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. 2015 Feb 16;179(3):529–538. doi: 10.1111/cei.12479

Regulatory T cell levels and cytokine production in active non-infectious uveitis: in-vitro effects of pharmacological treatment

B Molins *,†,✉, M Mesquida *, R W J Lee ‡, V Llorenç *, L Pelegrín *, A Adán *
PMCID: PMC4337685  PMID: 25354724

Abstract

The aim of this study was to quantify the proportion of regulatory T cells (Treg) and cytokine expression by peripheral blood mononuclear cells (PBMCs) in patients with active non-infectious uveitis, and to evaluate the effect of in-vitro treatment with infliximab, dexamethasone and cyclosporin A on Treg levels and cytokine production in PBMCs from uveitis patients and healthy subjects. We included a group of 21 patients with active non-infectious uveitis and 18 age-matched healthy subjects. The proportion of forkhead box protein 3 (FoxP3)+ Treg cells and intracellular tumour necrosis factor (TNF)-α expression in CD4+ T cells was determined by flow cytometry. PBMCs were also either rested or activated with anti-CD3/anti-CD28 and cultured in the presence or absence of dexamethasone, cyclosporin A and infliximab. Supernatants of cultured PBMCs were collected and TNF-α, interleukin (IL)-10, IL-17 and interferon (IFN)-γ levels were measured by enzyme-linked immunosorbent assay (ELISA). No significant differences were observed in nTreg levels between uveitis patients and healthy subjects. However, PBMCs from uveitis patients produced significantly higher amounts of TNF-α and lower amounts of IL-10. Dexamethasone treatment in vitro significantly reduced FoxP3+ Treg levels in PBMCs from both healthy subjects and uveitis patients, and all tested drugs significantly reduced TNF-α production in PBMCs. Dexamethasone and cyclosporin A significantly reduced IL-17 and IFN-γ production in PBMCs and dexamethasone up-regulated IL-10 production in activated PBMCs from healthy subjects. Our results suggest that PBMCs from patients with uveitis express more TNF-α and less IL-10 than healthy subjects, and this is independent of FoxP3+ Treg levels. Treatment with infliximab, dexamethasone and cyclosporin A in vitro modulates cytokine production, but does not increase the proportion of FoxP3+ Treg cells.

Keywords: IL-10, regulatory T cell, TNF-α, uveitis

Introduction

Whether endogenous or associated with a systemic disease, non-infectious uveitis causes up to 10% of cases of legal blindness in developed countries and may result in significant visual impairment in untreated patients 1,2. Corticosteroids remain the first-line choice of systemic therapy and may be accompanied by other immunosuppressants 3, but treatment success is variable and it is estimated that up to a third of patients with uveitis are unable to achieve disease control at tolerable corticosteroid doses 4,5. In uveitis patients who do not respond to this conventional immunosuppression, biological agents such as tumour necrosis factor (TNF)-α antagonists are being used as rescue therapy.

The proinflammatory cytokine TNF-α is an important mediator of intra-ocular tissue damage in uveitis patients as well as in different models of experimental autoimmune uveitis 6–9. Its inhibition improves visual outcomes significantly 10 and reduces clinical inflammation by also decreasing circulating levels of other proinflammatory cytokines 11. Moreover, besides its anti-inflammatory properties, TNF-α antagonists also exert immunomodulatory effects on adaptive immune responses, as they alter interleukin (IL)-10 expression in CD4+ T cells 12 and regulatory T cell (Treg) levels in some autoimmune diseases 13,14.

The effect on Treg cells may have clinical relevance in uveitis, as a reduction in Tregs has been reported in patients with active non-infectious uveitis 15, ocular Behçet's disease 16 and inactive Vogt–Koyanagi–Harada (VKH) disease 17. Nevertheless, some contradictory results have been observed, as increased levels of Tregs have been also associated with active ocular Behçet's disease 18, and in another study VKH patients did not show reduced levels of Tregs compared to healthy subjects 19. These discrepancies may arise from the different strategies used to identify Treg populations 20 as multiple phenotypes of Tregs have been described, and there are several T cell populations with regulatory properties 21. Treg populations are defined by cell surface markers, mechanism of action and tissue of origin. The characterization of the transcription factor forkhead box protein 3 (FoxP3) as a key regulator of regulatory cell function has been valuable in identifying Tregs 22,23, and it is widely accepted that FoxP3 Tregs, arising either spontaneously from the thymus or induced peripherally after infections, play a central role in controlling immune activity against self-antigens 20.

