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Increased, but Functionally Impaired, CD14+ HLA-DR–/low Myeloid-Derived Suppressor Cells in Psoriasis: A Mechanism of Dysregulated T Cells

Open ArchivePublished:January 22, 2016DOI:https://doi.org/10.1016/j.jid.2015.12.036
      The clinical extent of psoriasis pathology is regulated in part by defects in immune networks, including a defect in the suppressive actions of regulatory T cells. Recently, CD14+ HLA-DR–/low monocytic myeloid-derived suppressor cells (Mo-MDSCs) have been shown to suppress T-cell activation as one of their suppressive mechanisms. However, little is known about the role of Mo-MDSCs and their functional relationship to T-cell suppression in relation to human chronic immune-mediated inflammatory diseases, including psoriasis. Despite psoriasis being a hyperinflammatory condition, Mo-MDSCs were elevated in psoriatic patient peripheral blood mononuclear cells compared to nonpsoriatic healthy controls (2.6% vs. 0.9%, P < 0.002). Freshly isolated psoriatic Mo-MDSCs directly suppressed CD8 T-cell proliferation less efficiently than healthy control Mo-MDSCs. In addition, psoriatic Mo-MDSCs expressed reduced surface expression of programmed cell death protein 1 compared to healthy controls. Additional in vitro assays also demonstrated that psoriatic and control Mo-MDSCs both induce regulatory T-cell conversion from naïve T effector cells, but, importantly, the regulatory T cells induced by psoriatic Mo-MDSCs displayed decreased suppressive functionality. These results suggest that aberrations in psoriatic Mo-MDSCs prevent proper suppression of effector T-cell expansion and hamper the immune system’s ability to correctly self-regulate.

      Abbreviations:

      iTreg (induced regulatory T cell), MDSC (myeloid-derived suppressor cell), Mo-MDSC (monocytic myeloid-derived suppressor cell), PBMC (peripheral blood mononuclear cell), PD-1 (programmed cell death protein 1), PD-L1 (programmed death-ligand 1), ROS (reactive oxygen species), Treg (regulatory T cell)

      Introduction

      Psoriasis is a highly prevalent (1–3% of Caucasian populations) chronic immune-mediated inflammatory disease of the skin that is modified by susceptibility genes and environmental triggers. In addition to an enormous negative impact on quality of life, psoriasis has co-morbidities, including destructive psoriatic arthritis, stigmatization, depression and anxiety, inflammatory bowel disease, lymphoma, obesity, metabolic syndrome-associated conditions, and increased risk of early death from cardiovascular disease (
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      ), psoriatic Mo-MDSCs display a decreased suppressive profile compared to Mo-MDSCs from healthy control subjects. Whereas Mo-MDSC–mediated conversion of naïve effector T cells into induced regulatory T cells (iTregs) was observed in psoriatic as well as control Mo-MDSCs, converted Tregs from psoriatic origin displayed diminished suppression of autologous proliferating CD8 T cells. These observations suggest an effect of psoriasis on circulating myeloid monocytic cells and a previously unrecognized cellular contributor to compromised control of immune regulation in psoriasis patients, as well as a potential target for immune restitution.

