Psoriasis patients exhibit impairment of the high potency CCR5+ T regulatory cell subset

Abstract

CCR5 expression on CD4⁺CD25^highFoxp3⁺ regulatory T cells (Tregs) has been reported to be crucial for limiting Th1 inflammation associated with autoimmunity and bacterial infections. We inquired whether abnormalities in chemokine receptors expressed on Tregs might be involved in the psoriatic pathogenesis. Indeed, the proportion of CCR5⁺Treg was 58.8% in healthy individuals (n=9), whereas only half as many CCR5⁺Treg cells were found in psoriatic individuals (29.1%, n=8, p<0.01). The flow-enriched control CCR5⁺Tregs consistently exceeded the suppressive capacity of unsorted Tregs in autologous MLR assays (n=5, p<0.05) showing that CCR5⁺Treg subset is a high potency regulatory T cell population. Interestingly, psoriatic CCR5⁺Treg cells exhibited significantly less migratory capacity toward CCR5 ligands MIP-1β and RANTES in vitro compared to CCR5⁺Treg controls (n=3, p<0.05). Our data demonstrate that psoriatic CCR5⁺Tregs cells are numerically-, functionally- and chemotactically-deficient compared to controls and may pose a triple impairment on the ability of psoriatic Tregs to restrain inflammation.

1. Introduction

Human regulatory T cells (Tregs) are considered the immune system guardians against development of autoimmune disorders, being responsible for maintaining self-tolerance and playing an important role in limiting autoreactive and pathogenic cells. Tregs are characterized by expression of CD4 and high expression of CD25 with concomitant expression of the Forkhead box P3 (Foxp3⁺) transcription factor and lack of expression of the Interleukin-7 receptor subunit alpha (IL7R-α or CD127). Tregs were originally discovered when mice containing mutations in the Treg master transcription factor Foxp3 resulted in a scurfy phenotype that immunologically resembled human patients with immune dysregulatuion, polyendocrinopathy, enteropathy and X-linked syndrome (IPEX) disease (1), (2).

Recently, several studies have demonstrated that various chemokine receptors including CC chemokine receptor 5 (CCR5) are constitutively expressed on Tregs in humans and likely provide Tregs with a competitive advantage over naive T cells to migrate more efficiently to the periphery (3–6). In addition to regulatory cell trafficking CCR5 also participates in control of effector functions of memory/effector T lymphocytes, macrophages, and immature dendritic cells (DCs). CCR5 is a G-Protein Coupled Receptor (GPCR) that binds the chemokines CCL5 (RANTES), CCL3 (MIP-1α) and CCL4 (MIP-1β) and is expressed on effector T cells that home toward peripheral sites of inflammation (7). Deficiency in CCR5 leads to a reduced T cell infiltration in parasitic infections such as Trypanosoma cruzi (8, 9) and Toxoplasma gondii (10), as well as viral infections (10).

Several human autoimmune diseases exhibit a dysregulated T cell response in which the activity of pathogenic effector T cells (Teff) is inadequately controlled by naturally occurring CD4⁺ CD25^high Foxp3⁺ CD127⁻ Tregs (11, 12). In those cases, functional defects appear in Teff as well as Treg populations which lead to a loss of tolerance and subsequent activation and differentiation of pathogenic Th1 and Th17 cells (13, 14). Psoriatic Tregs are deficient in suppressing not only autoimmune Teffs but also normal (healthy) Teffs as shown in previous “criss-cross experiments” (15). We also previously demonstrated that, as shown in the mouse (16) IL-6 could reverse the function of healthy human Tregs (17) Interleukin-6, which is highly elevated in psoriatic tissue, was demonstrated to induce Stat3 signaling prior to subsequent T cell activation, which results in the loss of functional Treg suppression (18). However, the underlying mechanisms responsible for the suboptimal suppressive activity of psoriatic Treg remain incompletely defined. Recently CCR5 expression has been shown to be important for high potency Treg cells in a mouse model (19)

In the current manuscript, we show that the presence of the chemokine receptor CCR5 on the surface of regulatory T cells is associated with Tregs that exhibit enhancement of their suppressive potential in vitro. Indeed, Treg cells with higher CCR5 expression are superior suppressors of activated Teff cells and exhibit higher levels of the transcription factor Foxp3 as measured by flow cytometry and Real-Time PCR. Unfortunately for psoriasis patients CCR5 expression on Tregs is decreased compared to healthy controls, leading to a reduction in the CCR5⁺ Foxp3^high high potency Treg population. Chemotaxis assays confirmed that CCR5⁺ Tregs isolated from healthy controls display superior migration toward the CCR5 ligands MIP1β and RANTES compared to CCR5⁺ Tregs from psoriasis patients. These results suggest that impaired CCR5 likely contributes to the long-term failure of psoriatic Tregs to competently suppress the expanding effector cell response.

