Introduction
Rheumatoid arthritis (RA) is a chronic inflammatory and deforming joint disease that affects approximately 1% of adults. Although the precise etiology of RA has not been clearly ascertained, numerous studies support the concept that autoreactive T cells play a central role in the initiation and maintenance of the disease [
1]. Advanced RA is treated with TNF-α inhibitors such as Infliximab or Embrel, however a significant proportion of patients do not respond [
2]. These patients have shown some improvement following treatment with Abatacept, a clinically approved CTLA4-Ig, which is believed to inhibit antigen presenting cell (APC) co-stimulation of T cells by high affinity binding to CD80/86 [
3]. Clinical responses induced by the co-stimulatory blockade support the rationale for targeting this pathway. In addition to the CD80/86-CD28 interaction, co-stimulation of T cell responses occurs through the CD40-CD154 interaction between APCs and T cells. CD40 signaling has been demonstrated to be critical in the initiation and progression of the rodent model of RA, collagen induced arthritis (CIA) [
4]. It has been demonstrated that overexpression of CD154 (CD40L) on T cells correlates with higher disease activity [
5], which is confirmed by studies showing treatment of mice with agonistic anti-CD40 Abs at the time of CIA induction exacerbates disease [
6]. Conversely, administration of antagonistic anti-CD154 monoclonal antibody (mAb) prior to induction of CIA ameliorates the disease [
7]. Suppression of the CD40-CD154 interaction has been shown to actually induce generation of T regulatory (Treg) cells [
8]. Despite promising preclinical data, translation of CD40/154 blockade approaches has proved difficult due to the expression of CD154 on platelets, which causes risk of thromboembolic events. Accordingly novel methods of manipulating this interaction without evoking platelet reactions are needed.
Since the DC acts as the most potent APC, we have previously used siRNA to manipulate expression of immunological genes in antigen pulsed DCs to either upregulate or suppress immune responses in a specific manner [
9,
10]. However, ex vivo cellular manipulation is impractical for widespread use. In addition, numerous autoantigens are involved in clinical autoimmune diseases, thus adding another layer of complexity in terms of clinical development. Since CTLA4-Ig mediated co-stimulatory blockade induces remission of autoimmunity, we sought to determine whether a temporary suppression of CD40 expression by administration of siRNA may induce immune modulatory effects on RA that predispose towards reduction of immunity towards the autoantigen. Such an approach is based on the concept that a transient interruption of ongoing T cell activation during the initiation and progression of the autoimmune process may allow the host to default to a state of tolerance to the autoantigen.
In this study, we used a hydrodynamic protocol to systemically administer siRNA targeting CD40 in mice before and after administration of autoreactive antigen. We demonstrated antigen-specific immune modulation, as well as both inhibition of arthritic disease. These data support the possibility of temporary immune modulation in the context of autoimmunity.
Materials and methods
Animals
Male DBA/1 LacJ and BALB/c mice (The Jackson Laboratories, Bar Harbor, ME, USA), five weeks of age, were kept in filter-top cages at the Animal Care and Veterinary Services Facility at the University of Western Ontario, according to the Canadian Council for Animal Care Guidelines. Mice were fed food and water ad libitum and allowed to settle for two weeks before initiation of experimentation, which had ethical approval from the university review board.
CIA Model
DBA/1 LacJ mice, seven weeks of age, were intradermally immunized (Day 0) at the base of the tail with 200 μg of bovine type II collagen (CII) (Sigma-Aldrich, St. Louis, MO, USA) with complete Freund's adjuvant (CFA) (Sigma). On Day 21 after priming, the mice received an intraperitoneal booster injection with 200 μg. Mice were examined visually three times per week for the appearance of arthritis in the peripheral joints, and an arthritis score index for disease severity was given as follows: 0 - no evidence of erythema and swelling; 1 - erythema and mild swelling confined to the mid-foot (tarsals) or ankle joint; 2 - erythema and mild swelling extending from the ankle to the mid-foot; 3 - erythema and moderate swelling extending from the ankle to the metatarsal joints; 4 - erythema and severe swelling encompassing the ankle, foot, and digits. The maximum possible score per mouse was 16. The severity of the arthritis was also determined by the quantification of the paw swelling measured with a dial gauge caliper. Scoring was done by two independent observers, without knowledge of the experimental and control groups.