      Results

      Mo-MDSCs are increased in the peripheral blood of patients with psoriasis

      Based on previously published reports on other autoinflammatory illnesses and the overactivity of signal transducer and activator of transcription-3–mediated signaling in psoriasis, which modulates MDSC, we hypothesized that the frequency of Mo-MDSCs in psoriatic patients would be abnormal compared to healthy controls (
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      • Bachmann O.
      • Westendorf A.M.
      • et al.
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      ,
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      • Takatsuki S.
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      Circulating myeloid-derived suppressor cells are increased and activated in pulmonary hypertension.
      ). Patient demographics for subjects participating in this study are listed in Table 1. Representative images demonstrating the calculation of Mo-MDSCs as a percentage of CD14+ selected cells from blood of healthy control or psoriatic individuals are shown in Figure 1a and b , respectively. The average proportion of Mo-MDSCs presented as a percentage of total CD14+ cells is shown in Figure 1c (23.4% in psoriasis vs. 9.7% in controls, n = 22 and n = 18, respectively, P < 0.002), representing an elevated level in 63% of psoriasis patients. We also determined the proportion of CD14+ HLA-DR-/low Mo-MDSCs calculated as a percentage of total peripheral blood mononuclear cells (PBMCs) as shown in Figure 1d for psoriatic patients versus healthy controls (2.6% vs. 0.9%, n = 17 and 19, respectively, P < 0.002). CD14+ bead-enriched cells from both healthy controls and psoriatic patients were CD33+, CD11b+ (100%), and CD15 (0%) (see Supplementary Figure S1a and b, respectively, online). To determine the relative intensity of HLA-DR on CD14+ HLA-DR–/low populations, we further analyzed the median fluorescence intensity and found that psoriatic patients displayed a significantly lower mean median fluorescence intensity than controls (21,486 vs. 27,107, n = 16 and n = 13 respectively, P < 0.02; Figure 1e).
      Table 1Patient demographics
      Type of patient
      PsoriaticHealthy controls
      No. of patients analyzed2218
      Age (y)45 (21–74)30 (25–54)
      Gender64% males, 36% female72% male, 28% female
      Race/ethnicity21 Caucasian, 1 African American11 Caucasian, 4 African American, 2 Asian, 1 other
      Current treatment21 none, 1 topical steroidNone
      Psoriasis area and severity index10 (0–29) ± 8NA
      Abbreviation: NA, not applicable.
      Values are given as number, median (range), or median (range) ± standard deviation.
      Figure 1
      Figure 1Frequency of monocytic myeloid-derived suppressor cells (Mo-MDSCs) is increased in psoriatic patients. (a, b) Representative flow cytometry panels for isotype-based quantification of Mo-MDSCs from peripheral blood mononuclear cells (PBMCs) subjected to CD14 magnetic bead selection of (a) healthy control subjects and (b) psoriasis patients. (c) Cumulative data representing the frequency of HLA-DR–/low cells among CD14+ cells selected by magnetic separation from 18 healthy controls and 22 psoriatic patients, respectively (P < 0.002). (d) Cumulative data representing the frequency of CD14+ HLA-DR–/low population in 17 healthy donors and 19 psoriatic patients determined by flow cytometry measuring expression of CD14+HLA-DR–/low among PBMCs (P < 0.002). (e) Average HLA-DR median fluorescence intensity (MFI) of CD14+ HLA-DR–/low cells from 13 healthy controls and 16 psoriasis patients (P < 0.02). (f) Correlation analyses among psoriasis severity assessed by psoriasis area and severity index (PASI) and percentage of circulating Mo-MDSCs among CD14+ monocytes revealed a significant positive correlation (n = 22, r = 0.72, P = 0.00091).
      We next asked whether the number of circulating Mo-MDSCs is correlated with psoriasis severity. Indeed, the psoriasis area and severity index (PASI) for each patient correlated significantly with circulating Mo-MDSCs among CD14+ cells (r = 0.72, R2 = 0.53, P = 0.00091; Figure 1f). Although psoriasis treatments may affect the number of circulating Mo-MDSCs, only psoriasis patients washed out or naïve to treatment were enrolled in the current study.

