Background: The use of regulatory or immature dendritic cells (DCs) as tools for modulating experimental rheumatoid arthritis is very recent. Tumour necrosis factor (TNF)-stimulated DCs have been shown to restore tolerance in experimental autoimmune encephalomyelitis and collagen-induced arthritis (CIA).
Objective: We investigated the capacity of short-term lipopolysaccharide (LPS)-stimulated DCs pulsed with type II collagen (CII) to induce tolerance against established CIA.
Methods: Bone marrow-derived DCs were generated in the presence of granulocyte monocyte colony-stimulating factor (GM-CSF). After CIA induction, mice were injected at day 35 with a single dose of 4- or 24-h LPS-stimulated DCs that had been loaded with CII (4hLPS/CII/DCs or 24hLPS/CII/DCs). Arthritis progression was monitored by clinical and histological evaluations.
Results: Flow cytometry of 4hLPS/CII/DCs showed intermediate CD40 and CD86 expression, lower than that of 24hLPS/CII/DCs (fully mature) and higher than that of CII/DCs (immature). A functional assay showed that 4hLPS/CII/DCs display increased endocytosis ability with respect to 24hLPS/CII/DCs, indicating a semimature state. The single inoculation of 4hLPS/CII/DCs in mice with established CIA reduced disease severity significantly over time. Histological evaluation of mice treated with 4hLPS/CII/DCs revealed diminished inflammatory synovitis, cartilage damage and fibrosis. Co-cultures of DCs with splenocytes from CIA mice showed that collagen-specific interferon (IFN)γ production was dramatically inhibited by 4hLPS/CII/DCs. 4hLPS/CII/DCs were high IL10 producers, which could explain the inhibition of arthritis progression in mice receiving this treatment because neither antibodies nor regulatory CD4+CD25+Foxp3+ T lymphocytes were demonstrated to be involved.
Conclusion: Short-term LPS-modulated DCs inoculation interferes with CIA progression when loaded with CII.
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In rheumatoid arthritis (RA) pathogenesis, there is consensus that the autoimmune process involves altered self-antigen presentation by antigen-presenting cells (APCs) or dendritic cells (DCs) leading to the activation of auto-reactive CD4+ T lymphocytes.1 Antigen-activated CD4+ T lymphocytes are the main mediators of inflammation and articular damage through the secretion of interleukin (IL)-17, which activates the release of IL1, IL6 and tumour necrosis factor (TNF) by macrophages.2 3
Among several arthritogenic antigens, the participation of endogenous proteins such as type II collagen (CII) is well demonstrated.4 Moreover, inoculation of the genetically susceptible DBA/1 mouse strain with bovine CII has allowed the development of the collagen-induced arthritis (CIA) murine model, the most extensively applied experimental model of RA.5
Current data indicate that DC functions are related to the stage of their maturation. After an antigenic stimulus, immature DCs become fully mature DCs, as shown by upregulation of major histocompatibility complex (MHC) and costimulatory molecules, such as CD80 and CD86.6 Unlike mature DCs, immature DCs are poor APCs, and they have been found to have antigen-specific regulatory properties.7 Thus, DCs play a central role in orchestrating the immune response and inducing tolerisation of immune responses against self and non-self antigens.8
To promote tolerance, DCs must capture, process and present self antigens in the “steady state”, an immature form of differentiation involved in the induction of peripheral T cell tolerance.9 10 Therefore, the generation of tolerogenic or regulatory DCs in the laboratory has become the focus of new therapies for inhibiting the unwanted immune responses responsible for autoimmunity.11 In order to induce and keep a regulatory phenotype of DCs, several pharmacological strategies have been used, including agents such as IL10, TNF and the vasoactive intestinal peptide (VIP), among others.12–15
Regulatory DCs have been used to restore tolerance in experimental autoimmune encephalomyelitis (EAE) and CIA.13 14 Hence, repetitive injections of DCs matured with TNF-induced antigen-specific protection of mice from EAE13 and CIA.14
In this work, we investigate the ability of immature DCs, short-term lipopolysaccharide (LPS)-stimulated DCs (semimature DCs) and long-term LPS-stimulated DCs (fully mature DCs), all of them collagen-pulsed, to induce tolerance in mice with established CIA. Our data confirms that DCs can be differentially modulated by using a wide spectrum of biological agents, but most importantly, it indicates a strong influence of the duration of stimulus.