Taking the above considerations into account, the goal of the present work was to evaluate circulating FoxP3+ Treg levels and cytokine production by peripheral blood mononuclear cells (PBMCs) in patients with active non-infectious uveitis. Furthermore, we also aimed to evaluate the in-vitro effect of the synthetic corticosteroid dexamethasone, the commonly used conventional second-line immunosuppressive agent cyclosporin A (CsA) and the TNF-α antagonist infliximab on cytokine production, and FoxP3+ Treg levels in PBMCs from uveitis patients and healthy subjects.

Patients and methods

Patients

A total of 21 patients with a clinical diagnosis of active non-infectious uveitis (10 women and 11 men, median age 37 years, aged 21–60 years) were recruited from the Institut Clinic of Ophthalmology from the Hospital Clinic Barcelona (Spain) between May 2012 and June 2013. The diagnosis of active non-infectious uveitis followed the clinical criteria based on inflammatory cell reaction in the anterior chamber or vitreous as per standardization of uveitis nomenclature (SUN) and National Eye Institute (NEI) grading systems 24,25. Active chorioretinal lesions and vasculitis were evaluated by indirect ophthalmoscopy, fundus autofluorescence and fluorescein angiography. Any mentioned inflammatory sign (i.e. anterior chamber cell ≥0·5+, vitreous cells ≥0·5+, active retinal vasculitis or active chorioretinal lesions) was enough to be eligible. Infectious causes of uveitis were ruled out case by case on the basis of clinical signs and laboratory tests, including treponemic serological tests, tuberculosis interferon (IFN) gamma release assay (IGRA) and toxoplasmic serology and aqueous herpes group polymerase chain reaction (PCR), when appropriate. Data collected from patients included demographic information (age, sex), diagnosis classified by anatomical location according to the SUN criteria 25, systemic disease activity and previous systemic treatments.

As controls, 18 sex- and age-matched healthy subjects with no history of autoimmune disease were enrolled into the study. All participants provided informed consent and the research followed the tenets of the Declaration of Helsinki and was approved by the clinical experimentation Ethics Committee of the Hospital Clinic Barcelona.

PBMCs isolation and culture

PBMCs from uveitis patients and healthy subjects were obtained from fresh heparinized venous blood by Ficoll (Ficoll-Plaque Plus; GE Healthcare, Little Chalfont, UK) gradient centrifugation and washed twice with RPMI-1640 containing 2% heat-inactivated fetal calf serum (FCS). Isolated PBMCs were then cultured in RPMI-1640 with 10% FCS (PAA/GE Healthcare), 100 UI/ml penicillin (PAA/GE Healthcare), 0·1 mg/ml streptomycin (PAA/GE Healthcare), 100 IU/ml L-glutamine (PAA/GE Healthcare) and 20 mM HEPES buffer (PAA/GE Healthcare). Cells were seeded in 24-well plates (1·5 × 106 per well) and incubated overnight at 37°C in humidified air with 5% CO2 in the presence of infliximab (20 μg/ml), dexamethasone (1 × 10−6 M) or CsA (200 ng/ml). Doses were chosen based on preliminary dose–response experiments (Supporting information, Fig. S1). PBMCs were also seeded separately into 24-well plates coated with anti-CD3/anti-CD28 (5 μg/ml; eBiosciences, San Diego, CA, USA) to stimulate T cell proliferation and cultured for 72 h in the presence or absence of infliximab (20 μg/ml), dexamethasone (10−6 M) or CsA (200 ng/ml).

Treg determination by flow cytometry

FoxP3 levels in CD3+CD4+ cells from PBMCs which were rested overnight were determined by flow cytometry. A live/dead Fixable Dead Cell Stain (Invitrogen, Carlsbad, CA, USA) was used to exclude dead cells from the analysis, followed by incubation for 30 min on ice with AF700-CD3 (clone OKT3) and fluorescein isothiocyanate (FITC)-CD4 (OKT4) (eBiosciences). For intracellular staining, cells were then washed, fixed and permeabilized according to the manufacturer's instructions before adding phycoerythrin (PE)-FoxP3 (clone 236A/E7) (eBiosciences) for 30 min. Appropriate isotype controls were included in each experiment. Flow cytometric analyses were performed on a Fortessa flow cytometer (BD Biosciences, San Jose, CA, USA) with a total of 50 000 events recorded for each sample through a live CD3+CD4+ lymphocyte gate. Analysis of flow cytometry data was performed using FlowJo software (TreeStar, Inc., Ashland, OR, USA). Tregs were defined as CD3+CD4+FoxP3+ cells. The gating strategy is shown in Supporting information, Fig. S2.

