Introduction
The most salient feature of apoptosis is the lack of inflammatory responses or tissue damage. Several mechanisms of peripheral tolerance have been described to explain this lack of immune responses against apoptotic cell-derived antigens [
1,
2]. First, apoptotic cells themselves possess immunomodulatory properties by the release of transforming growth factor beta (TGFβ) stored in their cytoplasm [
3]. Then professional phagocytes, such as macrophages and some dendritic cell subsets [
1,
4], can also favor an immunomodulatory environment by the release of anti-inflammatory cytokines during apoptotic cell uptake. Such immunomodulatory milieu consists mainly of TGFβ and IL-10 [
5‐
7].
Recently, the role of TGFβ in immune tolerance has been highlighted by its direct and indirect effects on autoimmunity and inflammation [
6,
8]. Moreover, TGFβ is a key factor to convert peripheral naive CD4
+CD25
- T cells into CD4
+CD25
+Foxp3
+ regulatory T cells (Tregs),
in vitro [
9] as well as
in vivo [
8]. Also, the TGFβ signaling pathway has also been shown to be critical for natural Treg development [
10].
The feasibility of cellular therapy based on the immunomodulatory properties of apoptotic cells has already been evaluated in different experimental models to restore or induce immune tolerance. Indeed, apoptotic cell injection favors allogeneic hematopoietic cell engraftment, favors allograft heart survival and decreases acute graft-versus-host disease (for a review see [
11]). Moreover, spontaneous type I diabetes occurrence in NOD mice could be delayed by injection of apoptotic beta cells [
12]. These beneficial effects have been mainly related to TGFβ and/or Tregs [
11‐
13].
Although such an approach of apoptotic cell infusion has not yet been used directly in patients, the immunomodulatory properties of apoptotic cells may play a role in the tolerogenic effects of blood product transfusions [
14] or of extracorporeal photochemotherapy [
15,
16]. Indeed, the beneficial effects of extracorporeal photochemotherapy in the treatment of severe chronic or acute graft-versus-host disease have been associated with the significant number of the apoptotic cells generated during extracorporeal photochemotherapy [
15,
16]. While apoptotic cell instillation prevents and treats autoimmunity [
8] and inflammation in several experimental models [
6,
11,
13], the suppressive effect of apoptotic cell infusion on experimental arthritis is unknown.
Injection of Group A streptococcal cell wall (SCW) peptidoglycan–polysaccharide complexes induces an acute inflammation of the peripheral joints, followed by a chronic, erosive arthritis in susceptible rats. This corresponds to an animal model for rheumatoid arthritis (RA) [
17,
18]. The acute phase is clinically evident within 24 hours after injection of SCW and is characterized histologically by neutrophil infiltration into the synovium. The chronic erosive arthritic stage, on the other hand, is induced by T-cell-mediated and macrophage-mediated immune responses, characterized by accumulation of mononuclear cells with release of proinflammatory cytokines and erosive destruction of subchondral and periarticular bone and cartilage [
18‐
20].
Systemic administrations of IL-4, TGFβ or an inhibitor of nitric oxide have been shown to suppress pathogenesis of SCW arthritis [
19,
20]. Macrophage depletion could also suppress the chronic phase of the SCW-induced arthritis [
21]. Oral administration of SCW prior to systemic injection of SCW substantially prevents the joint swelling and destruction typically observed during both acute and chronic phases of the arthritis [
18]. The effect of oral tolerance on SCW arthritis was associated with an increase in circulating levels of TGFβ accompanied by a decrease in inflammatory cytokines and inhibition of the arthritic response [
18]. Because macrophages have been identified as pathogenic in SCW-induced RA and because TGFβ has a protective role on SCW-induced RA, we proposed to test the efficiency of apoptotic cell infusion to modulate the arthritic response.
Materials and methods
Animals, induction and monitoring of arthritis
Arthritis was induced in pathogen-free Lewis female rats (Charles River Laboratories, Wilmington, MA, USA) by intraperitoneal injection of Group A SCW peptidoglycan–polysaccharide complexes (30 μg rhamnose/g body mass; Lee Laboratories, Grayson, GA, USA) [
18]. Animals were housed in a specific pathogen-free rodent facility at the National Institute of Dental and Craniofacial Research, National Institutes of Health. All animal studies were performed according to National Institutes of Health guidelines for use and care of live animals and were approved by the Animal Care and Use Committee of National Institute of Dental and Craniofacial Research.