2. Materials and Methods

2.1 Cell isolation and culture of human dermal cells and PBMCs

All studies involving human subjects were approved by the Institutional Review Boards of Case Western Reserve University and University Hospitals Case Medical Center. Punch biopsies and/or peripheral blood samples were obtained from healthy adult volunteers or patients with moderate plaque psoriasis following informed consent. Primary dermal cells were isolated as previously described (20) and cultured in complete medium (RPMI 1640 containing 10% FBS (Cambrex), L-glutamine, penicillin, streptomycin from Cellgro, and 2-ME). PBMCs were prepared from peripheral blood as previously described (15) and adhered to plastic for 1 h to enrich for the non-adherent, lymphocyte-containing fraction. Dermal cell suspensions were prepared from lesional skin biopsies as described previously, with minor modifications (20). Viability was determined by trypan blue exclusion (70–90% range); CD4⁺ cells were separated from PBMC's by negative selection on midiMACS columns (CD4⁺ T cell isolation kit; Milteny Biotec) according to the manufacturer's instructions. After over-night incubation, CCR5⁺ CD25^high Foxp3 cells were sorted using a BD FACSAria flow cytometer.

2.2 Proliferation assays

CD4⁺ CD25⁻ Teff, 2×10⁴ cells were cultured 6–7 days in round-bottom, 96-well plates (Costar) in the presence or absence of various dilutions of Treg cells. 1×10⁵ Plastic-adherent 3000-rad irradiated PBMCs were used as APCs per well. Cells were pulsed with 1uCi/well [3H] thymidine for the last 16 h before harvesting on the day described.

2.3 Antibodies and flow cytometry

Surface markers on blood or skin cells were detected by incubation with CD4-APC and CD25-PE monoclonal antibodies (mAbs) (BD), followed by fixation in 4% paraformaldehyde. To detect intracellular antigens, cells were permeabilized using a perm/fix kit (BD Biosciences). Analytical samples were analyzed on a BD LSR II flow cytometer (BD Biosciences). The following chemokine receptor mAbs were used for analyzing sorted Tregs: anti-CD62L-PECy5, anti-CLA-FITC, anti-CCR4-Pecy7, anti-CCR5-PEcy5, anti-CCR6-APC, anti-CCR7-PECy7, anti-CCR8-APC, anti-CXCR3-PEcy5, anti-CXCR4-PEcy5, anti-CXCR5-APC, anti-CXCR6-APC, anti-β7-integrin-APC (all R&D).

2.4 Real-time quantitative RT-PCR

Total RNA was extracted from CD4⁺ CD25^high top 5% Treg of healthy and psoriatic volunteers using an RNeasy mini kit (Qiagen). RNA was reverse transcribed using 200U of Moloney murine leukemia virus (MMLV) reverse transcriptase (Invitrogen Life Technologies) according to the manufacturer's instructions. cDNA was amplified in the presence of specific primers and probes for Foxp3 or 18S rRNA and TaqMan Universal Master Mix in a 96-well microtiter plate format on an Abi PRISM 7700 Sequence Detection System (all from Applied Biosystems). Each PCR was performed in triplicate, using the following conditions: 2 min at 50C and 10 min at 95C, followed by 40 cycles of 15 s at 95C and 1 min at 60C. Copy numbers of samples were calculated against input copy numbers of plasmid standards for both target and 18S rRNA genes and were normalized with the copy numbers of the 18S rRNA gene.

2.5 Regulatory T cells Chemotaxis assay

Chemotaxis using Transwell plates (pore size, 5um) was conducted in vitro, and the migratory capacity of control and psoriatic CCR5⁺ Treg cells toward the CCR5 relevant chemokines MIP-1β and RANTES was measured. 1×10⁵ CCR5⁺ peripheral Tregs from psoriatic patients or healthy donors were loaded onto the upper transwell plates. The average number of CCR5⁺ Treg cells isolated from healthy controls that migrated toward MIP-1β or RANTES was counted and the averages were expressed as the ratio (chemotactic index) of the number of cells in the treated sample versus control (spontaneous migration)

2.6 Statistical analysis

Statistical analysis was performed using Student's t test. A value of p< 0.05 was considered significant.