Detection of intracellular cytokine expression

After overnight culture in the presence of the different drugs, PBMCs were stimulated for 4 h with a leucocyte activation cocktail (BD Pharmingen, San Jose, CA, USA) containing phorbol 12-myristate 13-acetate (PMA), ionomycin calcium salt and the protein transport inhibitor BD GolgiPlug™ (brefeldin A) to stimulate cytokine production. To analyse intracellular cytokine production, cells suspended in PBS were stained with a live/dead Fixable Dead Cell Stain (Invitrogen) to exclude dead cells. Cells were then suspended in PBS supplemented with 2% FCS, surface-stained with AF700-CD3 (clone OKT3) and FITC-CD4 (OKT4) (eBiosciences), fixed and permeabilized with a fixation and permeabilization solution (BD Cytofix/Cytoperm Fixation and Permeabilization Solution Kit; BD Biosciences) and stained for intracellular cytokine expression with PE anti-humanTNF-α (FastImmune; BD Pharmingen) and allophycocyanin (APC) anti-human IL-10 (clone JES3-19F1; BD Pharmingen. Flow cytometric analyses were performed on a Fortessa flow cytometer (BD Biosciences), with a total of 50 000 events recorded for each sample through a live CD3+CD4+ lymphocyte gate. Analysis of flow cytometry data was performed using FlowJo software (TreeStar, Inc.). The gating strategy for intracellular determination of IL-10 and TNF-α is shown in Supporting information, Fig. S3.

Determination of cytokine production

Supernatants from non-stimulated (overnight culture) and anti-CD3/anti-CD28-stimulated (72 h culture) PBMCs were collected, centrifuged to eliminate debris and frozen at −70°C until further analysis. TNF-α, IL-10, IL-17 and IFN-γ production was determined by enzyme-linked immunosorbent assay (ELISA) (Duoset; R&D Systems, Minneapolis, MN, USA), following the manufacturer's instructions.

Statistical analysis

Results were expressed as mean ± standard error of the mean (s.e.m.). Non-parametric analysis was performed using the Mann–Whitney U-test for comparison of unpaired data from control and uveitis groups and the Kruskal–Wallis test for comparison of paired data, unless otherwise specified. Statistical significance was set at P < 0·05. All calculations were performed using spss version 18·0 (SPSS, Inc., IBM Corporation, New York, NY, USA).

Results

Twenty-one patients with a clinical diagnosis of active non-infectious uveitis were included in the study. Table 1 summarizes different features of the study cohort. A variety of uveitis conditions were included: birdshot chorioretinopathy (three), intermediate uveitis (one), punctuate inner choroidopathy (one), Behçet's disease (six), pars planitis (two), VKH (two), sarcoidosis (three), ankylosing spondylitis (two) and idiopathic anterior uveitis (one). None of the patients had received biological drugs in the previous 12 months before their inclusion.

Table 1.

Characteristics of the cohort study†

Patient Age Sex Diagnosis Treatment
 1 39 F Birdshot Prednisone
 2 46 F Intermediate uveitis Prednisone
 3 37 M Punctate inner choroidopathy Prednisone
 4 60 F Birdshot Prednisone
 5 29 F Behçet Prednisone
 6 27 M Pars planitis No
 7 55 F Vogt–Koyanagi–Harada Prednisone
 8 42 F Behçet No
 9 34 M Behçet Prednisone
10 56 F Sarcoidosis Metotrexate, prednisone
11 27 M Sarcoidosis Prednisone
12 30 M Behçet Prednisone
13 21 F Vogt–Koyanagi–Harada No
14 41 M Ankylosing spondylitis No
15 37 M Ankylosing spondylitis Prednisone
16 58 F Sarcoidosis Prednisone
17 26 M Idiopathic uveitis No
18 43 M Behçet Prednisone
19 41 M Birdshot Prednisone
20 29 M Pars planitis No
21 30 F Behçet No
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†

At the time of sampling all patients had active disease (i.e. anterior chamber cell ≥0·5+, vitreous cells ≥0·5+, active retinal vasculitis or active chorioretinal lesions). M = male; F = female.