      Psoriatic Mo-MDSCs suppress less proliferating CD8 T cells than healthy controls

      Next, we tested whether Mo-MDSCs from psoriatic patients suppressed autologous T-cell responses at the same level as Mo-MDSCs isolated from healthy controls. CD14+ monocytes were selected by magnetic separation and sorted on lack of HLA-DR expression. Mo-MDSCs were added at a 1:2 ratio to autologous anti-CD2/CD3/CD28-stimulated e670-labeled CD8 T cells, and proliferation was analyzed by dye dilution assay (Figure 2a and b ). Psoriatic Mo-MDSCs have a statistically significant lower suppressive capacity toward their proliferating CD8 T cells compared to the suppression exhibited by healthy control Mo-MDSCs (106% proliferation vs. 47% proliferation, respectively, relative to their respective CD8 T cells stimulated without MDSC present [100%], P < 0.0001). When CD14+ HLA-DR+ monocytes were used as controls, they failed to suppress proliferation of the responding CD8 T cells, as expected (data not shown).
      Figure 2
      Figure 2Psoriatic peripheral blood monocytic myeloid-derived suppressor cells (Mo-MDSCs) are less potent direct inhibitors of T-cell proliferation. Magnetic bead-sorted CD14+ HLA-DR–/low (bottom 10%) purified from peripheral blood of normal volunteers and psoriatic patients were mixed directly 1:2 with autologous CD8 T cells. Proliferation was assessed by dilution of the far-red dye e670. (a) Representative suppression assays by dye dilution for control (top panel) and psoriasis (bottom panel) Mo-MDSCs compared to stimulated CD8 T cells without Mo-MDSCs or unstimulated CD8 T cells. (b) Normalized average proliferation from mixed Mo-MDSC–CD8 T cell co-cultures from 8 healthy volunteers (47%) and 9 psoriatic patients (106%) compared to basal proliferation of bead-stimulated healthy CD8 T cells alone (100%) and bead-stimulated psoriatic CD8 T cells alone (100%). (c) Representative criss-cross suppression assays mixing control Mo-MDSCs with psoriatic CD8 T cells or vice versa. (d) Normalized average proliferation from control from 4 healthy volunteers (66%) versus psoriatic Mo-MDSCs mixed with control CD8 T cells from 3 psoriatic patients (100%) compared to basal proliferation of bead-stimulated healthy or psoriatic CD8 T cells alone (100%). Whereas control Mo-MDSCs are effective at suppressing CD8 T-cell proliferation (first panel), psoriatic Mo-MDSCs fail to suppress CD8 T-cell proliferation (second panel).
      To address whether the defect in suppression in the psoriatic cultures lay within the MDSCs or the responding T cells (i.e., lack of suppression by MDSC vs. overproliferation by psoriatic CD8 T cells), we also undertook a criss-cross experiment using healthy Mo-MDSCs mixed with proliferating psoriatic CD8 T cells and vice versa (Figure 2c). Whereas healthy Mo-MDSCs effectively suppressed psoriatic CD8 T-cell expansion (Figure 2c, first panel and Figure 2d, first bar), psoriatic Mo-MDSCs failed to suppress healthy proliferating CD8 T cells (Figure 2c, second panel, and Figure 2d, second bar). Note that although the proliferation of stimulated CD8 T cells was slightly higher in psoriatic individuals, as we reported previously (
      • Sugiyama H.
      • Gyulai R.
      • Toichi E.
      • Garaczi E.
      • Shimada S.
      • Stevens S.R.
      • et al.
      Dysfunctional blood and target tissue CD4+CD25high regulatory T cells in psoriasis: mechanism underlying unrestrained pathogenic effector T cell proliferation.
      ), the difference in accelerated proliferation did not reach statistical significance (P = 0.07) and was not accountable for the observed decrease in suppression among psoriatic MDSCs, although this may contribute to the ultimate suppressive capacity of psoriatic MDSCs.

      Psoriatic Mo-MDSCs exhibit decreased levels of programmed cell death protein 1 and programmed death-ligand 1 compared to healthy control Mo-MDSCs