DC Cultures
DCs were generated from bone marrow progenitor cells as previously described [
11]. Briefly, DCs were cultured from bone marrow cells in six-well plates (Corning Inc., Corning, NY, USA) at 4 × 10
6 cells/well in 4 mL of RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg of streptomycin, 50 μM 2-ME, and 10% Fetal calf serum (FCS) (all from Invitrogen, Mississauga, Ontario, Canada). Recombinant granulocyte macrophage clony-stimulating factor (GM-CSF) (10 ng/mL; PeproTech, Rocky Hill, NJ, USA) and recombinant mouse interleukin 4 (IL-4) (10 ng/mL; PeproTech) were added to the culture medium as growth factors. All cultures were incubated at 37°C in 5% humidified CO
2. Non-adherent cells were removed after 48 h of culture (Day 2) and fresh medium was added every 48 h.
siRNA design and gene transfection
siRNA sequence (TGTTCCACTGGGCTGAGAA), specifically targeting the CD40 gene, was designed and synthesized by Dhmarcon (Dharmacon, Inc., Chicago, IL, USA). CD40 siRNA-expressing vectors were generated using the Silencer Express Kit (Ambion Inc, Austin, TX, USA).
DCs (106cells/well) were plated in a 12-well plate one day before transfection. On Day 6, 3 μl of 20 μM annealed CD40 siRNA and 5 μL of transfect reagent Genesilener (Gene Therapy Systems, San Diego, CA, USA) were separately diluted with 50 μL serum-free medium RPMI 1640, and then mixed rapidly and incubated at room temperature for five minutes to form a complex. The above siRNA-Genesilencer complex was added to the DCs. GL2 siRNA was used as a negative control. After four hours incubation, an equal volume of RPMI 1640 (500 μl) supplemented with 20% FCS, 20 ng/mL GM-CSF and 20 ng/mL IL-4 was added to the cells. Twenty-four hours after transfection, cells were collected to determine gene expression in DCs.
Administration of siRNA
DBA/1LacJ mice were treated with CD40 siRNA or control siRNA on Day -2 or Day 2 using a hydrodynamic method [
12]. In brief, 50 μg siRNA was diluted in 1 ml Phosphate buffered saline (PBS) and quickly injected into mice via tail veil in 10 s.
Mixed Leukocyte Reaction (MLR)
At Day 6 of culture, bone marrow-derived DCs from DBA/1 LacJ mice were transfected with CD40 siRNA or control siRNA. Transfected DCs were irradiated (3,000 rad) and seeded in triplicate in a flat-bottom 96-well plate (Corning) for use as stimulator cells. Spleen T cells from BALB/c mice were isolated by gradient centrifugation over Ficoll-Paque (Amersham Pharmacia Biotech, Montreal, Quebec, Canada) and added as responders (2 × 105 cells/well). The mixed lymphocytes were cultured at 37°C for 72 h in 200 μL of RPMI 1640 supplemented with 10% FCS, 100 U/ml of penicillin, and 100 μg/mL of streptomycin and pulsed with 1 μCi/well of 3H-labelled thymidine (Amersham Pharmacia Biotech) for the last 18 h of culture. Finally, cells were harvested onto glass fiber filters (Perkin Elmer Life Science, Turku, Finland), and the radioactivity incorporated was measured using a Wallac Betaplate liquid scintillation counter (Beckman, Fullerton, CA, USA). Results were expressed as mean counts per minute of triplicate cultures ± SEM.
