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Abstract
Background Systemic sclerosis (SSc) is an autoimmune disease and accumulating evidence suggests a role for Toll-like receptor (TLR)-mediated activation of dendritic cells (DCs).
Objective To map TLR-mediated cytokine responses of DCs from patients with SSc.
Methods 45 patients with SSc were included. Patients were stratified as having diffuse cutaneous SSc (dSSc) or limited cutaneous SSc (lSSc) according to the extent of skin involvement, and further divided into those with late (>3 years) or early disease (<2 years). DCs were stimulated with ligands for TLR2, TLR3, TLR4, TLR7/8 or combinations. Plasma samples were collected from patients with SSc (n=167) and measured for interleukin 6 (IL-6), tumour necrosis factor α (TNFα), IL-12, IL-10 and interferon γ.
Results Stimulation of DC subsets from patients with early lSSc and dSSc with ligands for TLR2, TLR3 or TLR4 resulted in higher secretion of IL-6 and TNFα compared with those having late disease or healthy controls. Remarkably, the production of IL-12 was lower upon stimulation with TLR ligands in most patients with SSc, whereas the secretion of IL-10 was very high in patients with the dSSc phenotype, particularly in those having early dSSc. The combination of various TLR ligands led to reduced cytokine secretion in all patients with SSc. Circulating levels of these cytokines further underscored the presence of differences between various SSc phenotypes.
Discussion The altered TLR-mediated activation of DCs may be responsible for Th2 skewed T-cell activation in SSc that may be orchestrated by fibrogenic T-cell cytokines, such as IL-4 and IL-13. DC targeting could thus offer new avenues for therapeutic intervention.
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Introduction
Systemic sclerosis (SSc) is a complex autoimmune disease characterised by excessive deposition of matrix molecules leading to fibrosis. The hallmark of SSc is skin fibrosis and fibrosis of internal organs, leading to considerable morbidity and premature death. Although the pathogenesis of SSc is not well understood, it is recognised that three seemingly disparate processes: vascular injury, immune activation and fibrosis, underlie its clinical appearance.1
Several studies in SSc have focused on the role of fibroblast activation, which is generally thought to underlie organ fibrosis. Indeed, seminal studies have shown that SSc fibroblasts explanted from lesional skin synthesise increased collagen in vitro compared with fibroblasts isolated from healthy controls.2 3 Although these studies suggested an intrinsic defect of fibroblasts in SSc, more recent studies have questioned this interpretation and propose that fibroblast abnormalities in SSc skin are at least partially dependent on local factors that stimulate the fibrotic phenotype. Mononuclear cell infiltration is one of the earliest events observed in involved skin from patients with SSc and these cells release potent regulators of inflammation and tissue remodelling, cytokines. Accumulating evidence suggests the involvement of a myriad of inflammatory mediators in SSc, such as interleukin 1α (IL-1α), IL-23, IL-6 and IL-17, none of which are made specifically by fibroblasts. In contrast, the main producers, or in the case of IL-17, inducer, of these mediators are professional antigen-presenting cells (APCs), of which dendritic cells (DCs) are the most influential. Moreover, transforming growth factor (TGF)β, a profibrotic cytokine thought to be of paramount importance in this condition, is highly secreted by DCs.