      To address potential mechanisms of Mo-MDSC suppression, we examined known functional mediators of MDSC suppression. Surface expression of programmed cell death protein 1 (PD-1), a molecule associated with suppression through release of IL-10 in monocytes (
      • Said E.A.
      • Dupuy F.P.
      • Trautmann L.
      • Zhang Y.
      • Shi Y.
      • El-Far M.
      • et al.
      Programmed death-1-induced interleukin-10 production by monocytes impairs CD4+ T cell activation during HIV infection.
      ) was assessed by flow cytometry. Interestingly, we found that Mo-MDSCs from psoriasis patients have a decrease in PD-1 expression compared to healthy Mo-MDSCs (13% vs. 28%, n = 14, n = 9 respectively, P = 0.028; Figure 3a and b ). In conjunction with this reduction in PD-1, IL-10 was produced by psoriatic MDSC at about half the level of control MDSC, with IL-10 levels of 25, 36 and 64 pg/ml in psoriasis Mo-MDSCs (Average expression, 42 pg/ml) and 101 and 109 pg/mL (Average expression 105 pg/ml) in control MDSC (see Supplementary Figure S2a online). The programmed death-ligand 1 (PD-L1), is associated with suppression of CD8 T cells by direct contact phagocytosis (
      • Kim Y.J.
      • Park S.J.
      • Broxmeyer H.E.
      Phagocytosis, a potential mechanism for myeloid-derived suppressor cell regulation of CD8+ T cell function mediated through programmed cell death-1 and programmed cell death-1 ligand interaction.
      ), and MDSC have also been described to suppress CD8 T-cell proliferation through expression of PD-L1 (
      • Kim Y.J.
      • Park S.J.
      • Broxmeyer H.E.
      Phagocytosis, a potential mechanism for myeloid-derived suppressor cell regulation of CD8+ T cell function mediated through programmed cell death-1 and programmed cell death-1 ligand interaction.
      ,
      • Noman M.Z.
      • Desantis G.
      • Janji B.
      • Hasmim M.
      • Karray S.
      • Dessen P.
      • et al.
      PD-L1 is a novel direct target of HIF-1alpha, and its blockade under hypoxia enhanced MDSC-mediated T cell activation.
      ). Although PD-L1 expression appeared heterogeneous in control MDSC (4/8 with significant expression), we noted a possible decrease in PD-L1 in psoriatic MDSC (2/14 with nearly significant expression, P = 0.055; Figure 3c and d).
      Figure 3
      Figure 3Psoriatic monocytic myeloid-derived suppressor cells (Mo-MDSCs) exhibit decreased PD-1 and PD-L1 surface expression. (a) Representative flow cytometry scheme for analysis of Mo-MDSC surface expression of PD-1. (b) Cumulative comparison of PD-1 expression on control versus psoriatic Mo-MDSCs (28% vs. 13% respectively, P = 0.028, based on nonparametric testing for equality of medians and equality of distribution, Mann-Whitney U test). (c, d) Representative flow cytometry scheme for analysis of Mo-MDSC surface and cumulative measurement of PD-L1 expression, respectively. Cumulative comparison of PD-L1 expression on control versus psoriatic Mo-MDSCs is shown in panel d (16% vs. 4%, P = 0.055, based on nonparametric testing for equality of medians and equality of distribution, Mann-Whitney U test). Although approximately 50% of the control patients (4/8) exhibit PD-L1 levels below the mean expression, the majority of psoriasis patients (12/14) exhibit less PD-L1 expression than the mean. PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1.
      We assessed the levels of reactive oxygen species (ROS) in Mo-MDSCs isolated from fresh PBMCs of psoriasis patients and healthy control volunteers. Psoriatic Mo-MDSC ROS oxidation of intracellular 2′,7′-dichlorofluorescein diacetate did not significantly differ from that of control MDSC (see Supplementary Figure S2b).

      Psoriatic and control Mo-MDSCs induce conversion from CD4+ effector T cells to induced Tregs

