An Imbalance between Frequency of CD4+CD25+FOXP3+ Regulatory T Cells and CCR4+ and CCR9+ Circulating Helper T Cells Is Associated with Active Perennial Allergic Conjunctivitis

Allergic conjunctivitis (AC) is one of the most common eye disorders in ophthalmology. In mice models, it has been suggested that control of allergic conjunctivitis is a delicate balance between Tregs and inflammatory migrating effector cells. Our aim was to evaluate the frequency of Tregs and the frequency of homing receptors expressing cells in peripheral blood mononuclear cells (PBMC) from patients with perennial allergic conjunctivitis (PAC). The analyses of phenotypic markers on CD4+ T cells and both soluble or intracellular cytokines were performed by flow cytometry. CD4+CD25+ cells were 15 times more frequent in PBMC from patients than HC; the vast majority of these CD4+CD25+ cells were FOXP3−, and most of CD4+ T cells were CCR4+ and CCR9+ cells. Upon allergen-stimulation, no significant changes were observed in frequency of Treg; however, an increased frequency of CD4+CCR4+CCR9+ cells, CD4+CD103+ cells and CD4+CD108+ cells with increased IL-5, IL-6, and IL-8 production was observed. These findings suggest an immune dysregulation in PAC, characterized by diminished frequency of Tregs and increased frequency of circulating activated CD4+ T cells; upon allergen-stimulation, these cells were expressing cell-surface molecules related to mucosa homing and were able to trigger an inflammatory microenvironment.


Introduction
Allergies represent the most frequent chronic diseases worldwide [1]; ocular allergy is one of the most common ocular conditions encountered in clinical practice. Allergic conjunctivitis (AC) includes a spectrum of a number of traditional overlapping conditions that range from intermittent to persistent signs and symptoms, and these are fluctuating in severity and presentation. AC could be as mild forms with transient inflammation, such as seasonal (SAC) and perennial allergic conjunctivitis (PAC), or as more severe persistent and chronic inflammatory forms such as vernal keratoconjunctivitis (VKC) and atopic keratoconjunctivitis (AKC) [2,3]. Allergic conjunctivitis is initiated by the predominant activation of CD4+ T cells to environmental allergens, culminating in a Th2 response with generation of IgE antibodies [4]. The CD4+ T cells from allergic patients are resistant to apoptosis and produce large amounts of IL-5 [5], favouring chronicity and perpetuating inflammation and relapsing-remitting symptoms. It is well known that in the chronic forms of allergic conjunctivitis CD4+ T cells are able to migrate to the ocular mucosa, maintaining the inflammatory process [6]. In mice models of ocular allergy, it has been demonstrated that CD4+CD25+FOXP3+ regulatory T cells (Tregs) influence the expression of immune-mediated allergic inflammation in conjunctiva, [7] counteracting inflammation through antiinflammatory cytokines such as TGF-and IL-10 [7,8].
Therefore, it appears that control of allergic conjunctivitis is a delicate balance between Tregs and inflammatory migrating effector cells. In humans, intraepithelial leukocytes in the ocular surface express human mucosal lymphocyte antigen (HML or CD103) in nonpathological conditions [9], whereas in chronic allergic status, infiltrating CD4+ T cells are CCR3+ and/or CXCR3+ cells [10,11] Nevertheless, the molecules involved in T-cell homing to conjunctiva, during the acute forms of AC in humans, are not fully studied yet. The aim of this study was to characterize the immunophenotypical features of circulating helper T cells, associated with Treg phenotype and homing receptors, in patients with perennial allergic conjunctivitis.