DCs are the professional APCs that control the balance between immunity and tolerance.4 In this light, several autoimmune conditions have been associated with aberrant function of DCs, including diabetes,5 multiple sclerosis,6 7 systemic lupus erythematosus8 and rheumatoid arthritis.9 To carry out their surveillance function, DCs express a plethora of pattern recognition receptors, among which the Toll-like receptor (TLR) family has been the most thoroughly studied. TLRs belong to the family of pattern-recognition receptors, which were first identified to recognise microbial components, known as pathogen-associated patterns. However, increasing evidence indicates that TLRs can also be activated by numerous ‘host-derived’ agonists (so-called endogenous ligands or alarmins).10 This latter observation underlies the recent increased interest in TLRs and their potential implication in numerous autoimmune syndromes (reviewed by Roelofs et al 11). We previously demonstrated that endogenous TLR4 ligands are more abundantly expressed in the circulation of patients with SSc, further supporting a role for TLR-mediated immune activation in this disease.12 Finally, the conundrum that surrounds the induction of TGFβ suggests a potential role for TLRs in SSc. The trigger(s) that drives TGFβ production and/or activation is largely unknown. However, recent publications suggest a role for the ‘milieu interior’ that triggers APCs to secrete TGFβ, probably designed to dampen immune responses and limit tissue damage. For example, zymosan, a ligand for TLR2 was shown to induce IL-10 and TGFβ-expressing macrophages that could induce immunological tolerance in vitro.13 In addition, TLR4 has been shown to be crucial in the development of hepatic fibrosis.14 Thus, while the emerging picture of the pathogenesis of SSc is one of enormous complexities; many observations suggest a role for DCs and TLR-mediated DC activation in this condition.
We show here that the stimulation of DCs from patients with SSc with single TLR ligands results in the increased production of various cytokines compared with that seen by DCs from healthy controls. Interestingly, TLR-stimulated IL-12 secretion was lower in patients with SSc with early disease, whereas IL-10 production was markedly increased. Stimulation with combinations of TLR agonists resulted in a much lower secretion of inflammatory mediators by DCs from patients with SSc compared with that in healthy controls. Again, IL-10 secretion was higher in DCs from patients with SSc, suggesting that TLR agonists in patients with SSc might stimulate strong Th2 skewing. These observed aberrant TLR responses may have an important role in the production of profibrotic mediators such as IL-13.
Patients and methods
Patient collection
For in vitro experiments, 28 patients presenting at the Arthritis Center, Boston Medical Center and 17 patients visiting the Radboud University Nijmegen Medical Center (RUNMC) were included in the study. All patients met the American College of Rheumatology preliminary criteria for the classification of SSc.15 For in vitro experiments, patients were subdivided as having limited cutaneous SSc (lSSc, n=24) or diffuse cutaneous SSc (dSSc, n=21) according to the extent of their skin involvement (table 1 for clinical characteristics).16 A further subdivision was made between early and late disease based upon the duration of disease listing as early dSSc (edSSc) for those having a disease duration <2 years and late dSSc (ldSSc) for those who had disease for longer than 3 years. As a comparator group, 22 healthy controls were studied. For the measurement of circulating levels of tumour necrosis factor α (TNFα), IL-6 and interferon γ (IFNγ) plasma from healthy controls (n=28) and 168 patients with SSc (early limited cutaneous SSc (elSSc), n=43; late limited cutaneous SSc (llSSc), n=68; ldSSc, n=34; edSSc, n=23) from the Boston University area, RUNMC area and Lund University Hospital area were analysed (table 2 for clinical characteristics). Treatment was monitored after the diagnosis of SSc and until inclusion in the study. Blood samples were obtained with informed consent under local institutional review board approval.
Clinical characteristics of patients with SSc included in cell-based experiments
Clinical characteristics of patients with SSc included for serological measurements
Isolation and culture of monocytes, monocyte-derived DCs, myeloid BDCA-1 (CD1c) DCs
Peripheral blood mononuclear cells were isolated from heparinised venous blood by density-gradient centrifugation over Ficoll–Paque (Amersham Bioscience, Diegem, Belgium). Monocytes and myeloid DCs (mDCs) were obtained using CD14 and BDCA-1 microbeads, respectively (Miltenyi Biotec, California, USA). How monocyte-derived DCs (moDCs) were generated has been extensively described by our group.
Flow cytometric analysis of CD14, CD86 and major histocompatibility complex (MHC) expression on moDCs
Phenotypical analysis of moDCs was performed using standardised flow cytometry protocols as described previously.17 DCs were characterised by staining with monoclonal antibodies against human CD14 (Miltenyi Biotec), CD86 (BD Biosciences, New Jersey, USA), major MHC-II DR/DP (clone Q1514). Cells were analysed with a fluorescence-activated cell sorter (FACSCalibur; BD Biosciences) and analysed with FlowJo 8.7.3 (TreeStar, Oregon, USA) for the proportion of positive cells and the mean fluorescence intensity relative to cells stained with the relevant IgG isotype controls.
