Th1 Response and Systemic Treg Deficiency in Inclusion Body Myositis

Objective Sporadic inclusion body myositis (sIBM), the most frequent myositis in elderly patients, is characterized by the presence muscle inflammation and degeneration. We aimed at characterizing immune responses and regulatory T cells, considered key players in the maintenance of peripheral immune tolerance, in sIBM. Methods Serum and muscle tissue levels of 25 cytokines and phenotype of circulating immune cells were measured in 22 sIBM patients and compared with 22 healthy subjects. Cytokine data were analysed by unsupervised hierarchical clustering and principal components analysis. Results Compared to healthy controls, sIBM patients had increased levels of Th-1 cytokines and chemokines such as IL-12 (261±138 pg/mL vs. 88±19 pg/mL; p<0.0001), CXCL-9 (186±12 pg/mL vs. 13±7 pg/mL; p<0.0001), and CXCL-10 (187±62 pg/mL vs. 13±6 pg/mL; p<0.0001). This was associated with an increased frequency of CD8+CD28− T cells (45.6±18.5% vs. 13.5±9.9%; p<0.0001), which were more prone to produce IFN-γ (45.6±18.5% vs. 13.5±9.9%; p<0.0001). sIBM patients also had a decreased frequency of circulating regulatory T cells (CD4+CD25+CD127lowFOXP3+, 6.9±1.7%; vs. 5.2±1.1%, p = 0.01), which displayed normal suppressor function and were also present in affected muscle. Conclusion sIBM patients present systemic immune activation with Th1 polarization involving the IFN-γ pathway and CD8+CD28− T cells associated with peripheral regulatory T cell deficiency.


Introduction
Idiopathic inflammatory myopathies are a group of acquired muscular disorders including polymyositis (PM), dermatomyositis (DM), immune-mediated necrotizing myopathies (IMNM), and sporadic inclusion body myositis (sIBM). sIBM is a rare disease, which differs from the other idiopathic inflammatory myopathies because it affects more frequently elderly subjects. Muscle weakness progression in sIBM is generally very slow and refractory to immunosuppressants [1]. The muscle inflammatory pattern in PM and sIBM is very similar, with upregulation of class I major histocompatibility complex (MHCI) molecules on the surface of muscle fibres, and muscle infiltration by cytotoxic CD8 + T cells, suggesting an autoimmune responses against muscle fibers [2]. Furthermore, the histological feature of sIBM includes presence of amyloid deposits, presumably linked to degenerative process [3]. The relative pathophysiological importance of inflammatory mechanisms is controversial in the genesis of this disease [4].
Additionally, little is known about the overall activation state of the immune system in sIBM, especially for what concerns natural regulatory T cells (Tregs) that play a key role in autoimmunity.
Thus, defining the pattern of immune activation and the balance of Treg vs. T effector responses in sIBM could lead to the development of targeted immune treatments and may provide insights into the interactions of immune system and muscle degeneration, which characterize the disease.
We observed in sIBM patients a systemic immune activation with Th1 polarization involving IFN-c pathway and CD8 + CD28 2 T cells associated with peripheral Tregs deficiency, suggesting that therapeutic approaches targeting effectors Th1 T cells and sparing or increasing Treg number and function might be of interest.

Patients and Samples
We studied patients with a definite diagnosis of sIBM based on pathological criteria [5], i.e. presenting inflammatory infiltrates with non-necrotic fibres infiltrated by lymphocytes, rimmed vacuoles, amyloid deposits or degeneration-related proteins evidenced by TDP43 (ProteinTech Group) and P62 (clone: 3/ P62 LCK ligand, BD Bioscience) immunostaining [6]. None of the subjects had received immunosuppressive or immunomodulatory drugs during the 6 months before inclusion in the study.
Sera and peripheral blood mononuclear cells (PBMCs) of 22 sIBM patients were compared to age-and sex-matched healthy subjects (n = 22) free of inflammatory/autoimmune diseases, chronic viral infection (human immunodeficient virus, or hepatitis B and C virus), past history of cancer or active cancer, and who were not receiving any immunosuppressive or immunomodulatory drugs. Frozen muscle biopsies from a subset of sIBM patients (n = 8) were studied by immunohistochemistry. Cytokine and chemokine levels in muscle lysates from sIBM patients (n = 8) were compared to those from non-myositis controls (patients with isolated myalgia and histologically normal muscle biopsy, n = 7).
In a second set of experiments, sera of 9 sIBM patients (different from those previously included) were tested and compared to sera (sampled at diagnosis before any therapeutic intervention) from other myositis patients, which included polymyositis (PM, n = 12, among them five had anti-Jo1 antibodies), dermatomyositis (DM, n = 12), and immune-mediated necrotizing myopathy associated with anti-SRP antibodies (SRP IMNM, n = 13) based on ENMC criteria [7].
The local ethics committee Groupe Hospitalier Pitie Salpetriere approved the study protocol, all the patients were enrolled after giving their written informed consent, and it received the clinicalTrials.gov number: NCT00898989.
Intranuclear detection of FoxP3 was also performed using a commercially available kit (eBioscience). For the detection of Cell acquisition and analysis by flow cytometry were performed with an FC500 cytometer (Beckman Coulter). Data were analysed with CXP software (Beckman Coulter).