We used 7–8-week-old DBA1/lacJ (H2q) mice, obtained from Jackson Laboratories (Bar Harbor, ME, USA) and maintained in accordance with international guidelines for animal care; protocols were approved by the University of Chile Bioethics Committee.
Bone marrow (BM)-derived DCs
BM cells were cultured for 7 days in medium containing granulocyte monocyte colony-stimulating factor (GM-CSF).16 17 After 6 days of culture, DCs were loaded for 24 h with bovine CII protein (Chondrex, Redmond, Washington, USA) (CII/DCs) or were left unloaded. Then, DCs were removed and used as immature DCs (untreated DCs) or they were stimulated with 1 μg/ml of LPS (Escherichia coli, serotype 026:B6, Sigma, St Louis, Missouri, USA) for 4 h (CII loaded: 4hLPS/CII/DCs or unloaded: 4hLPS/DCs) or for 24 h (CII loaded: 24hLPS/CII/DCs or unloaded: 24hLPS/DCs), respectively.
Antibodies and fluorescence-activated cell-sorting (FACS) analysis
Antibodies were purchased from eBiosciences (San Diego, California, USA): fluorescein isothiocyanate (FITC)-labelled anti-CD11c, anti-CD86, anti-CD80, anti-MHC class II (AI/EI) and anti-CD40. Differentially treated DCs were incubated with the appropriate antibodies, fixed in paraformaldehyde and analysed by flow cytometry (FACSCalibur, Becton Dickinson, San Jose, California, USA).
DCs, 2×105, were incubated with 1 mg/ml of the endocytosis marker FITC-conjugated dextran (Molecular Probes, Leiden, The Netherlands) for 30 min at 37°C or at 4°C (control) and then analysed by FACS. Mean fluorescence intensity values within the gate for the different endocytically active stages were plotted.18
Cytokine determination in non-purified DCs
Cytokines were captured by specific anti-IL10 or anti-IL12p70 monoclonal antibodies (eBioscience), detected by biotinylated anti-cytokine antibodies and revealed by luminescence (Luminoskan Ascent; Thermo, Vantaa Finland).
Cytokine determination in purified DCs
DCs were washed in PBS/0.1% BSA and stained with FITC-labelled anti-mouse CD11c antibody (Becton Dickinson). CD11c+ cells were sorted in a FACSCalibur sorter (Becton Dickinson). Sorted CD11c+ cells (>98%) were differentiated from untreated DCs, 24hLPS/DCs and 4hLPS/DCs, and cytokine contents in supernatants were assayed by ELISA (eBioscience).
CIA induction and clinical evaluation of arthritis
Bovine CII protein was emulsified with complete Freund adjuvant, and then injected subcutaneously into the base of the tail (100 μg CII/mouse). Immunisation was boosted 3 weeks later by a subcutaneous injection of 100 μg of CII. Beginning 3 weeks after immunisation, mice were clinically examined 3 times each week. Arthritis severity was graded as follows: 0 = normal joints, 1 = 1 or 2 swollen joints, 2 = 2–4 swollen joints, 3 = >4 swollen joints and 4 = extreme swelling of the entire paw and/or ankylosis. An arthritis score was calculated by adding the scores for each paw/mouse.
Differentially treated DCs (5×105) were intraperitoneally administered to 4–6 mice per group by a single inoculation at day 35 after the first inoculation of CII.
DC and splenocyte co-cultures
Splenocytes from CIA mice were co-cultured with 4hLPS/CII/DCs, 24hLPS/CII/DCs, CII/DCs or untreated DCs for 7 days.19 Then, 100 000 cells, isolated from the co-cultures, were co-cultured with 20 000 differentially treated DCs in a 96-well enzyme-linked immunosorbent spot (ELISPOT) assay plate (Millipore, Billerica, Massachusetts, USA), coated with an anti-mouse IFNγ monoclonal antibody (eBiosciences). After adding a secondary anti-IFNγ antibody, the reaction was revealed with streptavidin-alkaline phosphatase (Mabtech, Cincinatti, Ohio, USA). Spots representing IFNγ secreted by a single cell were counted.