FoxP3+ regulatory T cell levels in peripheral blood from uveitis patients

As shown in Fig. 1, no differences in CD3+CD4+FoxP3+ levels were observed between patients and controls (4·01 ± 0·54 versus 3·72 ± 0·57%, P = 0·51). FoxP3+ Tregs were also measured in freshly isolated PBMCs and we did not observe differences in the percentages between freshly isolated PBMCs and PBMCs cultured overnight (Supporting information, Fig. S4).

Fig 1.

Fig 1

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Detection of CD3+CD4+forkhead box protein 3+ (FoxP3+) cells in peripheral blood mononuclear cells (PBMCs) from uveitis patients and healthy subjects. Percentage of CD3+CD4+FoxP3+ cells in non-activated PBMCs from uveitis (n = 21) and healthy subjects (n = 18) cultured overnight. Results are expressed as mean ± standard error of the mean.

Because it has been suggested that patients with Behçet disease may have altered levels of Tregs, we analysed FoxP3 levels in the subgroup of patients with Behçet disease (n = 6) and compared them with the control group, without observing differences (3·53 ± 1·10% versus 3·72 ± 0·57% CD4+ T cells, P = 0·40).

Subgroup analysis according to immunotherapy

No differences were observed when conducting a subgroup analysis of FoxP3+ Treg levels based on the treatment patients were receiving at the time of blood draw (Supporting information, Table S1).

Cytokine production by PBMCs from uveitis patients

Non-activated PBMCs

We then evaluated the concentration of the proinflammatory cytokines TNF-α, IL-17 and IFN-γ and the immunoregulatory cytokine IL-10 in the supernatants of non-activated PBMCs from uveitis and healthy subjects after overnight culture. Analysis by ELISA showed significantly increased levels of TNF-α (P < 0·05) and reduced levels of IL-10 (P < 0·05) in uveitis patients (Fig. 2a,b) compared to healthy subjects. Secretion of IFN-γ and IL-17 was very low and below detection levels in the majority of samples.

Fig 2.

Fig 2

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Tumour necrosis factor (TNF)-α and interleukin (IL)-10 production by non-activated peripheral blood mononuclear cells (PBMCs) and CD4+ T cells in uveitis patients and healthy subjects. PBMCs were cultured overnight and secreted TNF-α (a) and IL-10 (b) were analysed by enzyme-linked immunosorbent assay (ELISA) (n = 11). In selected samples PBMCs were stimulated with phorbol myristate acetate (PMA), ionomycin and brefeldin to induce intracellular cytokine production. Expression of intracellular TNF-α and IL-10 in CD4+ T cells from healthy subjects (n = 5) and uveitis patients (n = 3) was measured by flow cytometry. Graph shows the percentage of CD3+CD4+ T cells expressing TNF-α and IL-10 (c). Results are expressed as mean ± standard error of the mean. Statistical analysis was performed by Kruskal–Wallis test (*P < 0·05).

To determine whether the altered production of TNF-α and IL-10 was attributed to the population of CD4+ T cells, intracellular staining of PBMCs was performed in selected samples. Whereas IL-10 expression in CD4+ T cells was very low or not detected in most of the analysed samples, intracellular TNF-α expression in CD4+ T cells was significantly higher in uveitis patients than in healthy subjects (P < 0·05, Fig. 2c).

Activated PBMCs

Based on the assumption that T cells are activated in the context of inflammation in vivo, we sought to test the effect of T cell activation in vitro by engaging the T cell receptor with immobilized anti-CD3/anti-CD28. Activated PBMCs from uveitis patients produced significantly higher amounts of TNF-α than those from healthy subjects (1238 ± 305 versus 2003 ± 496 pg/ml, P < 0·05). By contrast, the decreased IL-10 secretion in PBMCs from uveitis patients was not maintained after activation and no statistically significant differences were observed in the secretion of IFN-γ and IL-17 of PBMCs from uveitis or control subjects (see also Fig. 5).

Fig 5.

Fig 5

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Effect of in-vitro treatment with infliximab, dexamethasone and cyclosporin A (CsA) on cytokine secretion by activated peripheral blood mononuclear cells (PBMCs) of uveitis patients and healthy subjects. Effect of in-vitro treatment on TNF-α (a), IL-10 (b), IL-17 (c), and IFN-γ (d) secretion by anti-CD3/anti-CD28 activated PBMCs from healthy and uveitis subjects cultured for 72 h (n = 6). Secreted cytokine concentrations were determined by enzyme-linked immunosorbent assay (ELISA) and results were expressed as mean ± standard error of the mean. Statistical analysis was performed by Wilcoxon test (*P < 0·05 and **P < 0·01 versus baseline of control PBMCs, #P < 0·05 and ##P < 0·01 versus baseline of uveitis PBMCs).