Acute and chronic joint pathology was clinically monitored and the articular index was determined, as previously described [
17,
19]. Briefly, the degree of joint swelling was monitored using a plethysmometer (UGO Basile, Varese, Italy). Radiographs taken with direct exposure (1:1) on X-Omat TL Kodak film using 60-kV, 345-mA, 60-s exposure by a Faxitron X-ray machine (Faxitron X-ray Corporation, Buffalo Grove, IL, USA) were evaluated for soft tissue swelling, joint space narrowing, bone erosions and deformity. On days 25 and 26 after SCW immunization, joints were harvested and fixed with neutral 10% formalin, extracted, embedded in paraffin and cut into 5 μm sections for H & E staining.
Preparation of apoptotic cells
Rat thymocytes were gamma-irradiated (1,500 rad) and cultured in complete DMEM medium at 5% carbon dioxide and 37°C for 4 to 6 hours as previously described [
22]. This culture allowed apoptotic changes to occur. Cells were 90 to 95% apoptotic as determined by Annexin-V staining and 7-Aminoactinomycin D exclusion before washing with PBS and intraperitoneal injection into the indicated rats at 2 × 10
8 cells per animal at the same time as SCW (two different injections). This corresponds to the early apoptotic state, as indicated by 7-Aminoactinomycin D exclusion. Cells were 70 to 80% apoptotic 3 hours after irradiation and were 90 to 95% apoptotic 6 hours after apoptosis induction.
Flow cytometry
The spleens, inguinal and mesenteric lymph nodes were removed aseptically and single-cell suspensions were prepared. Peripheral T cells were also analyzed after retro-orbital bleeding and red cell lysis with ACK lysing buffer (Biowhittaker, Walkersville, MD, USA). One to 5 × 105 cells were resuspended in PBS (Biowhittaker) containing 1% BSA (Irvine, Santa Ana, CA, USA). For surface staining, cells were incubated with FITC-conjugated anti-rat CD4 (Caltag, San Francisco, CA, USA) and allophycoyanin-conjugated anti-CD25 mAbs (BD Biosciences, San Jose, CA, USA) on ice for 30 minutes. After two washes with PBS-% BSA, cells were prepared for intracellular phycoerythrin-labeled Foxp3 mAb staining according to the manufacturer's recommendations (eBiosciences, San Diego, CA, USA). Cells were then resuspended in 0.5 ml PBS-1% BSA for analysis by flow cytometry (FACSCalibur®; BD Biosciences) using CellQuest Pro® software (BD Biosciences).
Cell culture and cytokine assays
Peritoneal macrophages were obtained 4 days after disease induction from peritoneum cavity exudates. Briefly, after four washes with cold PBS of the peritoneum cavity of each rat, enriched macrophage suspension was adjusted to 1 × 106 cell/ml and cultured with or without lipopolysaccharide (LPS) stimulation (50 ng/ml) in complete DMEM medium containing 10% (vol/vol) heat-inactivated FBS, 2 mM glutamine, 15 mM Hepes, 1% nonessential amino acids, 1 mM sodium pyruvate, penicillin (100 μg/ml), streptomycin (50 μg/ml) and 50 μM 2-mercaptoethanol (all from Biowhittaker). Supernatants were then collected at 24 hours and tested for TNF by ELISA (BioLegend, San Diego, CA, USA) following the manufacturer's instructions. Rats were blood punctured in the retro-orbital sinus at days 1, 4, 6 and 11 for total TGFβ quantification in the plasma after a 1/20 dilution by ELISA (Promega, Madison, WI, USA) following the manufacturer's instructions.
Statistical analysis
Group comparisons of parametric data were made by Student's t test. We used the Mann-Whitney rank-sum test for nonparametric data. We assessed score comparisons between groups by one-way analysis of variance, and when significant differences were found we used Dunn's method to identify differences compared with the control group. We performed statistical analyses with SigmaStat 3.11 software (Systat Software, Richmond, CA, USA). We tested data for normality and variance, and considered P < 0.05 significant. Statistical analysis was assessed when the number of experimented animals or conditions was sufficient.
Discussion
Apoptotic cell injection has been previously shown to induce a transient immunosuppressive environment, sufficient in animal models to reduce inflammation [
6,
13] or to favor tolerance toward allo-antigens [
13,
22] or self antigens [
8]. RA is an autoimmune disease characterized by a lack of apoptosis leading to hyperplasia of the synovial lining. The macrophage is one of the principal cell types that contribute to the pathogenesis of RA, since macrophage depletion suppresses the chronic phase of SCW-induced arthritis [
21]. This is why we decided to infuse apoptotic cells in a RA model: providing apoptotic cells to macrophages may change their proinflammatory behavior. In the present article, we showed that apoptotic cell injection prevents macrophages from SCW-induced TNF secretion. In addition, apoptotic cell infusion leads to an increased of Tregs in the draining lymph nodes. This was associated with a decrease in the symptoms and the severity of SCW-induced arthritis.