      We next investigated the capacity of psoriatic and control Mo-MDSCs to generate iTregs. CD2/CD3/CD28 bead-stimulated effector CD4 T cells isolated from healthy controls were mixed with healthy control or psoriatic Mo-MDSCs (1:2) for 3 days, and iTregs (CD4+) were detected using CD25+ and forkead box p3+(Foxp3+) staining (see Supplementary Figure S3 online). Supplementary Figure S3 shows the results of two independent experiments for the conversion of iTreg by either psoriatic or healthy control Mo-MDSCs compared to CD2/CD3/CD28 bead-stimulated effector CD4 T cells alone. Cumulative results demonstrate that normal and psoriatic Mo-MDSCs are capable of inducing iTregs compared to CD4 T cells with beads only (12.4% vs. 2.7%, n = 10, P = 0.0051; Figure 4a).
      Figure 4
      Figure 4Psoriatic and normal monocytic myeloid-derived suppressor cells (Mo-MDSCs) induce regulatory T cell (Treg) forkhead box p3 (Foxp3) expression, and psoriatic Mo-MDSC–induced Tregs are dysfunctional. (a) CD14+ HLA-DR–/low cells were co-cultured with effector T cells (CD4+ CD25 bottom 20%) for 3 days. Staining for Foxp3 was performed after the 5 day co-culture with Mo-MDSCs from either healthy control volunteers (triangles) or psoriasis patients (circles). Cumulative results of the percentage of induced CD4+ CD25+ Foxp3+ T cells (iTregs) are shown for Mo-MDSC–induced iTreg versus CD4+ CD25 effector T cells alone stimulated with anti-CD2/CD3/CD28 antibody-coated beads. (b) Psoriatic or healthy control subject Mo-MDSCs were mixed with autologous CD4+ CD25 T cells that were previously stimulated with anti-CD2/CD3/CD28 antibody-coated beads for 3 days to generate iTregs. The iTregs were then co-cultured with proliferating autologous CD8 T cells for 5 days. CD8 T-cell proliferation was assessed by dilution of the far-red dye e670. Healthy control Mo-MDSC–induced iTregs suppressed proliferating CD8 T cells effectively (44% proliferation, n = 5), whereas psoriatic Mo-MDSC–induced iTregs did not suppress CD8 T-cell proliferation (83% proliferation, n = 4, P = 0.009).

      Psoriatic Mo-MDSC–iTregs have decreased suppressive capacity compared to healthy control Mo-MDSC–induced iTregs

      iTregs induced by Mo-MDSCs from psoriatic or healthy control subjects were tested for functionality using an in vitro suppression assay. Results show that psoriatic Mo-MDSC–iTregs were minimally to nonsuppressive toward proliferating CD8 T cells (83% proliferation, n = 4; Figure 4b, middle bar), whereas iTregs induced by healthy control Mo-MDSCs effectively suppressed CD8 T-cell proliferation by >50% (44% of control proliferation, n = 5; Figure 4b, last bar). Preliminary findings indicate that this did not appear due to aberrant expression of either cytotoxic T lymphocyte-associated protein-4 (CTLA-4) or glucocorticoid-induced tumor necrosis factor receptor-family related gene (GITR) on the psoriasis MDSC-iTregs (see Supplementary Figure S4a and b online).

      Mo-MDSCs and psoriasis skin

      Mo-MDSCs may have limited ability to enter skin, as both control and psoriasis MDSC expression of skin-homing chemokine receptors such as CCR4 and CCR10 is quite limited (see Supplemental Figure S2c and d). Despite this, Mo-MDSCs were visualized (Figure 5a) in psoriatic skin using a combination of mouse-anti-human CD14 (Abcam, Cambridge, MA) and Alexa Fluor 647-conjugated goat anti-mouse IgG (Life Technologies, Grand Island, NY) as well as FITC-conjugated mouse anti-human HLA-DR (BD Biosciences, San Jose, CA). Monocytes positive for CD14-Alexa Fluor 647 but negative for HLA-DR–FITC expression (white arrows) were marked as Mo-MDSCs and can be visualized in the dermis of psoriasis patients as well as in the dermal epidermal junction region (dashed white line). Double-positive CD14+ HLA-DR+ monocyte/macrophage lineage cells (orange arrows) and CD14 HLA-DR+ dendritic cell lineage and potentially activated endothelium endothelial (gray arrows) cells in psoriasis tissue. Mo-MDSC cells are also detectable in healthy control skin, although quantitative differences between psoriasis and control tissue are not noted (Figure 5b).
      Figure 5
      Figure 5In situ localization of monocytic myeloid-derived suppressor cells (Mo-MDSCs) in psoriatic and healthy control skin. Monocytes positive for CD14-Alexa Fluor 647 but negative for HLA-DR–FITC expression (white arrows) were marked as Mo-MDSCs and can be visualized in the dermis of (a) psoriasis or (b) healthy control patients as well as in the dermal epidermal junction region (indicated by the dashed white line), compared to double-positive CD14+ HLA-DR+ (orange arrows) or CD14 HLA-DR+ (gray arrows) cellular infiltrates into psoriasis tissue. Bar = 0.2 mm.