Subgroup analysis according to immunotherapy

A subgroup analysis of cytokine secretion was performed based on the treatment that patients were receiving. IL-10 production in non-activated PBMCs from uveitis patients treated with prednisone was higher than in those without treatment (15·0 ± 4·1 versus 0 pg/ml, P < 0·05 Fig. 3). IL-10 secretion by non-activated PBMCs from uveitis patients who were not receiving any treatment was below detection levels (n = 3), whereas PBMCs from uveitis patients treated with prednisone (n = 8) produced similar amounts of IL-10 to those from healthy subjects. Conversely, IL-17 production in activated PBMCs was significantly higher in PBMCs from patients without treatment than in those from patients treated with prednisone (Supporting information, Fig. S5).

Fig 3.

Fig 3

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Subgroup analysis of interleukin (IL)-10 secretion. The IL-10 concentration in supernatants of non-activated peripheral blood mononuclear cells (PBMCs) from uveitis patients who were not receiving any treatment (n = 3), non-activated PBMCs from uveitis patients treated with prednisone (n = 8) and non-activated PBMCs from healthy subjects (n = 11) was determined by by enzyme-linked immunosorbent assay (ELISA). Results are expressed as mean ± standard error of the mean. Statistical analysis was performed by Kruskal–Wallis test (**P < 0·005 versus control subjects and prednisone-treated uveitis patients).

Effect of pharmacological treatment in vitro on cytokine production

Non-activated PBMCs

We then aimed to evaluate whether in-vitro treatment with infliximab (20 μg/ml), dexamethasone (10−6 M) or CsA (200 ng/ml) had any effect on cytokine secretion by PBMCs. For this purpose, PBMCs from uveitis and healthy subjects were cultured overnight in the presence of the different drugs. As seen in Fig. 4a, overnight treatment with infliximab and dexamethasone significantly reduced TNF-α secretion by non-stimulated PBMCs from control and uveitis subjects, whereas CsA did not. However, none of the treatments resulted in any significant effect on IL-10 production (the increase in CsA-treated cells from healthy subjects did not reach statistical significance) (Fig. 4b). IFN-γ and IL-17 were not determined, given their low expression in basal conditions. The effect of the different drugs on intracellular expression of TNF-α by CD4+ T cells showed that in both uveitis and healthy subjects dexamethasone and CsA reduced intracellular expression of TNF-α significantly, whereas infliximab had no effect (Fig. 4c).

Fig 4.

Fig 4

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Effect of in-vitro treatment with infliximab, dexamethasone and cyclosporin A (CsA) on tumour necrosis factor (TNF)-α and interleukin (IL)-10 production by non-activated peripheral blood mononuclear cells (PBMCs) and CD4+ T cells of uveitis patients and healthy subjects. PBMCs were cultured overnight and the effect of the different drugs on secreted TNF-α (a) and IL-10 (b) were analysed by by enzyme-linked immunosorbent assay (ELISA) in 11 uveitis patients and 11 healthy subjects. In selected samples PBMCs were stimulated with phorbol myristate acetate (PMA), ionomycin and brefeldin to induce intracellular cytokine production. Expression of intracellular TNF-α and IL-10 in CD4+ T cells from healthy subjects (n = 5) and uveitis patients (n = 3) was measured by flow cytometry. Graph shows the percentage of CD3+CD4+ T cells expressing TNF-α (c). Results are expressed as mean ± standard error of the mean. Statistical analysis was performed by Wilcoxon test (*P < 0·05 versus baseline of control PBMCs and CD3+CD4+ T cells, #P < 0·05 versus baseline of uveitis PBMCs, **P < 0·001 versus baseline).

Activated PBMCs

To assess the effect of T cell activation on cytokine secretion, PBMCs were stimulated with anti-CD3/anti-CD28 for 72 h in the presence of the different drugs. As seen in Fig. 5a, TNF-α secretion was greatly reduced when PBMCs were treated with any of the analysed drugs. Regarding IL-10, neither infliximab nor CsA was able to modulate IL-10 secretion by PBMCs (Fig. 5b). However, dexamethasone increased IL-10 production significantly by PBMCs from healthy subjects (P < 0·05). Conversely, in-vitro treatment with dexamethasone and CsA reduced IFN-γ and IL-17 secretion significantly and infliximab reduced IFN-γ secretion, but not IL-17, as seen in Fig. 5c,d.