Stimulation of moDCs and mDCs with TLR ligands
Freshly isolated monocytes, mDCs and day 6 moDCs were re-plated in a concentration of 0.5×106 DCs/ml and either transferred to 24-well (1 ml) or 96-well (100 µl) culture plates for monocytes and mDCs or to 96-well (100 µl) culture plates for monocytes, and mDCs. The culture medium consisted of RPMI-1640 Dutch modification (Invitrogen Life Technologies) supplemented with 10% fetal calf serum and antibiotic–antimycotic agent (Invitrogen Life Technologies, Breda, The Netherlands) in the presence of IL-4 (500 U/ml; Schering-Plough) and granulocyte monocyte colony-stimulating factor (800 U/ml; Merck, New Jersey, USA). Cells were then stimulated with TLR agonists for 24 h for the collection of supernatants. TLR agonists were used at the following concentrations unless otherwise described: pLPS (TLR4, 100 ng/ml, Escherichia coli; Sigma-Aldrich, The Netherlands), R848 (TLR7/8, 2 µg/ml; InvivoGen, France), Pam3Cys (TLR2, 5 µg/ml; EMC Microcollections, Germany) and poly(IC) (TLR3, 25 µg/ml; InvivoGen).12 17 E coli lipopolysaccharide was double purified at our laboratory according to the phenol–water extraction method to remove any contaminating proteins before use.
Measurement of cytokines
Levels of IL-10, TNFα, IL-12p70 and IL-6, were measured in the supernatants using commercially available kits (Bio-Rad, Veenendaal, The Netherlands), according to the manufacturer's instructions. Cytokine levels were measured and analysed with the Bio-Plex system (Bio-Rad). The sensitivity of the cytokine assay was <5 pg/ml for all cytokines measured. IL-6, TNFα, IL-12, IL-10 and IFNγ were measured in the plasma of patients with SSc and healthy controls using the Bio-Plex system.
Statistical analysis
Values are shown throughout the paper as mean±SEM. Proportions of lymphocyte subpopulations were tested for normal distribution using the Shapiro–Wilk W test and compared using statistical analyses appropriate for normal (Student t test) or non-normally distributed results (Mann–Whitney U test) where appropriate. Differences were considered significant for *p<0.05, **p<0.01 and ***p<0.001. All statistical analyses were performed using Graphpad Prism (GraphPad Prism 4.0 by Graph Pad software, La Jolla, California, USA). Notably, monocytes, moDCs and mDCs from patients with SSc showed a very low and comparable level of spontaneous production of the cytokines tested. Therefore, in all figures, the data are corrected for spontaneous production (basic culture conditions without stimulation).
Results
TLR-mediated stimulation of freshly isolated monocytes from patients with SSc indicates an altered response compared with healthy controls
Monocytes provide an easy means to screen for potential alterations in the myeloid lineage because they are easily accessible in relatively large quantities. To test for dysregulated monocyte signalling in SSc, we positively selected monocytes using magnetic bead isolation and stimulated them with specific agonists for TLR2 (Pam3Cys), TLR3 (poly-IC) or TLR4 (LPS) for 24 h and measured the proinflammatory mediators IL-1β, IL-6 and TNFα. Monocytes from patients with SSc (n=6) secreted significantly higher levels (pg/ml) of IL-6 then those from healthy controls (n=6) upon stimulation with Pam3Cys (8518±1050 vs 4134±1425, p=0.01 (mean±SD)), LPS (9176±480 vs 7606±937, p=0.03) and poly-IC (320±50 vs 189±40, p=0.06). Likewise, the production of IL-1β, TNFα and IL-10 after TLR-mediated stimulation showed similar trends, although small sample sizes precluded definitive conclusions (data not shown). Altogether, these data suggested that TLR responses might be different between patients with SSc and healthy controls and prompted us to define these differences in DCs more carefully.