Tissue sections were cut and stained with haematoxylin/eosin. A morphometric analysis was performed evaluating all joints in the paws with the most evident clinical damage. Synovial membrane leukocyte infiltration was determined as an index (%), calculating the proportion of the area corresponding to cellular nuclei.20 The loss of articular cartilage by destruction was graded on a scale of 0–5 (0 = normal cartilage, 1 = minimum damage in chondrocytes, 2 = moderate chondrocyte death, 3 = moderate chondrocyte death and loss of articular surface, 4 = active chondrocyte death and articular surface damage and 5 = maximum chondrocyte death and cartilage erosion). Fibrosis was evaluated by a score ranging from 0–3 (0 = normal tissue, 1 = 1–3 fibrous layers, 2 = 4–10 fibrous layers or 1–4 fibrous foci and 3 = >5 fibrous foci).
Antigen-specific antibodies in serum by ELISA
Microtitration plates, sensitised with bovine CII protein (Chondrex), were incubated with mice serum and then treated with anti-mouse-specific subclass antibodies.
Differences in disease severity were analysed by the Mann–Whitney U test. A one-way analysis of variance (ANOVA) was used for other comparisons. A p value <0.05 was considered statistically significant with a 95% CI.
Influence of LPS on DC maturation
DCs were obtained from BM cells after 7 days of culture in medium containing GM-CSF, with a yield of approximately 60%. Figure 1A shows that these cells expressed CD11c, a characteristic marker for DC populations, and CD11b specific for myeloid lineage.
To study the response of DCs to various maturation stimuli, DCs obtained from DBA1/lacJ mice were previously loaded with bovine CII or left unloaded, and then subjected to LPS for 4 or 24 h as mentioned above. Untreated DCs were used as immature controls. After the different stimuli and incubation times, the cells were harvested and analysed by FACS for the expression of MHC class-II and costimulatory molecules (CD40 and CD86) (fig 1B). For all stimulatory or loading treatments, DCs showed equivalent levels of MHC class II. Interestingly, 4hLPS/CII/DCs displayed a lower level of CD40 and CD86 expression than 24hLPS/CII/DCs (p<0.01). However, these levels were similar to those of untreated DCs, which suggest that 4hLPS/CII/DCs correspond to a transition between immature and mature stages of phenotypic differentiation.
The functional ability of DCs to take up the endocytosis marker FITC–dextran was also examined, although none of the cells analysed demonstrated significantly differential endocytosis abilities (fig 1C). Untreated DCs and CII/DCs displayed the highest levels of endocytosis compared with 4hLPS/CII/DCs, which showed a diminished endocytosis level, indicating a semimature transition state of these cells. 24hLPS/CII/DCs and 24hLPS/DCs both showed a diminished endocytotic ability, characteristic of functionally mature DCs. Thus, phenotypic and functional data indicate that 4hLPS/CII/DCs represent a semimature stage of DCs with the ability to present CII to naïve T lymphocytes.
Using non-purified DCs, we detected that 4hLPS/CII/DCs and 4hLPS/DCs showed a higher IL10 production than 24hLPS/CII/DCs (p<0.05) and untreated DCs (p<0.01) (fig 2A). All LPS-stimulated DCs showed a higher IL12 release when compared to untreated DCs (p<0.01), while no significant differences were detected between 4hLPS/DCs or 24hLPS/DCs loaded or unloaded with CII. In purified DCs, we observed that 4hLPS/DCs released higher IL10 levels than 24hLPS/DCs (p<0.001), while the IL12, IL6 and TNF levels released by 4hLPS/DCs were significantly lower than those released by 24hLPS/DCs (p<0.001) (fig 2B).
Modulation of CIA by immunisation with 4-h LPS-treated DCs loaded with collagen
To evaluate the ability of the DCs to modulate CIA in an antigen-specific manner, they were pulsed with CII. After inducing active CIA, DBA1/lacJ mice were immunised at day 35 with differentially treated DCs. The progression and onset of the disease in the groups treated with CII/DCs, 4hLPS/DCs or 24hLPS/CII/DCs were similar to those observed in the control mice group immunised with CII/CFA alone (fig 3A,B) (p>0.05). By contrast, mice treated with 4hLPS/CII/DCs displayed significantly less-severe clinical disease as compared to the other groups over time (fig 3A,B) (p<0.001). Additionally, the injections with untreated DCs and 24hLPS/DCs were unable to modify the natural progression of CIA (data not shown). In addition, two injections (at days 28 and 35) with 4hLPS/CII/DCs resulted in significantly less severe clinical disease as compared to the other groups over time (p<0.001). Although two 4hLPS/CII/DC injections interfered slightly more with CIA progression than one injection, no significant differences were observed.