2.1.
Patients. 21 individuals (12 males and 9 females, mean age 11.3 years, range 5-17) with active perennial allergic conjunctivitis (PAC) were studied. Perennial allergic conjunctivitis diagnosis was based on clinical history (mean disease duration 3.5 (SD 3.1) years) and eye and physical examination. Seven healthy volunteers were used as controls (4 males and 3 females, mean age 10.2 years, range 7-15). All participants gave their informed/assent consent for blood sampling after written information was provided. The study adhered to the ethical principles of the Declaration of Helsinki, the E11 Statements of International Conference of Harmonisation (E11-ICH), and was approved by the Institutional Ethics Committee Board at the Institute of Ophthalmology "Fundación Conde de Valenciana", Mexico City.  ) were separated on a Ficoll density gradient by  centrifugation at 1700 rpm for 30 min at room temperature. After centrifugation, the cells in the interface were collected, washed twice, and counted using a handheld automated cell counter (Millipore Co., Billerica, MA, USA), and viability was assessed by eosin dye exclusion.

Immunofluorescence Staining of Cell Surface Markers.
Double or triple-colour staining was performed on PBMC by direct immunofluorescence, using APC-or PECy5-mAb anti-CD4 and either FITC-and/or PE-labelled mAbs against CD25, CD103, CD108, CCR4, CCR7, or CCR9. Briefly, 2 × 10 5 cells were suspended in 20 L PBS supplemented with 0.2% bovine serum albumin and 0.2% sodium azide (PBA) and were incubated with fluorochrome-labelled mAb for 30 min at 4 ∘ C. After incubation, the cells were washed twice with PBA, fixed with 1% p-formaldehyde, and analysed by flow cytometry.

Immunofluorescence Staining of Intracellular Markers.
Stimulated or nonstimulated PBMC were washed with PBA and stained with APC-or PECy5 labelled mAbs against CD4 and/or PE labelled mAbs against CD25 for 30 min. After washing, the cells were fixed with 4% p-formaldehyde in PBS for 10 min at 4 ∘ C. The cells were washed twice with PBS and permeabilised with saponin buffer (0.1% saponin and 10% BSA in PBS) by shaking gently for 10 min at room temperature. The cells were then incubated with FITClabelled anti-human FOXP3 antibodies and/or PE-labelled anti-human IL-5. In all cases isotype-matched controls were used.

Cell
Cultures. PBMC were cultured in 96-well flat bottomed cell culture plates (Costar, Cambridge, MA, USA) at 2 × 10 5 cells/well in RPMI-1640 medium supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, 50 g/mL gentamicin, and 0.5% heat-inactivated fetal calf serum and incubated at 37 ∘ C in a 5% CO 2 humidified chamber. After 24 h the culture medium was removed, and fresh culture medium supplemented with 10% heat-inactivated fetal calf serum and Der p (7.5 g/mL) were added. After 7 days of culture, the cells were harvested and processed to measure intracellular FOXP3 expression, and homing receptors on cell surface by flow cytometer. The Con A mitogen (2 g/mL) was used as a cell stimulation positive control. Supernatants were collected and stored at −70 ∘ C to determine soluble cytokines. In order to determine intracellular IL-5, four hours before antigen or polyclonal cultures ended, brefeldin-A was added (10 g/mL), and at the end of the incubation period the cells were harvested and were processed to immunofluorescence staining as described above.

Flow Cytometric Analysis.
All cells were analysed for the expression of phenotypic markers on a FACScan flow cytometer (Becton Dickinson, San Jose, CA) using cell quest software, and 10000 events were counted. To analyse the staining of cell-surface markers, the lymphocytes were first gated by their physical properties (forward and side scatter), then a second gate was drawn based on immunofluorescence characteristics of the gated cells, assessing fluorescence intensity by histograms. To determine Tregs the cells were gated on FSC-SSC dot plot, then the lymphocytes were gated on CD4+ T cells in a SSC-CD4 dot plot, then CD4+ cells were selected, and a CD4-CD25 dot plot was created to select, the double positive CD4+CD25+ T cells; finally to analyse intracellular FOXP3 staining on CD4+CD25+ T cells a histogram was created to analyse the mean fluorescence intensity (MFI) of FOXP3+ cells. Data are presented as dot-plots or histograms. Control stains were performed using isotype-matched mAb of unrelated specificity. Background staining was <1% and was subtracted from experimental values.