moDCs from different SSc phenotypes display an augmented but differential inflammatory response towards TLR ligands
To investigate whether alterations in innate immunity and DC function might underlie some of the clinical distinctions between these disease subsets, we stimulated moDCs from these four SSc subgroups and healthy controls with ligands for TLR2 (Pam3Cys), TLR3 (poly-IC), TLR4 (LPS) and TLR7/8 (R848). After 24 h of co-incubation, secretion of the inflammatory mediators TNFα, IL-6, IL-12 and IL-10 was measured in culture supernatants. Interestingly, DCs from patients with edSSc produced significantly more TNFα and IL-6 upon stimulation with TLR2, TLR3 or TLR4 compared with those DCs from all other SSc phenotypes or healthy controls (figure 1A, B). Although DCs from patients with elSSc also showed a trend towards increased production of these inflammatory mediators upon stimulation with TLR2 and three ligands, no statistical significance was reached.
Single Toll-like receptor (TLR) ligand-mediated stimulation of monocyte-derived dendritic cells (moDCs) results in an aberrant cytokine pattern between patients with systemic sclerosis (SSc) in comparison with healthy controls. In these experiments moDCs were cultured from healthy controls (n=22), patients with early limited cutaneous SSc (elSSc, n=10), late limited cutaneous SSc (llSSc, n=14), early diffuse cutaneous SSc (edSSc, n=11) and late diffuse cutaneous SSc (ldSSc, n=10) and subsequently stimulated with ligands specific for TLR2 (Pam3Cys), TLR4 (LPS), TLR3 (poly-IC) and TLR7/8 (R848). After 24 h of incubation, interleukin 6 (IL-6) (A), tumour necrosis factor α (TNFα) (B), IL-12 (C) and IL-10 (D) were measured in the collected supernatants.
In contrast to the augmented TNFα and IL-6 secretion upon TLR2, 3 and 4 ligands by DCs from patients with edSSc, the secretion of IL-12 upon stimulation with TLR4 or TLR7/8 was significantly lower than for controls (figure 1C). DCs from patients with elSSc also showed decreased IL-12 secretion upon TLR4 or TLR7/8 stimulation. Notably, DCs obtained from patients with llSSc or ldSSc secreted comparable levels of IL-12 production to healthy donors.
The secretion of IL-10 by DCs from patients with dSSc, both those having early as well as late disease, was markedly increased upon stimulation with TLR2 and TLR4 ligands (figure 1D). However, DCs from patients with ldSSc secreted consistently less IL-10 than DCs from edSSc upon TLR2 or TLR4 stimulation. In addition, TLR3 ligand also induced some IL-10 production in edSSc. Together these data clearly indicate that the secretion of several key immunoregulatory cytokines is differently regulated in DCs derived from patients with lSSc compared with dSSc and also depends upon disease duration.
Stimulation of SSc moDCs with a combination of TLR2/3 and TLR3/4 ligands leads to a shift in classical Th1/Th2 cytokine production
It is likely that multiple TLR ligands are recognised simultaneously in vivo, emphasising the importance of studying the effects of combinations of TLR agonists. To further explore this in SSc, we stimulated moDCs with combinations of TLR2 and TLR4 (TLR2/4), TLR2 and TLR3 (TLR2/3), TLR2 and TLR7/8 (TLR2/7/8) and TLR3 and TLR4 (TLR3/4) agonists. Consistent with the recent literature, TLR2 agonists inhibited the secretion of IL-6 and TNFα induced by TLR4 from DCs from healthy controls (p values <0.001) (Gerosa et al18 and unpublished data). In contrast, the addition of TLR2 ligands did not markedly inhibit the TLR4-stimulated secretion of IL-6 and TNFα in patients with elSSc and edSSc, resulting in markedly higher levels in these patients than in those with llSSc and ldSSc disease and healthy controls (figure 2A,B). Although the combination of TLR2/4 ligands in patients with elSSc and edSSc led to TNFα and IL-6 secretion levels that were higher then those seen in DC supernatants from healthy controls, all other TLR ligand combinations led to a markedly blunted production of IL-6, TNFα in all patients with SSc (p values all <0.001) (figure 2A,B). The secretion of IL-12 closely followed the pattern of IL-6 and TNFα with the only difference that the combination of TLR2 and TLR4 agonist resulted in similar levels in all patients tested (figure 2C). Intriguingly, and in contrast with these latter observations, combinations of TLR2/4, TLR2/3 and TLR3/4 ligands led to a markedly higher production of IL-10 in patients with the dSSc phenotype, especially those having early disease (figure 2D). Notably, the combination of TLR2 and TLR7/8 ligands did not induce cytokine production in any of the investigated groups.