T Cell Suppression Assays
The T cell suppression assays was performed as previously described [8]. Briefly, Tregs (CD3 + CD4 + CD25 + CD127 low cells) were FACS sorted (Aria flow cytometer, BD Biosciences) and mixed at various cell ratios with autologous effector T cells (CD3 + CD4 + CD25 2 cells) in the presence of irradiated allogeneic mononuclear cells as stimulator cells. Cell proliferation was determined by incorporation of tritiated thymidine (3H-Tdr, Amersham, Buckinghamshire, UK) and measured using a bcounter (counter-WALLAC).
The results were expressed as percentage inhibition using the following formula: (1 2 (experimental counts per min/control counts per min) 6 100%.

Statistical Analyses
Data are presented as mean (6SD) for continuous variables and as percentage for qualitative variables. Fisher's exact test was used to compare qualitative variables, and non-parametric Mann-Whitney and Wilcoxon tests were used to compare continuous variables. A p-value ,0.05 was considered significant. Statistical analyses were performed using GraphPad Prism version 4.0 and Instat version 3.0 for Windows (GraphPad Software, San Diego, CA, USA).
For cytokine and chemokine multivariate analyses, a first quality control check step allowed identifying variables and samples for which the number of available data was insufficient. As describe before [10], differentially-expressed cytokines were used to perform principal components analysis (PCA) and unsupervised hierarchical clustering on normalised patient data. Based on the statistically significant cytokine vector (i.e. signature), a model using Linear Discriminant Analysis was built to predict the membership of a test sample to the patient or control group. For all data-processing steps and statistical and multivariate analyses, we used R software (http://www.r-project.org/).

Analysis of Cytokines and Chemokines Levels in sIBM Patients Compared to Healthy Subjects Shows a Th1 Polarization of Immune System
In order to characterise immune responses in sIBM patients, cytokines and chemokines levels were measured in serum from 22 sIBM patients (67.9610.3 years; 9 males, 13 females). Patients' characteristics are reported in Table S1.
Conversely, we observed no difference in the levels of Th2 cytokines (IL-4, IL-5, IL-10 and IL-13), either in serum or after ex vivo stimulation of PBMCs (data not shown), or Th-17 signature markers. The serum levels of IL-17 and the percentage of circulating Th-17 cells (CD4 + CCR6 + IL-17 + T cells) were similar in both sIBM and controls, and few IL-17 + cells were detected in muscle infiltrates in only 5 of the 12 sIBM patients tested (among these 5 patients, 2.561.6% of CD4 + cells were positive for IL-17, Figure S1). In muscle lysates from sIBM patients, only CXCL-10 levels were significantly increased compared to normal muscle (2.362.8 pg/mg vs. 0.1560.05 pg/mg, p = 0.02), while IL-1RA and CCL-2 were increased only in some sIBM patients (data not shown).
The five serum markers increased in sIBM patients included mostly Th1 cytokines and chemokines: CXCL-9, CXCL-10, IL-12, CCL-2, and IL-1RA. These five markers allowed a good segregation of sIBM patients from healthy age-matched controls ( Figure 2. A-B), enabling us to build a predictive model with an accuracy of 83%.
In another set of analysis including 9 sIBM patients (different from those included in the first set) we compared cytokines results to other myositis controls (DM, PM, and SRP IMNM patients). Significant variation of cytokine levels in sera such as IL-1RA or CCL-2 (Table 1) was observed but did not allow segregating sIBM patients from others myositis controls because of overlap between groups ( Figure S2).

Th-1 Lineage is Associated with an Increased Frequency of Circulating Late Effector Memory T Cells in sIBM Patients
In an attempt to define the phenotype of the cells involved in the immune response engaged in a Th-1 lineage, FACS analysis were performed on fresh PBMCs from sIBM and matched healthy controls (age-matched to account for immunosenescence [11,12]).

sIBM Patients Exhibit Decreased Frequency of Circulating Tregs with Normal Suppressive Functions
We tested Tregs, which are involved in the maintenance of peripheral tolerance [14]. We observed a lower percentage of natural Tregs (CD4 + CD25 + CD127 low FOXP3 + T cells, Figure 5. A-D) in sIBM patients compared to controls (5.261.1% vs. 6.961.7%; p = 0.01, Figure 5. D). The functional suppressive effect of Tregs from sIBM patients on the proliferation of autologous T cells was similar to that observed in controls ( Figure 5. E, n = 4).
We also examined the presence of Foxp3 + cells in sIBM muscle sections ( Figure 5. H). These cells represented 18.767.4% of all CD4 + cells. Co-expression of FoxP3 and CD4 by cells infiltrating muscle was confirmed with double immunofluorescence staining ( Figure 5. I).