3. Results

3.1 Expression levels of various chemokine receptors on normal and psoriatic CD4⁺ CD25^high and Foxp3⁺ Tregs from peripheral blood

We previously demonstrated that regulatory T cells isolated from psoriatic patients exhibited a decreased capacity to suppress expanding effector T cell population in allo-MLR assays compared to healthy controls (15). In order to characterize the possible mechanisms responsible for the previously observed decrease in psoriatic Treg suppressive capacity, we compared CD4⁺ CD25^high Foxp3-expressing Tregs isolated from healthy controls and psoriatic patients for the expression of several common immunological surface receptors including CD62L, CLA, CCR5, CCR4, CCR6, CCR7, CCR8, CXCR3, CXCR4, CXCR5, CXCR6 and β7-integrin. Primarily, expression of CCR5 appears decreased on psoriatic patient Tregs (29.1 ± 3.0%, n=8) compared to controls (58.8 ± 3.2% n=9), p<0.01 (Fig. 1) although the MFI of CCR5 on Tregs was comparable among psoriatic and healthy control regulatory T cells (Supplemental Figure S1). By contrast, no statistically significant expression differences were observed among the several other chemokine receptors examined.

Figure 1. Reduction of CCR5 Positive Regulatory T Cells in Patients with Psoriasis.

Expression levels of various chemokine receptors on both control and psoriatic regulatory T cells (Treg). CD4⁺ (negative bead selection) T cells defined by CD25 and FoxP3. CD25^highFoxP3⁺ Treg cells were gated and analyzed further for expression of the indicated chemokine receptors (3,000 positive events/chemokine). CCR5 expression was significantly decreased by 50% in psoriatic (n=8) Treg cells compared to control (n=9) Treg cells (p<0.01). No significant expression differences were observed on other chemokine receptors investigated.

3.2 The CCR5⁺ Treg subset exhibits higher Foxp3 expression and functional suppressive activity

To determine whether diminished CCR5 expression on Tregs may mark a functionally impaired subset, we examined the levels of Foxp3 expression in CCR5⁺ Tregs by flow cytometry and quantitative PCR. Total RNA from sorted CCR5⁺ Tregs (n=3) was reverse transcribed and amplified in the presence of primers specific for Foxp3 and 18S ribosomal RNA. Unsorted Tregs showed statistically significant lower levels of Foxp3 mRNA expression compared to CCR5⁺ Tregs (Fig. 2a).

Figure 2. CCR5 positive Treg cells express higher levels of mRNA and protein FoxP3.

A) Cumulative data (n=5) demonstrate a statistically significant higher mRNA FoxP3 expression level of CCR5⁺ Treg cells compared to that of both pooled Treg and CCR5⁻ Treg cells. In agreement, CCR5⁻ Treg cells showed significantly less FoxP3 mRNA levels compared to pooled Treg cells. B) Peripheral blood CD4⁺ T cells were analyzed for CD25 expression, and cells with the high levels of CD25 were selected. CD4⁺CD25^high T cells (black dots) were further defined by [FoxP3 and CCR5] (left) and [FoxP3 and CXCR3] (right). CCR5 expression (MFI 45.2) showed a trend toward higher Foxp3 levels, whereas the expression of another chemokine receptor CXCR3 (MFI: 51.4) in Tregs showed no relative increase in Foxp3.

Furthermore, intracellular flow cytometry on control CD4⁺ CD25^high Tregs appear to show that the CCR5+ subset of Tregs also expresses higher levels of Foxp3 (MFI 45.2). By contrast, the expression of another chemokine receptor, CXCR3 (MFI 51.4), in Tregs showed no relative increase in Foxp3 (Fig. 2b).

We next examined the functional suppressive capacity of CCR5⁺ Tregs versus a pool of unsorted CD4⁺CD25^high Tregs. CCR5-expressing Tregs demonstrated a higher suppressive capacity compared to the unsorted Treg pool. Shown for comparison is a representative Treg from a non-psoriatic individual (Fig. 3a, shown as individual cpm values compared to a Treg pool from the same individual) as well as cumulative data from five separate individuals (Fig. 3b, shown as reduction in percentage suppression).

Figure 3. Control CCR5⁺ Treg cells are functionally high potency regulatory T cells in normal yet still dysfunctional in psoriasis Tregs.

A) Cumulative data demonstrate a statistically significant enhancement of suppressive function of Treg cells when we compare CCR5⁺ Treg cells to the Treg cell-pool at any ratio ranging from 1:1 to 1:4 (p<0.05). xxxB) Control CCR5⁺ Tregs showed increased suppressive capabilities compared to pooled Tregs. C) Psoriatic CCR5⁺ or pooled Tregs display decreased suppressive capacity compared to normal control Tregs. Furthermore, no increased suppressive capacity was observed fro CCR5+ psoriatic Treg cells compared to pooled Tregs. Black and white bars represent CCR5⁺ Treg or pooled Tregs respectively.