Simultaneous stimulation of monocyte-derived dendritic cells (moDCs) with multiple Toll-like receptor (TLR) ligands potentiates cytokine secretion in healthy controls but inhibits this response in patients with systemic sclerosis (SSc). In these experiments moDCs were cultured from healthy controls (n=22), patients with early limited cutaneous SSc (elSSc, n=10), late limited cutaneous SSc (llSSc, n=14), early diffuse cutaneous SSc (edSSc, n=11) and late diffuse cutaneous SSc (ldSSc, n=10) and subsequently stimulated with ligands specific for combinations of TLR2/4, TLR2/3, TLR2/7/8 and TLR3/4. After 24 h of incubation, interleukin 6 (IL-6) (A), tumour necrosis factor α (TNFα) (B), IL-12 (C) and IL-10 (D) were measured in the collected supernatants.
Phenotypical analysis shows no differences between moDCs from healthy controls and patients with SSc
The level of maturation of moDCs is reflected by the cytokine profile and by the expression of various surface markers. Since we observed a markedly different cytokine pattern between moDCs from SSc clinical subsets upon TLR-mediated stimulation, we investigated whether these observations might be the result of an altered moDC phenotype in SSc subsets. To this aim, the expression of CD14, CD86 and MHC-II was investigated on moDCs from healthy controls (n=10), and patients with elSSc (n=5), llSSc (n=6), edSSc (n=6) and ldSSc (n=7) using flow cytometry (data not shown). We saw no clear difference in the expression of these markers between groups or on the number of positive cells or on the mean fluorescence intensity, suggesting that the altered phenotype of moDCs from patients with SSc cannot be detected using these markers of DC maturity.
TLR-mediated stimulation of mDCs
Since controversy exists in the literature about the relevance of in vitro cultured DCs, we also studied cytokine secretion from freshly isolated mDCs. Consistent with our observations for in vitro-generated DCs, mDCs from patients with elSSc and edSSc produced significantly more IL-6 and TNFα upon TLR2-, TLR3-, TLR4- and TLR7/8-mediated stimulation compared with those from healthy controls and patients with llSSc and ldSSc. This observation closely parallels the findings for moDCs (compare figure 3A,B with figure 1A,B). In addition TLR2-, TLR3- and TLR7/8-stimulated mDCs from all groups (including controls) secreted relatively lower levels of TNF than in vitro matured moDCs (figure 4B).
Freshly isolated myeloid-derived dendritic cells (mDCs) stimulated with Toll-like receptors (TLRs) display a similar secretion pattern to that of moDCs. mDCs were positively isolated with MACS bead selection after which they were cultured for 24 h. Immediately after isolation, the mDCs were stimulated with ligands specific for TLR2 (Pam3Cys), TLR4 (LPS), TLR3 (poly-IC) and TLR7/8 (R848). In these experiments, mDCs were cultured from healthy controls (n=10), patients with early limited cutaneous systemic sclerosis (elSSc, n=8), late limited cutaneous SSc (llSSc, n=6), early diffuse cutaneous SSc (edSSc, n=8) and late diffuse cutaneous SSc (ldSSc, n=9). After 24 h of culture, interleukin 6 (IL-6) (A), tumour necrosis factor α (TNFα) (B), IL-12 (C) and IL-10 (D) were measured in the collected supernatants using Bio-Plex assays.