      Discussion

      Treatments of psoriasis patients that achieve a stably rebalanced immune system with relatively durable treatment-free remissions remain a major challenge in dermatology (
      • Nestle F.O.
      ,
      • Nestle F.O.
      • Kaplan D.H.
      • Barker J.
      ). Escape of T helper type 17 (Th17) inflammatory pathways from natural regulatory mechanisms, such as the reduced functionality of regulatory T cells in psoriasis (
      • Sugiyama H.
      • Gyulai R.
      • Toichi E.
      • Garaczi E.
      • Shimada S.
      • Stevens S.R.
      • et al.
      Dysfunctional blood and target tissue CD4+CD25high regulatory T cells in psoriasis: mechanism underlying unrestrained pathogenic effector T cell proliferation.
      ), has emphasized the intricate cellular immune regulation responsible for balancing inflammatory response with timely resolution in healthy skin. Identifying mechanisms of immunoregulatory dysfunction in psoriasis are expected to result in new therapeutic approaches that leverage the ability of the immune regulatory balance to be restored and then maintained.
      Mo-MDSC modulation of Treg production, direct suppression of T-cell activation, and myeloid differentiation status in the background of psoriatic pathology have not been examined to date. Currently, the best characterization of human Mo-MDSCs are cells expressing CD14 with low or absent HLA-DR expression, a recognized shortcoming, albeit the only currently available discriminating marker combination (
      • Gabrilovich D.I.
      • Nagaraj S.
      Myeloid-derived suppressor cells as regulators of the immune system.
      ).
      In this study we show that psoriatic Mo-MDSCs exhibit a capacity to convert naïve T cells into iTregs compared to healthy Mo-MDSCs, although the psoriatic Mo-MDSC–converted iTregs have diminished suppressive functions, an observation that provides insight to our previously published report that Tregs in psoriasis are not decreased in number but have diminished suppressive capacities (
      • Goodman W.A.
      • Levine A.D.
      • Massari J.V.
      • Sugiyama H.
      • McCormick T.S.
      • Cooper K.D.
      IL-6 signaling in psoriasis prevents immune suppression by regulatory T cells.
      ,
      • Sugiyama H.
      • Gyulai R.
      • Toichi E.
      • Garaczi E.
      • Shimada S.
      • Stevens S.R.
      • et al.
      Dysfunctional blood and target tissue CD4+CD25high regulatory T cells in psoriasis: mechanism underlying unrestrained pathogenic effector T cell proliferation.
      ). However, it is possible that rather than creating iTreg cells, the Mo-MDSC co-cultured CD4 T cells may have differentiated directly into another lineage (e.g., T helper type 2) that would also be suppressive and may account for the observed inhibitory effect on CD8 T cells seen afterward. Importantly, it has also been recently shown that Mo-MDSCs attract Tregs in a C-C chemokine receptor type 5-dependent manner in murine skin (
      • Schlecker E.
      • Stojanovic A.
      • Eisen C.
      • Quack C.
      • Falk C.S.
      • Umansky V.
      • et al.
      Tumor-infiltrating monocytic myeloid-derived suppressor cells mediate CCR5-dependent recruitment of regulatory T cells favoring tumor growth.
      ), an observation that complements our recent results demonstrating that psoriatic patient Tregs have decreased expression of C-C chemokine receptor type 5, which could explain the decreased number of high potency C-C chemokine receptor type 5+ Tregs in the skin (
      • Soler D.C.
      • Sugiyama H.
      • Young A.B.
      • Massari J.V.
      • McCormick T.S.
      • Cooper K.D.
      Psoriasis patients exhibit impairment of the high potency CCR5(+) T regulatory cell subset.
      ).
      In this study, we also show that Mo-MDSC numbers in psoriatic patients are increased compared to healthy controls, as reported for other skin conditions such as chronic contact eczema (