CII-specific T cell response
T cell responses to CII in subsequent groups of mice were measured by 3H -thymidine incorporation in the presence of CII. T cells were isolated from spleen and lymph node by gradient centrifugation over Ficoll-Paque (Amersham Pharmacia Biotech). Unfractionated lymph node cells were cultured in 96-well plates at a concentration of 2 × 105 cells/well for 72 h in the presence or absence of CII antigen. Cells were cultured for two days, and then cells were pulsed with 1 μCi of 3H-thymidine (Amersham Pharmacia Biotech) for another 18 h of culture. Cells were harvested onto glass fiber filters, and incorporated radioactivity was quantified using a Wallac Betaplate liquid scintillation counter.
Anti-CII antibody measurement
CII-specific Abs were detected using a standard indirect ELISA in which 500 ng of CII was absorbed to each well of a 96-well microtitre plate. Serial dilutions of immune mouse serum were added to the appropriate wells in duplicates and incubated overnight at 4°C. Dilutions of serum were 1:100 to 1:10,000. To develop the ELISA, horseradish peroxidase-conjugated goat anti-mouse IgG Fc and ortho-phenylenediamine dihydrochloride substrate buffer (Sigma) were used. An OD into each well was measured at 450 nm wavelength in an ELISA plate reader (Bio-Rad, Hercules, CA, USA).
Cytokine quantification
CD40 siRNA- or control siRNA-transfected DCs derived from DBA/1 LacJ mice were cultured with the allogeneic (BALB/c) T cells or alone for 48 h. The supernatants were collected and examined for cytokines IL-2, IFN-γ and IL-4) by ELISA. Cytokine-specific ELISA (Endogen, Rockford, IL, USA) was used for detecting cytokine concentrations in culture supernatants according to the manufacturer's instructions using a Benchmark Microplate Reader (Bio-Rad Laboratories Ltd., Mississauga, ON, Canada).
Histology
Paws from experimental and control groups of freshly dissected mice were removed and joint tissues were immersion-fixed in buffered formalin in 0.15 M PBS (pH 7.4) and decalcified with 5.5% EDTA. The specimens were processed for paraffin embedding in paraplast (BDH, Poole, Dorset, UK) as routine procedure. Serial paraffin sections throughout the joint were cut at 5 μm thickness on a microtome. Sections were stained with haematoxylin and eosin (H&E) for assessment of histological damage. The severity of joint damage was scored in a blinded manner as follows: 0 = normal, 1 = moderate infiltration with discrete erosions, 2 = severe infiltration and moderate erosions with preserved joints structure, and 3 = severe erosions with loss of joint architecture.
Flow cytometry
Phenotypic analysis of DC was performed using flow cytometry on a FACScan (Becton Dickinson, San Jose, CA, USA). The cells were stained with FITC-, PE- or PE-cy5-conjugated mAbs against surface markers associated with DC maturation, these include: anti-mouse CD11c, anti-mouse CD40, anti-mouse CD80 (Cedarlane Laboratories, Mississauga, ON, Canada). Tregs were analyzed by triple staining with mAb against with Foxp3-FITC, CD25-PE, and CD4-CY5, followed by flow cytometry analysis. Ig of the same isotype was used as a control.
Statistical analysis
Data are expressed as mean ± SEM. Differences between different groups of mice were compared using the Student t-test for gene expression, antibody, cytokine and histological scores or the Mann-Whitney U test for nonparametric data. A P-value less than 0.05 was considered significant.
Discussion
This report is the first to our knowledge to describe systemic inhibition of CD40 by siRNA as a means of interrupting autoimmune processes in order to up-regulate the natural tendency of the immune system to re-equilibrate tolerogenic mechanisms. We observed that administration of siRNA targeting CD40 could induce in vivo inhibition of proliferative and cytokine recall response to the joint antigen CII, as well as prophylactically inhibit disease. Mechanistically it appeared that induction of Treg cells was associated with suppression of autoimmunity.