Circulating cytokine patterns suggest a different M1/M2 profile in systemic sclerosis (SSc) phenotypes. Circulating levels of interleukin 6 (IL-6) (A), tumour necrosis factor α (TNFα) (B), IL-12 (C), IL-10 (D), interferon γ (IFNγ) (E) and IL-13 (F) were measured in plasma samples from healthy controls (n=28), patients with early limited cutaneous SSc (elSSc, n=43), late limited cutaneous SSc (llSSc, n=68), early diffuse cutaneous SSc (edSSc, n=34) and late diffuse cutaneous SSc (ldSSc, n=23) originating from three independent international cohorts.
In accordance with the results seen for moDCs, mDCs from patients with elSSc and edSSc secreted less IL-12 upon TLR4 stimulation than with healthy controls, or patients with llSSc or ldSSc (compare figure 3C with figure 1C). The striking stimulation of IL-12 by TLR7/8 seen in moDCs was not seen in fresh mDCs from healthy controls or any of the patient groups (figure 4C).Thus, we could not confirm the striking decrease in IL-12 secretion seen upon TLR7/8 by moDC from patients with elSSc and edSSc (figure 1C).
Notably, as seen in moDCs, mDCs from patients with dSSc produced significantly more IL-10 upon TLR2- or TLR4-mediated stimulation (compare figure 3D with figure 1D). This difference reached statistical significance in both early and late dSSc but was more pronounced in those patients with the edSSc phenotype (figure 3D). mDCs stimulated with TLR3 and TLR7/8 ligands produced little IL-10 and was similar between all groups investigated.
Circulating cytokine levels reflect TLR-mediated cytokine secretion by DCs in SSc phenotypes
To test the relationship between in vitro TLR-stimulated cytokine secretion and in vivo cytokine production, we tested the circulating levels of IL-6, TNFα, IL-12, IL-10 and IFNγ in a large set of patients with SSc in comparison with healthy controls. Both IL-6 and TNFα levels were significantly increased in SSc (all clinical phenotypes, p<0.001) compared with healthy controls. Cytokine levels observed in elSSc and edSSc were higher (p<0.01) compared with those with disease duration >2 and >3 years for diffuse and limited SSc, respectively (figure 4A,B). In line with our observations regarding the production of IL-12 and IL-10 by moDCs and mDCs from patients with SSc, the production of IL-12 was higher in longstanding disease than in those having early disease, whereas the levels of IL-10 were markedly higher in those patients with early diffuse (p<0.0001) and late diffuse (p<0.001) disease than in all other patients tested (figure 4C,D). As a hallmark cytokine of the Th1 responses, we also measured IFNγ levels in patients with SSc. IFNγ was increased in patients with lSSc—elSSc (p=0.003) and even higher in patients with llSSc phenotype (p<0.0001)—but not in those with dSSc (figure 4E). Thus, the increase of circulating several levels of the cytokines upon TLR stimulation of DCs further suggests the presence of Th1/Th2 difference in SSc phenotypes and mirrors the pattern from moDCs and mDCs derived from comparable SSc phenotypes (figure 4F).
Discussion
Although a role for TLRs has been examined in many inflammatory conditions including autoimmune diseases, their role in SSc has not been subjected to similar scrutiny. Here, we show that monocytes, moDCs and mDCs and from patients with SSc display an aberrant cytokine pattern compared with those from healthy controls. Interestingly, patients with early progressive disease had a more pronounced production of IL-6 and TNFα upon single-ligand TLR stimulation, whereas their counterparts with longstanding disease showed a similar production to that of healthy controls. Together with the observation that patients with early SSc produce much less IL-12—especially those with edSSc—and produce markedly higher levels of IL-10, this suggests that early in the disease an augmented inflammatory response exists that is directed towards the Th2 axis.