Statistical Analysis. Mann-Whitney tests or Wilcoxon
Rank Signed test was used to detect significant differences. The analysis was performed with Graphpad Prism software v.5.0. Differences were considered statistically significant when the test yielded values less than 0.05.

Frequency of Chemokine Receptors on Peripheral Blood
Mononuclear Cells. To determine whether chemokine receptor expression was associated with a particular T helper cell traffic molecule in patients with PAC, CCR4, CCR7, and CCR9 was measured on PBMC. Results are summarized in Table 1. It was observed that CCR4+ cells were 1.9 times more frequent on PBMC from patients with PAC than in HC ( = 0.004). Most of the CCR4+ cells were CD4+ T cells, and CD4+CCR4+ cells were 1.8 times more frequent in patients with PAC than in HC ( = 0.03) (Figures 2(a) and 2(d)). We did not observe differences in frequency of CCR7+ or CCR7− cells neither on PBMC nor on CD4+ cells among groups (Figures 2(b) and 2(e)). The CCR9+ cells were 4.2 times more frequent on PBMC from patients with PAC than in HC ( = 0.01), and the CD4+ T cells expressing CCR9 were 2.5 times more frequent in patients with PAC than in HC ( = 0.01) (Figures 2(c) and 2(f)) ( Table 1).

Cytokines after Der p-Stimulation.
The levels of secreted cytokines IL-1b, IL-6, IL-8, IL-10, IL-12p70, and TNF-were determined in culture supernatants after Der p-stimulation. IL-6 and IL-8 were significantly increased when compared with nonstimulated cells ( = 0.01 and = 0.04, resp.). Results are depicted in Table 3. In order to know if    Der p-stimulation induced early secretion of IL-5 in CD4+ cells, we performed intracellular evaluation of IL-5 in CD4+ T cells from patients with PAC. We observed 10.3 times more frequency of CD4+IL-5+ cells after Der-p stimulation when compared with RPMI alone (Mean 15.5% SD 3.6 versus Mean 1.4% SD 3, resp.; = 0.02) (Figure 6).