Histological examination of joints, as shown in fig 4A, revealed that control CIA mice had more severe inflammatory synovial microscopic changes. Morphometric analysis indicated a significantly lower (p<0.005) inflammatory cell infiltration index for 4hLPS/CII/DCs mice compared to joints from control CIA mice or from 24hLPS/CII/DCs mice (fig 4B). The group treated with 4hLPS/CII/DCs showed a similar leukocyte distribution in the inflammatory cell infiltrate including macrophages, lymphocytes and neutrophils as compared to CIA and 4hLPS/DCs groups (data not shown). The evaluation of cartilage damage indicated that mice treated with 4hLPS/CII/DCs showed significantly less damage than the CIA group, the 24hLPS/CII/DCs group or the group inoculated with 4hLPS/DCs (fig 4C) (p<0.01). The fibrosis damage analysis revealed that mice inoculated with 4hLPS/CII/DCs displayed a moderate fibrosis score when compared with the other groups (fig 4D) (p = 0.05).
Inhibition of splenocyte IFNγ production by 4-h LPS-treated DCs loaded with collagen
In order to evaluate the inhibitory activity of 4hLPS/CII/DCs on the effector cells involved in CIA induction, splenocytes from CIA mice were co-cultured with differentially stimulated DCs and the IFNγ secretion was assessed. As depicted in fig 5, splenocytes co-cultured with 4hLPS/CII/DCs showed a dramatically diminished IFNγ production when compared to splenocytes co-cultured with 24hLPS/CII/DCs, CII/DCs or untreated DCs (p<0.001). All naïve DCs were able to induce IFNγ production by splenocytes (including 4hLPS/DCs and 24hLPS/DCs). However, DCs/CII and 24hLPS/CII/DCs were able to induce a higher IFNγ production than CII-unloaded DCs (p<0.05) and than 24hLPS/DCs (p<0.01), unlike 4hLPS/CII/DCs.
Additionally, in order to investigate whether the injection with differentially treated DCs could affect the mouse antibody immune response, we measured CII-specific IgG1, IgG2a and IgG2b mouse antibodies in serum obtained from mice at day 70, after CIA induction. No differences were detected for IgG1, IgG2a or IgG2b mouse subclasses (data not shown).
Finally, in order to determine whether CD4+CD25+Foxp3+ regulatory T lymphocytes (Treg) were responsible for the effect of 4hLPS/CII/DCs injection on CIA progression, intracellular Foxp3 expression was assessed by FACS at day 36, 38, 42 and 49 after the injection of DCs treated with the different stimuli. Although not significant, we observed that the percentage of Treg cells was greater in spleen and lymph nodes from mice treated with 4hLPS/CII/DCs than other groups at day 42 and 49 (data not shown).
Different approaches for modulating DCs in an antigen-specific manner have been successfully developed, including the establishment of human and murine-modified DCs with potent immunoregulatory properties.21–23 In autoimmune disease models, vaccination with TNF-modulated DCs has been shown to prevent EAE and CIA.13 14 The use of VIP has succeeded in reducing the progression of CIA and EAE15 and significantly ameliorating the clinical and histopathological severity of murine colitis.24 More recently, the administration of LF 15-0195-treated DCs, a nuclear factor (NF)-κB inhibitor, has been shown to interfere with CIA, improving clinical score and reducing antibody response to the CII.25
In this study, we have demonstrated that DCs can affect the outcome of CIA in an antigenic-specific manner. Inoculation of mice with a single injection of 4hLPS/CII/DCs, after established CIA, led to less severe arthritis as determined by swollen joint severity and joint score. The histological evaluation allowed us to conclude that mice treated with 4hLPS/CII/DCs revealed significantly less inflammatory synovitis, pannus formation, cartilage damage and fibrosis compared with the other study groups. Additionally, the ex vivo antigen-specific IFNγ production blockade by 4hLPS/CII/DCs suggests that the inhibition of arthritis progression observed in mice treated with 4hLPS/CII/DCs could be a consequence of in vivo IFNγ production inhibition, responsible for damage observed in CIA. In addition to the report of Morita et al, who with a single injection of IL4-transduced DCs, reduced the incidence and severity of CIA, ours is the first report ever published using the inoculation of regulatory antigen-pulsed DCs as a therapeutic approach.26