Interestingly, CCR5⁺ Tregs populations showed an increased percentage of CTLA4, CD120b and GITR expression compared to CCR5⁻ Tregs populations based upon isotype (Supplement Figure S2, two controls and two psoriatic samples are shown). However, the MFI of each marker (CTLA-4, CD120b, and GITR) was not different among CCR5⁺ versus CCR5⁻ cells.

Among Tregs isolated from psoriasis patients, however, no difference was observed in suppressive capacity between CCR5⁺Tregs versus Treg pooled cells (Fig. 3C).

3.4 Dermal Tregs express higher CCR5 levels in healthy skin compared to psoriatic skin

Next we provide proof of concept demonstrating that CCR5⁺ Tregs in human psoriatic lesions versus CCR5⁺ Tregs elicited into healthy control skin using sensitization and subsequent challenge with the hapten di-nitrochlorobenzene (DNCB), which ultimately self-resolves are less infiltrating. To avoid the detection of effector T cells transiently expressing CD25, epidermal and dermal cell suspensions were rested for 48h before flow cytometric analysis. In vivo priming of normal skin with the 0.03% DNCB was used to elicit a regulatory T cell response. Infiltrating Tregs were obtained and compared to non-elicited (except by psoriasis) Treg cells isolated from a psoriatic plaque. CCR5+ (compared to isotype) cells were then assessed for the expression of Foxp3.

Interestingly, CD4⁺ CD25^high psoriatic Treg cells co-expressed CCR5 in only 3.6% of Tregs cells, compared to 39.6% expression in Foxp3⁺ Tregs cells recruited into healthy skin by hapten elicitation (Fig. 4).

Figure 4. Skin-infiltrating FoxP3⁺ Treg cells showed less CCR5 expression in psoriasis patient.

Dermal cells were extracted from both 0.03% DNCB elicited healthy control skin and psoriatic lesional skin, and analyzed for CCR5 expression levels on skin-infiltrating Treg cells using flow cytometry. In a representative experiment, approximately 40% of Treg cells isolated from healthy skin undergoing DNCB elicited inflammation expressed CCR5, psoriatic Treg cells exhibited approximately 10 times less expression (4%) of CCR5-expressing regulatory cells. Percentage of CCR5-expressing cells among total skin-infiltrating Tregs are represented in black in the x-axis. Isotype controls are shown in white.

3.5 Chemotaxis of CCR5⁺-Tregs is reduced in psoriatic patients

To determine if the expression of CCR5 affected the ability of Treg cells to migrate, we examined the ability of CCR5⁺ Treg from psoriatic versus healthy CCR5⁺ Treg control individuals to migrate toward known ligands of the CCR5 receptor such MIP-1β and RANTES. 1×10⁵ CCR5⁺ peripheral Tregs from psoriatic patients or healthy donors were loaded onto the upper transwell plates. The average number of CCR5⁺ Treg cells isolated from healthy controls that migrated toward MIP-1β or RANTES was 12.4 × 10³ ± 3.2%, and 19.8 × 10³ ± 2.1% (p<0.05), respectively, whereas CCR5⁺ Treg cells isolated from psoriatic individuals (open bars) showed significantly weaker chemotaxis to both MIP-1β and RANTES (4.3 × 10³ ±1.7%, and 7.7 × 10³ ± 2.2%, thousand cells, respectively, n=4, p<0.01), Fig 5.

Figure 5. Psoriatic CCR5⁺ Treg cells showed less migratory capacity toward CCR5 ligands.

Chemotaxis assay using transwell plates was conducted in vitro, and the migratory capacity of both control and psoriatic CCR5⁺ Treg cells toward the CCR5 relevant chemokines MIP-1β and RANTES was determined. Average cell counts of control CCR5⁺ Treg cells migrated toward MIP-1β and RANTES were 12.4 and 19.8×10³ cells, respectively, whereas psoriatic CCR5⁺ Treg cells (red bars) showed significantly weaker chemotactic responses to both MIP-1β and RANTES (4.3 and 7.7×10³ cells, respectively, n=4).

4. Discussion

Psoriasis is currently interpreted to be an inflammatory skin disease that has been demonstrated to be driven by T cells (20, 21). The psoriatic pathology is a unique source of cutaneous T cells and can provide valuable information regarding the interaction of regulatory T cells (Treg) with pathogenic effector T cells (Teff) in vivo.

