from Lenti-M3shRNA–treated CIA mice was significantly reduced compared with those from control Lenti-ncshRNA–treated mice (Fig. 4d–i, 4n). Additionally, TRAP staining of knee joint sections indicated that Lenti-M3shRNA–treated CIA mice had reduced osteoclast activity compared with Lenti-ncshRNA–treated CIA mice (Fig. 4j–l, 4o). Quantification analysis revealed that CRACM3 silencing reduced the activity of osteoclasts by 24.4%.

FIGURE 4.

Lenti-M3shRNA–mediated CRACM3 silencing inhibits the bone-resorption activity of mature osteoclasts in CIA mice. Bone marrow cells were isolated from normal control mice, Lenti-ncshRNA–treated CIA mice, and Lenti-M3shRNA–treated CIA mice. The cells were differentiated into osteoclasts by culturing with rmM-CSF (20 ng/ml) and RANKL (150 ng/ml) in tissue culture plates coated with a type I collagen film. After 6 d in culture and subsequent washing, the collagen film was digested with a solution of 0.2% collagenase A. Harvested cells were plated on chamber slides or bone slices and fed with rmM-CSF (20 ng/ml) and RANK (150 ng/ml) in α-MEM. (ac) TRAP staining of the cells, which were plated on chamber slides (original magnification ×100). (df) Bone slides were stained with Mayer’s hematoxylin and examined using light microscopy (original magnification ×100). The arrows represent resorption pits. (gi) Surfaces of bone slides were examined using scanning electron microscopy (original magnification ×500). (jl) Joint sections were examined using TRAP staining. Paraffin-embedded joint sections were stained using a TRAP/ALP Staining Kit and counterstained with hematoxylin. The sections were examined using light microscopy (original magnification ×400). Red field represents the TRAP+ area. (m) The total number of TRAP+ cells isolated from each mouse was counted. Results are mean ± SEM. *p < 0.05, two-sample t test assuming unequal variance (n = 3–5). (n) The quantification of the pit area on bone slides. Resorption pits were visualized by staining with Mayer’s hematoxylin. The area of resorbed pits was segmented from background objects and scored. The pit area is represented by the resorption pit area/bone slide. Results are mean ± SEM. *p < 0.05, two-sample t test assuming unequal variance (n = 3). (o) Quantification of TRAP+ area on joint slides. A red-field image showing the TRAP+ area was segmented from background objects and scored. The total area of the tissue slide was segmented using the gray-scale image (1 = 0.237 mm2). The TRAP+ area was divided by the area of total tissue sections to determine the TRAP+ area/mm2. Results are mean ± SEM. *p < 0.05, **p < 0.001 two-sample t test assuming unequal variance (n = 3). (p) RANKL levels in serum and joint protein extraction. The levels of RANKL in serum and protein extracts, which were obtained on day 35 postimmunization from joints of normal control mice, Lenti-ncshRNA–treated CIA mice, and Lenti-M3shRNA–treated CIA mice, were determined using a sandwich ELISA. Results are mean ± SEM. *p < 0.05, two-sample t test assuming unequal variance (n = 10–15).

FIGURE 4.

Lenti-M3shRNA–mediated CRACM3 silencing inhibits the bone-resorption activity of mature osteoclasts in CIA mice. Bone marrow cells were isolated from normal control mice, Lenti-ncshRNA–treated CIA mice, and Lenti-M3shRNA–treated CIA mice. The cells were differentiated into osteoclasts by culturing with rmM-CSF (20 ng/ml) and RANKL (150 ng/ml) in tissue culture plates coated with a type I collagen film. After 6 d in culture and subsequent washing, the collagen film was digested with a solution of 0.2% collagenase A. Harvested cells were plated on chamber slides or bone slices and fed with rmM-CSF (20 ng/ml) and RANK (150 ng/ml) in α-MEM. (ac) TRAP staining of the cells, which were plated on chamber slides (original magnification ×100). (df) Bone slides were stained with Mayer’s hematoxylin and examined using light microscopy (original magnification ×100). The arrows represent resorption pits. (gi) Surfaces of bone slides were examined using scanning electron microscopy (original magnification ×500). (jl) Joint sections were examined using TRAP staining. Paraffin-embedded joint sections were stained using a TRAP/ALP Staining Kit and counterstained with hematoxylin. The sections were examined using light microscopy (original magnification ×400). Red field represents the TRAP+ area. (m) The total number of TRAP+ cells isolated from each mouse was counted. Results are mean ± SEM. *p < 0.05, two-sample t test assuming unequal variance (n = 3–5). (n) The quantification of the pit area on bone slides. Resorption pits were visualized by staining with Mayer’s hematoxylin. The area of resorbed pits was segmented from background objects and scored. The pit area is represented by the resorption pit area/bone slide. Results are mean ± SEM. *p < 0.05, two-sample t test assuming unequal variance (n = 3). (o) Quantification of TRAP+ area on joint slides. A red-field image showing the TRAP+ area was segmented from background objects and scored. The total area of the tissue slide was segmented using the gray-scale image (1 = 0.237 mm2). The TRAP+ area was divided by the area of total tissue sections to determine the TRAP+ area/mm2. Results are mean ± SEM. *p < 0.05, **p < 0.001 two-sample t test assuming unequal variance (n = 3). (p) RANKL levels in serum and joint protein extraction. The levels of RANKL in serum and protein extracts, which were obtained on day 35 postimmunization from joints of normal control mice, Lenti-ncshRNA–treated CIA mice, and Lenti-M3shRNA–treated CIA mice, were determined using a sandwich ELISA. Results are mean ± SEM. *p < 0.05, two-sample t test assuming unequal variance (n = 10–15).