Sorted 4hLPS/DCs secreted lower levels of IL12p70, IL6 and TNF than those detected in 24hLPS/DCs, suggesting that 4hLPS/DCs could have the ability to polarise the immune response in a pro-tolerogenic fashion. However, 4hLPS/DCs and 4hLPS/CII/DCs showed a higher secretion pattern of IL10 (in sorted and non-sorted cells), a key cytokine in modulating and regulating in vivo and ex vivo immune responses, indicating that this mechanism may be responsible for CIA blockade observed in mice injected with 4hLPS/CII/DCs as a consequence of the IL10 inhibitory effect on IFNγ-producing T cells, responsible for CIA damage. These data agree with previous reports involving specific DC subsets and IL10 as mediators of tolerance by the induction of regulatory T cells or by anergy.27 28
Thus, in the EAE model, Menges et al found that vaccination with peptide-pulsed TNF-stimulated DCs led to the induction of IL10-producing CD4+ T cells and an inhibited generation of autoantigen-specific Th1 cells.13 Chorni et al demonstrated that the progressive reduction observed in EAE and CIA, after the inoculation of VIP-treated DCs, was mediated by IL10-producing T lymphocytes (Tr1).15 Additionally, Charbonnier et al have reported that repeated injections with immature DCs trigger the expansion of a novel CD4+ α-galactosylceramide/CD1d-nonrestricted/IL10-producing T cell regulatory population (CD49b+), able to protect mice from CIA.29 In our case, the evaluation of Treg after 4hLPS/CII/DCs injection showed that the percentage of CD4+CD25+Foxp3+ cells was greater in spleen and lymph nodes from mice treated with 4hLPS/CII/DCs than other groups at day 42 and 49, suggesting that Treg may be involved in the 4hLPS/CII/DCs-mediated CIA modulation. These results, together with our observation that 4hLPS/DCs expressed intermediate CD86 levels, are consistent with the finding reported by Yamazaki et al who demonstrate that DCs induced antigen-specific Treg from Foxp3(–) precursors. During this process, the CD86 expression proved to be essential for inducing IL2 production by T cells stimulated by DCs.30
Alternatively, the effect of 4hLPS/CII/DCs injection could have been mediated by a clonal anergy mechanism triggered by the 4hLPS/DCs, displaying regulatory properties. This hypothesis is consistent with the fact that 2 months after the DC injection, the 4hLPS/CII/DCs-inoculated mouse group showed CIA progression re-establishment in 100% of the animals (data not shown). There is previous evidence of the induction of anergic and regulatory T cells by plasmacytoid DCs and other DC subsets.31 32 Thus, immature human IL10 DCs have shown to induce antigen-specific anergy by the interaction of CTLA-4 on T lymphocyte33 and indoleamine 2,3 dioxygenase production by DC subsets.27
Although LPS has been identified as a maturation agent for DCs, there are reports demonstrating that, depending on the stimuli used to terminally mature BM-DCs, DCs synthesise a different pattern of cytokines and exhibit distinct T helper cell-driving potential.34 Thus, the combination of low doses of LPS and cholera toxin is able to induce IL10 production of immature DCs, characterised by enhanced expression of CD80 and CD86 but inhibited CD40 expression and inflammatory cytokines production.35 Therefore, our findings describing a phenotypic and functional intermediate stage for 4hLPS/DCs, in addition to their high IL10 production and in vivo modulating abilities, are in agreement with data previously reported.
Differentiation of naïve CD4+ T cells into IFNγ-producing Th1 cells is critical for the generation of IgG2a antibodies in mice. We showed that 4hLPS/CII/DCs injection was unable to modify the established Th1 pattern in CIA mice. Since no Th1/Th2 cytokine profiles were determined, this was only shown by the absence of differences in the IgG2a CII-specific antibodies in the different mouse groups, reflecting that our strategy failed to shift the established Th1 response, unlike treatments to prevent CIA.
Finally, while 4hLPS/DCs expressing a regulatory phenotype may display a transient stage of maturation with the ability to interfere in the cellular processes operating in autoimmune processes such as arthritis, the molecular mechanisms involved remain to be elucidated.
Funding: This study was supported by Fondecyt-Chile (104-0860 and 107-0553).
Competing interests: None declared.
Ethics approval: All protocols were approved by the University of Chile Bioethics Committee.
LS, OA, AE, JC-L, N R-C, DC, AA, RZ, F S-O and JCA are members of the Millennium Nucleus on Immunology and Immunotherapy – Chile (P04/030-F).