The CD40-CD154 interaction is involved in the initiation of CIA. The major approach to intervening in this interaction has been use of CD154-specific blocking mAbs, which prevent T cell priming. This therapy is highly effective in mouse models [
4,
18], although the overall benefit of interruption of CD40-CD154 association is reduced when Abs is administrated after disease establishment. However, anti-CD154 treatment prevents relapses of ongoing disease [
18]. Despite the great promise displayed for CD154 Abs in a mouse CIA model, a particular challenge in clinical use has been thromboembolism [
4,
18]. Another approach has been the use of blocking peptides. Unfortunately, this approach was restricted by the high concentrations needed for in vivo effects [
19]. Using mAbs against CD40 such as the anti-human CD40 antagonist mAb, ch5D12, showed promise in autoimmune disease. New intervention strategies that spare normal immune function while blocking damaging effects of CD40 signaling are desirable. RNA inference may provide a novel approach to blocking the CD40 signal.
In the area of tolerance induction, numerous approaches have been attempted, however, to our knowledge, none have passed registration trials. Strictly antigen-specific approaches are deficient because in many situations numerous autoantigens contribute to disease. In the context of RA, while CII is sufficient to induce an RA-like disease in animal models, multiple proteins have been implicated in the human RA [
1]. Examples of autoantigens in human RA include citrullinated fibrinogen, hsp47 and 60 [
20,
21]. The second problem is that induction of Ag-specific tolerance can only be achieved by limited means even if antigens are known; for example, oral tolerance [
22], intravenous tolerance, or as we previously published,
ex vivo pulsing of DCs with autoantigen. These methods are either not practical or weakly effective.
The method used in the current study is based on our previous observations that manipulation of CD40 on DC can be tolerogenic [
10,
23]. Accordingly, we sought to determine if systemic inhibition through flooding the body with siRNA can achieve a therapeutic effect. This method has several drawbacks: First, hydrodynamic administration is clinically impossible. Second, while we demonstrated inhibition of disease, complete tolerance was not achieved. Third, the possibility of non-specific immune suppression during administration of CD40 siRNA is a possibility. There are ways to address these concerns which will be studied in subsequent experiments. We previously reported successful administration of nanoparticles containing siRNA to DCs by the use of DEC-205 antibody coated immunoliposomes [
23]. By selectively delivering the immune modulatory siRNA to the targeted DCs, it may be feasible to circumvent the clinical barriers associated with hydrodynamic administration. While complete inhibition of disease was not observed, the finding that administration of siRNA even after disease initiation occurred was effective suggests that the temporary CD40 inhibition may have potent therapeutic effects. While it is tempting to suggest multiple administrations, this would risk the possibility of compromising immunity towards pathogenic agents, as seen with the TNF-inhibitors causing reappearance of infectious diseases such as tuberculosis. One method of avoiding the need for multiple immune suppressions may involve co-immunization of a recipient with autoantigens under the cover of CD40 siRNA. This may be one method of
recycling previously tried antigen-specific agents that demonstrated some efficacy in clinical trials but were not sufficient to warrant marketing approval.
It is recognized that CD40 is expressed by B cells, macrophages, and some T cells. The effects seen after inhibition of CD40 might be attributable to a direct effect on CD40 in B cells or an indirect effect through lower activation of T cells. We consider that one attractive possible mechanism by which systemic CD40 inhibition mediates immune modulation is through the up-regulation of Treg cells. We have demonstrated increased numbers of Treg cells in CD40 siRNA treated mice. It is conceptually appealing to ask whether these Treg cells arise due to the presence of autoantigen in the absence of co-stimulatory molecules. Indeed, DCs in RelB knockout mice, which lack CD40, have been demonstrated to induce infectious tolerance through induction of Treg cells [
24].
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
XiZ, MS, XuZ, FZ, HL, AS and MHW performed research. XiZ, TEM, RDI and WM analyzed data and wrote the paper; WM, RDI and XiZ designed the research.