Close modal

Because the osteoclasts responsible for bone resorption in CIA mice are induced by increasing levels of RANKL, we also assessed RANKL levels in serum and protein extract from joints. We found that Lenti-M3shRNA treatment in CIA mice inhibited the production of RANKL in serum and in knee joints by 65.02 and 25.71%, respectively (Fig. 4p). Together, these results demonstrate that, in addition to tissue inflammation, Lenti-M3shRNA treatment downregulates the bone destruction associated with CIA.

There have been important advances in our understanding of the pathogenesis of RA over the past decades that have led to an expanding array of anticytokine-based biological therapeutics targeting selective immune responses. Highlighted cellular targets, including B lymphocytes, T lymphocytes, and osteoclasts, have shown great therapeutic potential, and specific immune modulators with improved efficacy have been developed for the management of RA. In the current study, we showed that the global immunological responses in an experimental arthritis model are suppressed by targeting a single membrane molecule, CRACM3. Our results demonstrate that gene silencing with specifically designed CRACM3 shRNA led to a reduction in synovial inflammation and especially downregulated Th1-mediated autoreactive responses. In addition to the regulation of general inflammatory responses, the blockade of CRACM3 reduces subsequent bone loss as a result of decreases in osteoclast activity and RANKL levels. Therefore, our results suggest that CRACM3 may be the next molecular target for the development of biological therapeutics in RA management.

Because the main mechanism by which intracellular Ca2+ levels are increased involves store-operated Ca2+ entry (SOCE) through CRACs in most human immune cells, abnormal CRAC activities have been linked to a range of autoimmune diseases. In our previous cross-sectional study, the expression level and functional status of CRACM1 in peripheral naive CD4+ T cells from RA patients, osteoarthritis patients, and healthy donors were characterized. Functionally aberrant T cells from patients with active RA exhibited increased Ca2+ influx, in addition to the upregulated protein expression and function of CRACM1 (10). This result indicates that CRACs may represent a new molecular target for RA therapies.

Of the three homologs of CRAC, CRACM1 and CRACM3 are distributed widely in human and murine tissues, although the relative levels vary by tissue. In contrast, CRACM2 is more restricted in its distribution and is prominently found in kidney, lung, and spleen (11, 12). CRACM1, which is widely expressed in the body, is fundamental to nonexcitable cell physiology; a loss of CRACM1 causes SCID in humans and poor survival rates in gene-trapped mice. CRACM3 is considered compensatory for the loss of CRACM1 because its overexpression can partially restore Ca2+ influx in T cells from SCID patients (3). Although it remains an open question whether CRACM3 is a bona fide ion channel by itself or is just a subunit, the typically inwardly rectifying current–voltage relationship observed for endogenous Icrac via CRACM3, which is selective for Ca2+ over Na+, was noted in previous studies (13, 14). Although CRACM1 and CRACM2 appear to have very similar properties, CRACM3 differs in many ways and is linked to specific functions (15). For CRACM3, fast Ca2+-dependent inactivation is more prominent, whereas slow Ca2+-dependent inactivation is modest compared with CRACM1 and CRACM2. CRACM3 can be activated by 2-aminoethoxydiphenyl borate to a much weaker degree than can CRACM1 (13). Additionally, the expression of CRACM3, but not CRACM2, in the fibroblasts of patients with a point mutation in CRACM1 can partially restore SOCE (16). CRACM3 rescues Ca2+ signals after the knockdown of endogenous CRACM1, without affecting normal Ca2+ homeostasis (17). The knockdown of CRACM3 had no effect on the proliferation, migration, or platelet-derived growth factor promigratory effects observed in cultured vascular smooth muscle cells or human aortic smooth muscle cells (1820). All of the evidence indicates that CRACM3 can modulate SOCE more moderately and with lower side effects via systemic administration compared with CRACM1. This mode of action provides us a chance to partially downregulate Ca2+ signaling via a CRAC with milder side effects by silencing the CRACM3 gene in a murine RA model. In the current study, SOCE in the cells, which were pooled from spleen or knee joint synovial membranes, was partially blocked by CRACM3 gene silencing, as indicated by intracellular fura 2–AM. There was no effect on body weight, individual organ weights (data not shown), or survival rate in CRACM3-knockdown animals, in contrast to CRACM1-knockdown mice. These data suggest that the regulation of CRACM3 is safer than that of CRACM1 for clinical applications.