CCR5 is a G-Protein Coupled Receptor (GPCR) known to be a co-receptor for HIV during infection of helper T-cells that aids the virus to establish a chronic infection. CCR5 is expressed on myeloid as well as lymphoid cells, including monocytes, macrophages, dendritic cells and T-cells (22, 23). However, its relevance in skin homing and its function in human Tregs has not been described in detail. Importantly, CCR5⁺ Treg cells have been experimentally shown in the periphery to limit Th1 immune responses in murine inflammatory disease models including Leishmania major infection (24) and acute graft-versus-host disease (25, 26). Interestingly, a randomized placebo controlled trial on psoriasis patients treated with the CCR5 inhibitor SCH351125 was recently performed (27). This study found no statistically significant clinical improvement in psoriasis patients treated with the CCR5 inhibitor (50 mg twice daily, orally for 28 days) over a follow-up period of 4 weeks. Interestingly, this study did report that relative expression of CCR5 in proportion to the cells observed showed only a statistically significant difference between lesional and non-lesional CD3 cells in the epidermis. These observations suggest that therapeutics targeting CCR5 are not likely to reverse established disease but rather may be a preventive measure.

In the present study, we sought to assess the expression levels of CCR5 specifically on Tregs involved in psoriatic pathology. We provide evidence the CCR5⁺ subset of CD4⁺CD25^high Tregs was associated with increased suppression of T effector cells in healthy control individuals. Accordingly, we also demonstrate that CCR5⁺ Tregs express higher levels of Foxp3 mRNA as well as Foxp3 protein (Fig 2a and 2b). However, we do not believe that CCR5 itself leads to increased Treg function. We show that peripheral psoriatic Tregs express lower CCR5 levels compared to Tregs isolated from healthy controls (Fig 1). Further, we show that psoriatic Treg cells expressing CCR5 are still functionally deficient in suppression.

We have also demonstrated that skin-infiltrating psoriatic Foxp3⁺ Tregs express lower levels of CCR5 on their surface compared to Treg cells elicited in healthy skin in response to a contact sensitizing agent elicitation (~4 % compared to ~40%) in CCR5⁺ expression on Tregs (Fig 4). In the latter situation, DNCB elicitation provokes a self-resolving skin inflammatory condition over the course of approximately 10 days total time, whereas psoriasis skin represents an ongoing non-self-resolving inflammatory site. This difference in resolution may alter the timing of CCR5 expression or the micromilieu of the resolving lesion may differ from a non-resolving site such that recruitment of cells with varying levels of CCR5 expression may home to various inflammatory sites in a time dependent manner. For example, an acute inflammatory burst, such as that observed in a sensitization response may produce a cytokine gradient that attracts Treg cells expressing higher CCR5 levels. In contrast, a chronic inflammatory state, such as that of psoriasis skin may recruit Treg cells that have lower CCR5 expression. Because wound-healing responses are not generally different in psoriasis patients, the acute skin milieu may still be capable of eliciting CCR5^high Tregs, however the chronic tissue cytokine/chemokine micromileu does not appear to recruit these cells under static conditions. Chemotactic studies using the CCR5 ligands MIP-1β and RANTES confirmed that the psoriatic Tregs exhibit a decreased migratory potential to either compared to the increased migratory capacity of Tregs from healthy controls (Fig 5).

These results suggest a possible mechanistic explanation for dysfunction of the suppressive capacity of psoriatic Treg cells compared to healthy Treg potential for controlling effector T cell proliferation competently (15). One possible explanation is that lower CCR5 levels may be responsible for the psoriatic Tregs decreased homing towards inflammatory signals (such as RANTES and MIPβ) released by the psoriatic plaques. Furthermore, the lower levels of Foxp3⁺ in CCR5⁺ psoriatic Tregs could contribute to the inability of psoriatic Tregs to sufficiently suppress pathogenic effector T cell expansion within psoriatic plaques. The inability of psoriatic Tregs to act may lead to a plaque microenvironment wherein the psoriatic milieu biases effector or even regulatory T cells towards becoming pathogenic (Th17) cell types as described recently (28).

Highlights.

• Psoriatic Tregs express lower CCR5 levels compared to Tregs isolated from healthy controls

• Healthy CCR5⁺Tregs show superior T-cell suppressive functions

• Psoriatic skin Tregs express less CCR5 than Tregs in DNCB-elicited healthy skin

• Psoriatic CCR5⁺ Tregs exhibit decreased migratory potential compared to control CCR5⁺Tregs