Many studies have previously shown prominent Th2 immune skewing in SSc. For instance, plasma levels of IL-4, IL-13 and IL-10 were increased in SSc.19 20 Likewise, several studies have shown that T cells from affected sites such as skin21 22 and bronchoalveolar lavage fluid23 24 from patients with SSc have higher Th2-type cytokines. More recently, the existence of a skewed Th2 response in SSc was further substantiated by a study from Boin et al, in which specific T-cell surface markers were analysed displaying a Th2 phenotype.25 The pathways that underlie such a Th2 skewing have thus far not been identified. T-cell priming is orchestrated by DCs and is dependent upon the cytokines released by these DCs. Whereas IL-12 is a potent inducer of Th1 response, IL-10 in the absence of IL-12 primes Th2 responses. Our finding of comparable levels of circulating IL-12 levels early in disease followed by increased levels in late disease is consistent with an earlier report.26 The observation that DCs from patients with SSc, especially those obtained from patients with SSc having early disease, secrete less IL-12 and more IL-10 upon TLR-mediated activation indicates that altered TLR signalling and DC activation might at least partly explain these findings. Since some of the T-cell-derived cytokines released as a result of T-cell skewing, such as IL-13, can contribute to fibrosis, these results suggest that DC dysfunction may have a key role in SSc pathogenesis.
During normal life we constantly encounter various micro-organisms that are fought by an effective innate and adaptive immune response. In addition to exogenous TLR ligands, multiple endogenous TLR ligands are likely to be released after various modes of cell stress such as trivial trauma or exercise, for example. Thus, it is generally accepted that both endogenous and exogenous ligands bind TLRs, subsequently driving immune responses.27 28 In systemic lupus erythematosus endogenous TLR ligands are well-documented, including nucleic acid components against which autoantibody complexes form. Autoantibodies directed against DNA- and RNA-binding proteins also occur in SSc, and DNA- and RNA-binding proteins might in some cases act as TLR7 and or TLR9 ligands.29 Aside from these autoantibody-containing immune complexes, endogenous ligands have only been identified for TLR4, but may also exist for other TLRs. Recently, our group observed increased circulating TLR4 ligands in patients with SSc.12 30 Although the study size did not allow an analysis between patients with limited and diffuse SSc, these data at least suggest that increased TLR4 signalling may activate DCs in patients with SSc.
Although stimulation with single TLR ligands produced results that were generally consistent with observed alterations in circulating cytokine levels, ligand combinations indicated the complexity of the effects of TLR activation on DC biology. Stimulation of DCs from all clinical phenotypes with TLR ligand combinations including TLR2 resulted in unchanged production of IL-6, TNFα and IL-12, which was present in all SSc clinical phenotypes. On the contrary, the combination of TLR3 and TLR4 resulted in synergistically enhanced cytokine production especially in the healthy controls but unexpectedly not in early patient subgroups, as recently described.12 31 In contrast and most importantly, IL-10 secretion remained significantly increased in patients with the early diffuse SSc phenotype. These latter observations strongly suggest that patients with SSc, in general, have a diminished Th1 response upon the recognition of endogenous and exogenous TLR ligands and that patients with edSSc have a marked skewing of the immune response to Th2.
Altogether, the unrelenting and destructive progression of the fibrotic process in SSc remains a major medical challenge for which treatments are desperately needed. Here we provide novel insights into the different TLR responses by moDCs and mDCs from different SSc phenotypes. These observations provide clear impetus for pursuing studies directed at further elucidating the effects of different TLR ligands and DC-mediated activation of immune responses in this condition.
Acknowledgments
We are indebted to Anila Hussaini who carefully selected all the patients who participated in the study.
References
Footnotes
LvB and CP contributed equally to this work.
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Funding CP was funded by an unrestricted grant from ‘Het Landelijk Katholiek Reuma Centrum (LKRC)’. The Post-doctoral programme (TRDJR) was funded by the Termeulen Stipendium from the KNAW, Netherlands. The work presented here was partly funded by the VIDI laureate (TRDJR) from the Dutch Organisation of Research (NWO). Support was also provided by grants to RL from the National Institutes of Health (NIAMS U01AR055063) and an unrestricted grant from the American Society for Scleroderma Research.
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Competing interests None.
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Ethics approval This study was conducted with the approval of the local ethical committees University Nijmegen and Boston.
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Provenance and peer review Not commissioned; externally peer reviewed.
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Patient consent Obtained.