The CIA model generally has been used for RA studies because it has numerous immunological and pathological similarities to human RA. In the current study, we used the lentiviral system, one of the most potent tools for in vivo gene delivery, to administer M3shRNA to silence the CRACM3 gene. Compared with other delivery systems, which also could be used to achieve this goal, such as plasmids, atelocollagen systems, or adeno-associated systems, the lentiviral system has the advantages of high efficiency, low immunogenicity, and high cargo capacity (7). With the exception of heart and kidney, lentiviruses were delivered systemically to individual organs of CIA mice, and the expression and function of CRACM3 were successfully suppressed. The administration of Lenti-M3shRNA to CIA mice resulted in decreased Ag-specific Th1 and Th17 responses in both the periphery and the joints. The inflammatory responses during CIA progression also were strongly reduced. In addition to the general reduction in inflammation, our study demonstrated that Lenti-M3shRNA treatment might reduce the bone loss associated with RA by downregulating the activities of osteoclasts. CRAC-mediated Ca2+ entry may affect osteoclast differentiation and function directly and indirectly. The blockade of Ca2+ entry by knockdown of CRACs directly inhibits the induction of NFATc1 and other genes downstream of NFATc1 in osteoclastogenesis (21). An in vivo study using CRACM1-knockout mice also confirmed the importance of SOCE in osteoclast development: multinucleated osteoclast formation and mineralized tissue resorption were impaired in knockout mice (22). Indirectly, Lenti-M3shRNA may reduce RANKL levels and osteoclast numbers and protect CIA mice from bone loss by modulating the function of Th17 cells, which are critical in the development of osteoclasts and bone erosion (23). The decreased activation of osteoclasts in the current study may result, at least in part, from the suppression of Ag-specific IL-17 release in Lenti-M3shRNA–treated CIA mice.

In summary, our study provides in vivo evidence that the regulation of SOCE via CRACs using systemic delivery of Lenti-M3shRNA reduces the severity of arthritis, ameliorates symptoms, and prevents joint damage and bone resorption. Building on our previous clinical study in RA patients and an in vivo study (10), SOCE via CRACs was shown to play critical roles in a variety of autoimmune diseases, allergic diseases, and cancer. Therefore, CRACs have attracted considerable attention as therapeutic targets for the development of novel treatments in these fields. Because no reported small-molecule antagonist has been moved successfully into human clinical trials, the development of specific boDMARDs against human CRACs is anticipated. Functional mAbs against human CRACs have been generated and characterized for the neutralization of Ca2+ entry via CRACs by us and other investigators (24). Although further modifications of both the lentivirus-mediated gene-silencing system and Abs are necessary prior to translation to the clinical setting, we believe that the basic findings described in this article provide grounds for optimism regarding the application of CRAC regulators to human RA.

This work was supported by Grant-in-Aid for Research Promotion 054102020 from Ehime University.

The online version of the article contains supplemental material.

Abbreviations used in this article:

boDMARD

biological originator DMARD

CIA

collagen-induced arthritis

CII

bovine collagen type II

CRAC

store-operated Ca2+ release–activated channel

DMARD

disease-modifying antirheumatic drug

FLIPR

fluorometric imaging plate reader

Lenti-M3shRNA

CRACM3-shRNA–expressing lentivirus

Lenti-ncshRNA

ncshRNA-expressing lentivirus

MRI

magnetic resonance imaging

ncshRNA

negative control shRNA

qPCR

quantitative real-time PCR

RA

rheumatoid arthritis

RFU

relative fluorescence unit

rm

recombinant mouse

shRNA

short hairpin RNA

SOCE

store-operated Ca2+ entry

TRAP

tartrate-resistant acid phosphatase.

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The authors have no financial conflicts of interest.

Supplementary data