Discussion
The aetiology of sIBM, is poorly understood and the role of the immune system in the disease pathophysiology is not completely characterized.
In this study, we show that sIBM patients have increased levels of Th1 chemokines and cytokines (CXCL-9, CXCL-10 and IL-12) associated with an increased level of CD8 + CD28 2 T cells prone to produce large amounts of IFN-c. Moreover, Tregs are found within sIBM muscle infiltrates, and the percentage of peripheral Tregs is lower than in age-matched controls. Treg cells from sIBM patients were functional and remained able to suppress effector T cells.
While these findings reveal an ongoing immune response with a Th-1 lineage in sIBM patients, the apparent unbalance in the frequency of Tregs in peripheral blood may represent a feature of sIBM as it was described for other autoimmune disorders [14].
Conversely, in this study we did not observe Th17 polarization in sIBM patients, neither in blood nor in muscle biopsies, arguing against a possible pathophysiological role of Th17 cells in the disease. We and others reported increased serum levels of Th-1 chemokines CXCL-9 and CXCL-10 [17], which is in accordance with previous data reporting the presence of these chemokines in muscle of sIBM [18][19], associated with strong CXCR-3 expression in the majority of muscle-infiltrating T cells [19]. Th1 immune responses permitting CXCR3-mediated CD8 + memory T cells migration to inflamed tissues [17] is driven by CXCL-9 and CXCL-10, that are two IFN-c-inducible chemokines.
This overexpression of Th1 cytokines and chemokines is not specific of sIBM since, unlike previous studies [20], it does not permit to delineate disease-specific profiles and identify diagnostic patterns allowing the discrimination of sIBM from the other myositis.
We also observed a significant increase in the production of IFN-c by CD8 + T cells in sIBM patients. We characterized these cells as CD8 + CD28 2 T cells and as having a memory phenotype. Further analyses allowed us to detect a significant percentage of CD8 + CD28 2 T cells corresponding to the increased percentage of CD8 + LM since the loss of CD28 is associated with antigen recognition [13]. These results are in agreement with previous observations in sIBM patients showing an increased frequency of oligoclonal CD8 + CD28 2 T cells in the periphery with higher degranulation potential associated with an accumulation of clonally expanded CD8 + CD28 2 T cells within muscle infiltrates [2,21].
Here we also looked at the Treg compartment in our cohort of sIBM subjects. The absence of Treg due to mutations in the Foxp3 gene, which encodes for a key transcription factor governing Treg differentiation, causes a multiorgan autoimmune disease in humans known as IPEX syndrome [22]. Tregs play a key role in the control in peripheral immune tolerance [23]. To our knowledge, the Treg deficiency we observed in the blood of sIBM patients had never been reported, while a decrease in frequency of peripheral Tregs has been described in other forms of myositis, including DM and PM [24,25], as well as in other autoimmune diseases [26][27][28]. Although Foxp3 can be expressed by activated T cells that do not display suppressive functions [15], we confirmed, based on CD45RA expression [16], that FoxP3 + cells correspond to true Tregs and that the observed Tregs decrease concerned mainly aTregs. We also showed for the first time that Tregs from sIBM patients display normal function. This is suggestive of a systemic Treg deficiency, which could be due to a global quantitative Treg deficiency or to a Treg migration from the periphery to inflamed muscle, since we detected Foxp3 + cells in large number in sIBM muscle biopsies.
As activated T cells can be converted into Tregs in specific conditions [29], muscle-infiltrating Tregs could also correspond to induce Tregs.
As stated above, some Foxp3 + cells may correspond to activated T cells, but in this case Foxp3 expression would be below the detection limit of immunohistological methods [30]. We observed no functional deficit of circulating Tregs in sIBM patients, whereas impaired function has been observed in other autoimmune diseases such as multiple sclerosis [31]. A functional deficit of muscle-infiltrating Tregs cannot be excluded, but the number of cells that can be extracted from muscle is too low to perform ex vivo functional analyses.
In conclusion, in our study the analysis of serum cytokines and the immunophenotyping in peripheral blood and muscle in sIBM patients, highlights a systemic immune activation with Th1 polarization involving IFN-c pathway and CD8 + CD28 2 T cells associated with peripheral Tregs cell deficiency. We were not able to show to what extent this immune activation is specific to sIBM compared to other myositis. Nevertheless therapeutic approaches targeting effector Th1 T cells and/or sparing or increasing Treg numbers might be of interest in the treatment of sIBM.  patients. Non myositis control (blue dot line) is also represented. Values of each cytokines are expressed in pg/mL. B. Hierarchical clustering (Euclidean distance; complete linkage method) based on the expression pattern of the six cytokines significant in sIBM compare to myositis controls (G-CSF, IFN-a, IL-15, IL-1RA, CXCL-10 and CCL-2) across myositides patients including: sIBM patients, DM patients, PM patients and IMNM patients, do not permitted to segregated different groups. C. Projection by principal component analysis (PCA) of sIBM, DM, PM and IMNM patients using the expression levels of IFN-a, CCL-2 and CXCL10 according to the first two principal components (PC). PC1 and PC2 capture respectively 72% and 14% of the total variability. The three cytokines are highly correlated and do not allow to distinguish the four pathologies. (TIF)