Both acute and chronic joint inflammations were significantly inhibited by apoptotic cell injection – including reduction of swelling and decreased tissue and bone destruction. The synovial inflammatory cell infiltration and TNF production were also markedly suppressed. Our data indicate that delivery of apoptotic cells
in vivo even in the periphery may initiate anti-inflammatory mechanisms to antagonize the joint inflammatory response. Macrophages exposed by apoptotic cells seemed to be less efficient to induce and sustain SCW inflammation. Indeed, apoptotic cell injection acts in two different ways. First, apoptotic cells together with phagocytes that digest apoptotic cells in a very efficient manner induce an anti-inflammatory microenvironment. This first sequential event directly targets the acute phase induced by the SCW complexes and may prevent effector T-cell activation and migration to inflammatory sites such as joints and bones. The apoptotic cell injection-induced TGFβ increase also correlates with the Treg increase. Then, after the uptake of apoptotic cells, professional phagocytes such as macrophages become more resistant to inflammatory signals [
23,
25] and cytokines, as we observed here with the decrease of TNF secretion after LPS stimulation. This second sequential event targets phagocytes and may prevent occurrence of the chronic phase. This is in line with the work of Richards and colleagues showing that macrophage depletion alters the chronic phase of SCW-induced RA [
21].
Macrophages, after the uptake of apoptotic cells, may then release TGFβ- which we detected in the periphery very early after apoptotic cell infusion – and may contribute to the resistance of macrophages to LPS stimulation, as previously described [
23]. The reduction of circulating TGFβ in SCW-induced inflammation at day 1 and the slight increase in circulating active TGFβ at day 4 in apoptotic cell-treated animals at the time of articular index reduction also suggests a critical role for endogenous TGFβ in the control of inflammation. The Treg increase observed at day 4 in the inguinal draining lymph nodes of animals receiving apoptotic cells plus SCW also supports this point. The increase of TGFβ we observed in the circulation after apoptotic cell injection alone is in line with another experimental model, where apoptotic cells were induced
in vivo and led to a TGFβ increase for 4 days with a peak at 24 hours after apoptotic cell induction [
8]. As previously shown in tolerance induction by oral administration of SCW peptide [
18], TGFβ is mainly responsible for the prevention of the disease; apoptotic cell administration may induce a similar effect.
In addition to macrophages, immature dendritic cells may also uptake apoptotic cells and then may produce less IL-1β, IL-6 and TNF in response to LPS stimulation [
4] – all of these proinflammatory cytokines were found at elevated levels in RA patients or in collagen-induced arthritis mice. The effects may be ascribed at least in part to the TGFβ production by immature dendritic cells upon digestion of apoptotic cells [
26]. Moreover, IL-1β has been demonstrated as an important mediator of SCW-induced arthritis by promoting Th17 differentiation [
27]. One may speculate that apoptotic cell infusion by downregulating IL-1β production in responses to inflammatory signals [
4] controls Th17 response and subsequent arthritis development.
The second effect mediated by apoptotic cell injection may implicate the release of TGFβ, as described previously [
3,
13,
23]. The elevated concentration of TGFβ permits the Treg increase, preventing activation of specific T cells responsible for the chronic phase of arthritis. The fact that the Treg increase was observed in our model only in the lymph nodes draining SCW-induced pathology further supported this idea. In line with this notion, it has been shown that adoptive transfer of CD25
+ Tregs effectively decreases collagen-induced arthritis [
28]. Because Th17 cells has been suggested to be involved in the induction of arthritis in an experimental model of spontaneous arthritis [
29,
30], apoptotic cell injection may also increase T-cell polarization to Tregs instead of Th17 differentiation by increasing the TGFβ levels.
Conclusions
In the present article we have shown that apoptotic cell injection can significantly decrease the occurrence and the severity of SCW-induced RA. Apoptotic cell injection offers a tool to control and prevent macrophage-induced SCW inflammation. Apoptotic cell prevention of SCW-induced RA seems to be achieved sequentially: first after uptake of apoptotic cells by phagocytes, in particular macrophages that decrease their response to LPS; and then through a Treg increase in lymphoid organs, in particular in the draining lymph nodes, thus preventing and controlling SCW inflammation. These findings may provide insight into understanding the pathogenesis of chronic inflammation and autoimmune disease, and may also offer clues to manipulate Tregs and macrophages by apoptotic cell injection. The data are in line with our previous work suggesting the potential of apoptotic cells to treat ongoing autoimmune disease such as experimental autoimmune encephalomyelitis.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SP designed and performed most of the experiments and wrote the manuscript. PS participated in the writing of the manuscript. WJC initiated and directed the study, designed and performed some of the experiments and edited the manuscript. All authors read and approved the final manuscript.