Effect of pharmacological treatment in vitro on Treg levels

Because we observed a significant effect of the different tested drugs on cytokine secretion by PBMCs, we aimed to evaluate whether infliximab, dexamethasone and CsA were also able to modulate FoxP3 expression in CD4+ T cells. For this purpose, non-activated PBMCs from uveitis and healthy subjects were cultured overnight in the presence of the different drugs and Treg levels were analysed by flow cytometry. As seen in Fig. 6, in-vitro treatment with dexamethasone 1 × 10−6 M and CsA (200 ng/ml) reduced FoxP3+ Treg levels significantly in PBMCs from healthy and uveitis subjects (P < 0·05). In uveitis subjects, it was also observed that infliximab reduced intracellular FoxP3+ levels significantly in CD4+ T cells (P < 0·05).

Fig 6.

Fig 6

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Effect of in-vitro treatment of peripheral blood mononuclear cells (PBMCs) from healthy and uveitis subjects with infliximab, dexamethasone and cyclosporin A (CsA) on levels of CD3+CD4+ forkhead box protein 3+ (FoxP3+) cells cultured overnight (n = 11). Results are expressed as mean ± standard error of the mean. Statistical analysis was performed by Wilcoxon's test (*P < 0·05 versus baseline of control PBMCs, #P < 0·05 versus baseline of uveitis PBMCs).

Discussion

In the present work we evaluated FoxP3+ Treg levels and cytokine production by PBMCs in patients with active non-infectious uveitis. In order to investigate the cellular effects of the most commonly used drugs to treat non-infectious uveitis, we also studied their in-vitro effect on FoxP3+ Treg levels and cytokine production. Our results showed that TNF-α and IL-10 production by PBMCs rather than altered Treg levels were associated with active uveitis and that in-vitro treatment with infliximab, dexamethasone and CsA modulated cytokine production differentially in PBMCs from uveitis and healthy subjects, regardless of the activation state of PBMCs. Moreover, none of these drugs increased the proportion of FoxP3+ Treg cells in either healthy subjects or uveitis patients.

The role of Tregs in the control of inflammatory disease has gained considerable interest in uveitis as well as in other systemic autoimmune conditions 26,27. However, the role of Tregs as a uveitis biomarker is far from established, as contradictory findings have been reported. Some authors have found decreased levels of Tregs in active uveitis compared to inactive patients 15,17 or healthy subjects 16, while others have found no differences 19 or even increased levels 18. Our results here do not support the use of Tregs as a uveitis marker, as we did not observe differences in Treg levels between patients with active non-infectious uveitis and healthy subjects. The discrepancies in Treg levels observed in the different studies could be attributed partly to the heterogeneity of uveitis entities, the activity of the disease and the different strategies used to characterize Tregs. FoxP3 is the canonical transcription factor for naturally occurring Tregs and is enriched in human CD4+CD25hi T cells. However, because the CD4+CD25hi population does not necessarily capture all FoxP3+ cells, in this study we defined CD3+CD4+FoxP3+ as Tregs.

Although it is possible to interrogate and characterize cytokine and transcription factor expression in individual T cell subpopulations, our primary goal was to characterize the immune signature in a mixed population of mononuclear cells based on the assumption that this reflects more closely the multiple complex immune cell interactions that occur in vivo. Although we did not observe differences in Treg levels, we observed increased production of TNF-α and reduction of IL-10 in PBMCs from uveitis patients. IL-10 is an immunoregulatory cytokine that plays a central role in maintaining immune tolerance by inhibiting the proliferation and activity of many immune cells. Indeed, the administration of exogenous IL-10 ameliorates experimental autoimmune uveitis (EAU) and inhibits proliferation and IFN-γ production by mature uveitogenic effector T cells 28. Thus, the reduced levels of secreted IL-10 by PBMCs from uveitis patients could contribute to the inflammation seen in active uveitis. IL-10 is produced primarily by monocytes, certain subsets of activated T cells and B cells and also Tregs 29,30. Because we did not detect intracellular IL-10 in CD4+ T cells, we speculate that PBMC secretion of IL-10 is most likely to have been monocyte-derived. Conversely, we also observed increased levels of TNF-α in the supernatants of PBMCs and at the intracellular level in CD4+ T cells from uveitis patients, consistent with the fact that TNF-α is a proinflammatory cytokine that plays a key role in intra-ocular inflammation 8,9, thus enhancing the inflammatory response in uveitis patients. We also studied the effect of T cell activation in vitro on cytokine production in uveitis and healthy subjects, and this showed similarly that TNF-α secretion was enhanced and IL-10 was reduced in PBMCs from uveitis patients, demonstrating that this observation is independent of the activation state of T cells. Conversely, IL-17 and IFN-γ production was not significantly higher in uveitis patients than in healthy subjects, which could be due to our small sample size, as previous reports have shown increased production of these cytokines in uveitis patients 31. Nevertheless, we observed an increased production of IL-17 in PBMCs from uveitis patients compared to those from uveitis patients treated with prednisone.