Discussion
Allergic conjunctivitis is an inflammation of the conjunctiva secondary to an immune response caused by contact with an allergen at the bulbar or tarsal conjunctiva in a previously sensitized individual [12]. Two types of AC have been described,    the acute forms and the chronic forms; acute forms included SAC and PAC and are the most frequent types of AC and are clinically characterized by itching, redness, and tearing [2,3,12,13]; VKC and AKC are the chronic forms and they could lead to permanent visual impairment due to persistent inflammation [13,14]. In the chronic forms of AC, allergenmediated inflammation is maintained by infiltrating CD4+ cells to conjunctiva [15]; migration of effector cells (T cells and non-T cells) is dependent of CCR3 and CXCR3 expression [10,11]. In acute forms of AC, molecules involved in CD4 recruitment have not been enough studied. In mice models of AC, induction of CD4+CD25+FOXP3+ regulatory T cells suppresses effector-cell activation through synthesis of IL-10 and TGF-b [7]; nonetheless, the frequency of Tregs in patients with AC has not been described yet.
In this work we analysed the frequency of circulating Tregs and the frequency of cells expressing molecules involved in T-cell homing to mucosa inflammation, in patients with perennial allergic conjunctivitis. Our results are in accordance with other authors [16][17][18][19] that have reported changes in frequency of CD4+CD25+ T cells and CD4+CD25+FOXP3+ regulatory T cells in atopic diseases, such as asthma, rhinitis, and atopic dermatitis [16][17][18][19]. These authors suggest that changes in the frequency of Tregs or impairment of their regulatory capacity could be associated with the activation of allergic status. In this work, the majority of CD4+CD25+ cells were FOXP3− cells; and after allergen-stimulation, no differences were observed among the frequency of FOXP3+ cells and FOXP3− cells; this result is relevant since it is well known that IL-6 is able to suppress Treg differentiation [20]. It is possible that after encountering with the antigen, allergen-specific CD4+ T cells from PAC patients would be able to secrete IL-6, as it was observed after in vitro stimulation, interfering with CD4+CD25+FOXP3+  differentiation, since it has been described that Treg cell differentiation requires antigen stimulation by engagement T cell receptor to induce FOXP3 expression [21]. Supporting this idea, the induction of FOXP3 by Con A-stimulation could be explained because of the polyclonal activation through mannose ligands on PBMC by Con A [22,23]; nevertheless, FOXP3+ cells induced by Con A are mainly NnTregs, a different T cell subset of Tregs [24,25].
On the other hand, it has been described that CD25 is the alpha subunit of IL2R and its expression has been related to 8 Clinical and Developmental Immunology activation status of T cells [26]; interestingly, in this study, the frequency of PBMC expressing this cell surface marker was significantly elevated in PAC patients group in contrast to healthy individuals, suggesting that PBMC and circulating CD4+ T cells are in an activation status, as similarly reported in other allergies like asthma [27,28] rhinitis [28], and dermatitis [29].
A differential expression of chemokine receptors was observed in patients with PAC; most CD4+ T cells were CCR4+ cells, and CCR9+ cells. These findings are remarkable, as CCR4 is known to modulate T-cell migration to sites of allergic-mediated inflammation in asthma and rhinitis [30,31]; CCR4+ cells are also an important source of IL-4 and other Th2 cytokines [30,31]. CCR9 is a molecule expressed on antigen-experienced memory T cells and it was described as a chemokine marker related to mucosalhoming [32]. It is possible that circulating CD4+CCR4 and/or CD4+CCR9+ cells observed in PBMC from our patients are cell-subsets in transit to conjunctiva, since after allergen-stimulation increased significantly the percentage of CD4+CCR4+CCR9+ cells. Remarkably, IL-4 is required for CCR9 imprinting on CD4+ T cells [33], and it is recognized that IL-4 and IL-5 are induced after Der p stimulation in allergic patients and promote allergic status [5].
HML-1 or CD103 ( 7 integrin) was first described as a molecule related to mucosal migration [34], and the vast majority of intraepithelial lymphocytes are CD103+ cells [9]. HML-1 could be induced by epithelial cells on activated lymphocytes [35] and has been implicated in epithelial T-cell retention through binding to E-cadherin [36]. In the present study, upon allergen specific-stimulation it was observed an increased percentage of CD4+CD103+ T cells. The CD4+CD103+ cells have been proposed as regulatory T cell subset [37]. Likewise, after Der p stimulation, the percentage of CD108+ cells were significantly increased. The CD108 (Sema7a) is a glycosylphosphatidylinositol-anchored semaphorin and has been described as a molecule that initiates T-cell-mediated inflammatory response through interaction with alpha1beta1 integrin [38]. On the other hand, it has been proposed that CD108 exists as a complex with TCR and/or CD3 on T cell surface and after activation inhibits T cell signalling and decreases proliferation [39]. In this context, whether the expansion of these two subsets, CD4+CD103+ cells or CD4+CD108+ cells, is related to mucosa-homing or corresponds to regulatory T cell subsets trying to counterbalance inflammatory CCR4+CCR9+ cell subsets and IL-6/IL-8 secretion is not known and needs further investigation.
The data shown here could lead to new perspectives in the treatment of the most frequent ocular condition seen by ophthalmologists and allergo/immunologists; CCR4 and CCR9 molecules could be used as potential targets for biological therapy in patients with PAC, as it has been proposed for asthma and the monoclonal anti-CCR4 antibody [40]. Taken together, it is possible that the interaction of CCR4, CCR9, and possibly CD103 and CD108 with their ligands secreted or expressed on activated endothelial cells and conjunctiva favours the selective adhesion of a circulating CD4+ T cell subset with an activated phenotype and the ability to respond to antigens, driving the immune-response to the ocular mucosa and inducing a proinflammatory microenvironment related to Th2 perennial allergic conjunctivitis.