In the current work we also aimed to elucidate potential mechanisms associated with the clinical benefits of some of the most commonly used drugs to treat non-infectious uveitis. For this purpose we chose one corticosteroid (dexamethasone), one conventional second-line immunosuppressant (CsA) and one TNF-α antagonist (infliximab). We observed a significant reduction in TNF-α secretion by PBMCs in both uveitis and healthy subjects after treatment with the different drugs. Dexamethasone and CsA, but not infliximab, also induced a significant reduction of intracellular production of TNF-α in CD4+ T cells and decreased secreted IL-17 and IFN-γ significantly in PBMCs from healthy and uveitis subjects. Moreover, dexamethasone treatment was able to induce IL-10 production in stimulated PBMCs from healthy subjects but not in uveitis patients. These findings could be attributed to possible steroid refractivity in some uveitis patients in which dexamethasone would be unable to increase IL-10 production 32.

In order to elucidate whether the effect of the different drugs on the modulation of TNF-α and IL-10 could be related to an increase in FoxP3+ Tregs we also analysed the in-vitro effect of infliximab, dexamethasone and CsA on levels of FoxP3+ Tregs. We found that the treatment in vitro with the different drugs did not increase Treg levels. On the contrary, dexamethasone and CsA treatment even decreased Treg levels in PBMCs from both uveitis and healthy subjects. Our observation that infliximab was unable to increase FoxP3+ Treg levels differs with the findings of Calleja et al. 33, who observed increased Treg levels in uveitis patients after treatment with a TNF-α antagonist (adalimumab). Nevertheless, the effect they reported could be attributed to another mechanism that increased Treg levels indirectly in the periphery of the studied patients. Similarly, another study 16 reported an increase of Treg levels in patients with Behçet disease treated with infliximab as well as an increase in Tregs after in-vitro treatment with infliximab. The main difference with our study relies on the fact that we used PBMCs instead of purified CD4+ T cells. These observations suggest that the clinical benefits associated with the treatment with these drugs may be associated with other mechanisms beyond a direct effect on Treg induction.

The limitations of our study include the heterogeneity of uveitic conditions studied and that most of our patients were receiving corticosteroid or immunosuppressant treatment. Despite these concerns, the results presented herein suggest that TNF-α and IL-10 production by PBMCs rather than altered FoxP3+ Treg levels are associated with active uveitis and that in-vitro treatment with infliximab, dexamethasone and CsA regulates cytokine production by PBMCs. In addition, although dexamethasone is known to further suppress the immune response by increasing IL-10, as seen in our healthy controls, this effect was lost in PBMCs from our uveitis patients with active disease. None of the pharmacological treatments used increased the proportion of FoxP3+ Treg cells.

Acknowledgments

The authors are indebted to the Citomics Unit of the IDIBAPS for the technical help. This work was supported by the Ministry of Science and Innovation of Spain, ‘Instituto de Salud Carlos III’, ‘Fondo de Investigación Sanitaria’ (PI13/00217). R. W. J. L. is supported by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.

Disclosure

R. W. J. L. is an inventor on a US patent (application no. 61/919 404), which includes a calcineurin inhibitor-based antibody–drug conjugate for the treatment of inflammatory diseases. He also consults on behalf of the University of Bristol for Roche-Genentech. The other authors have no financial disclosures.

Author contributions

B. M. designed the study, performed the experiments and wrote the paper; M. M. performed experiments and collected data; R. W. L. analysed data and wrote the paper; V. L. and L. P. collected data; and A. A. designed the study, reviewed the paper and provided funds.

Supporting Information

Additional Supportingporting information may be found in the online version of this article at the publisher's web-site:

Fig. S1. Dose–response experiments to test the effect of increasing concentrations of infliximab (a), dexamethasone (b) and cyclosporin A (CsA) (c) on tumour necrosis factor (TNF)-α secretion by non-activated peripheral blood mononuclear cells (PBMCs) cultured overnight.

Fig. S2. Regulatory T cell (Treg) gating strategy. A live/dead Fixable Dead Cell Stain (Pacific Blue) was used to exclude dead cells from the analysis (a). Lymphocyte population was gated based on forward-/side-scatter (FSC/SSC) distribution (b) and afterwards CD3+ cells were selected (c), to finally define Tregs as CD3+ CD4+forkhead box protein 3+ cells (d).

Fig. S3. Intracellular tumour necrosis factor (TNF)-α and interleukin (IL)-10 gating strategy. A live/dead Fixable Dead Cell Stain (Pacific Blue) was used to exclude dead cells from the analysis (a). Lymphocyte population was gated based on size and granularity by forward-/side-scatter (FSC/SSC) distribution (b) and on CD3+ CD4+ expression (c) to finally determine intracellular expression of IL-10 (y-axis) and TNF-α (x-axis) (d).

Fig. S4. Regulatory T cells (Tregs) were also measured in freshly isolated peripheral blood mononuclear cells (PBMCs) and no differences were observed in the percentages between freshly isolated PBMCs and PBMCs cultured overnight (n = 5, P = 0 22).

Fig. S5. Subgroup analysis of interleukin (IL)-17 secretion. The IL-17 concentration in supernatants of activated peripheral blood mononuclear cells (PBMCs) was determined by enzyme-linked immunosorbent assay (ELISA). Activated PBMCs from uveitis patients who were not receiving any treatment (n = 3) produced significantly increased amounts of IL-17 compared to those treated with prednisone (n = 8) and those from healthy subjects (n = 11). Results are expressed as mean ± standard error of the mean. Statistical analysis was performed by Kruskal–Wallis test (*P < 0 05 versus prednisone-treated uveitis patients).

Table S1. Subgroup analysis of forkhead box protein 3+ regulatory T cell (Treg) levels according to patients' immunotherapy.

cei0179-0529-sd1.zip (3.5MB, zip)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1. Dose–response experiments to test the effect of increasing concentrations of infliximab (a), dexamethasone (b) and cyclosporin A (CsA) (c) on tumour necrosis factor (TNF)-α secretion by non-activated peripheral blood mononuclear cells (PBMCs) cultured overnight.

Fig. S2. Regulatory T cell (Treg) gating strategy. A live/dead Fixable Dead Cell Stain (Pacific Blue) was used to exclude dead cells from the analysis (a). Lymphocyte population was gated based on forward-/side-scatter (FSC/SSC) distribution (b) and afterwards CD3+ cells were selected (c), to finally define Tregs as CD3+ CD4+forkhead box protein 3+ cells (d).

Fig. S3. Intracellular tumour necrosis factor (TNF)-α and interleukin (IL)-10 gating strategy. A live/dead Fixable Dead Cell Stain (Pacific Blue) was used to exclude dead cells from the analysis (a). Lymphocyte population was gated based on size and granularity by forward-/side-scatter (FSC/SSC) distribution (b) and on CD3+ CD4+ expression (c) to finally determine intracellular expression of IL-10 (y-axis) and TNF-α (x-axis) (d).

Fig. S4. Regulatory T cells (Tregs) were also measured in freshly isolated peripheral blood mononuclear cells (PBMCs) and no differences were observed in the percentages between freshly isolated PBMCs and PBMCs cultured overnight (n = 5, P = 0 22).

Fig. S5. Subgroup analysis of interleukin (IL)-17 secretion. The IL-17 concentration in supernatants of activated peripheral blood mononuclear cells (PBMCs) was determined by enzyme-linked immunosorbent assay (ELISA). Activated PBMCs from uveitis patients who were not receiving any treatment (n = 3) produced significantly increased amounts of IL-17 compared to those treated with prednisone (n = 8) and those from healthy subjects (n = 11). Results are expressed as mean ± standard error of the mean. Statistical analysis was performed by Kruskal–Wallis test (*P < 0 05 versus prednisone-treated uveitis patients).

Table S1. Subgroup analysis of forkhead box protein 3+ regulatory T cell (Treg) levels according to patients' immunotherapy.

cei0179-0529-sd1.zip (3.5MB, zip)

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