      • Marhaba R.
      • Vitacolonna M.
      • Hildebrand D.
      • Baniyash M.
      • Freyschmidt-Paul P.
      • Zoller M.
      The importance of myeloid-derived suppressor cells in the regulation of autoimmune effector cells by a chronic contact eczema.
      ) and squamous cell carcinoma (
      • Vasquez-Dunddel D.
      • Pan F.
      • Zeng Q.
      • Gorbounov M.
      • Albesiano E.
      • Fu J.
      • et al.
      STAT3 regulates arginase-I in myeloid-derived suppressor cells from cancer patients.
      ). We also show that HLA-DR median fluorescence intensity on CD14+ cells is decreased in psoriatic patients compared to healthy controls, a characteristic that may have relevance in the antigen presentation machinery of monocytes, albeit its exact clinical significance at this point is poorly understood. The apparent suppression of HLA-DR expression in CD14+ cells isolated from psoriasis patients may reflect increased production of immature monocytes from the bone marrow due to the chronic inflammation associated with psoriasis. Similar changes in HLA-DR have been described for Mo-MDSCs from cancer patients as well (
      • Kitano S.
      • Postow M.A.
      • Ziegler C.G.
      • Kuk D.
      • Panageas K.S.
      • Cortez C.
      • et al.
      Computational algorithm-driven evaluation of monocytic myeloid-derived suppressor cell frequency for prediction of clinical outcomes.
      ). Alternatively, psoriasis may result in a decreased propensity for monocytic cells to undergo differentiation. We previously reported an increase in CD11b+ immature macrophages in psoriasis patients (
      • Cao L.Y.
      • Soler D.C.
      • Debanne S.M.
      • Grozdev I.
      • Rodriguez M.E.
      • Feig R.L.
      • et al.
      Psoriasis and cardiovascular risk factors: increased serum myeloperoxidase and corresponding immunocellular overexpression by Cd11b(+) CD68(+) macrophages in skin lesions.
      ), suggesting the micromilieu of psoriasis may favor immature status. Initially this result was counterintuitive because an increase in numbers would favor immune suppression and disease resolution. However, further characterization of psoriatic Mo-MDSCs revealed their suppressive functionality to be inferior compared to Mo-MDSCs isolated from healthy control subjects. An alternative possibility for this result may be that because we are performing an autologous model, psoriatic CD8 T cells are less sensitive to the suppressive activity of Mo-MDSCs compared to normal CD8 T cells. We compared the average proliferation of CD8 T cells isolated from either psoriatic patients (n = 9) or healthy control volunteers (n = 8) after CD2/CD3/CD28-bead stimulation. The difference in CD8 T-cell proliferation between psoriatic and control subjects was not statistically significant (P = 0.07), suggesting that the main defect may lie within the Mo-MDSCs.
      To address the potential mechanism behind the observed insufficiency of Mo-MDSCs, we examined molecules from pathways attributed to MDSC suppression, namely, expression of PD-1 and PD-L1, ROS, and IL-10 production. A reduction in expression of PD-1 was measured on psoriatic Mo-MDSCs, suggesting that IL-10 production by Mo-MDSCs is compromised, as PD-1 is known to induce IL-10 (
      • Said E.A.
      • Dupuy F.P.
      • Trautmann L.
      • Zhang Y.
      • Shi Y.
      • El-Far M.
      • et al.
      Programmed death-1-induced interleukin-10 production by monocytes impairs CD4+ T cell activation during HIV infection.
      ), thereby reducing the suppressive capacity of psoriatic Mo-MDSCs. This reduction in PD-1 and PD-L1 could be as a result of a systemic down-regulation of Mo-MDSCs. Indeed, although the levels of ROS in psoriatic and control Mo-MDSCs are comparable, less IL-10 may be produced by freshly isolated psoriatic Mo-MDSCs compared to healthy control Mo-MDSCs. Additionally, PD-L1 expression may be reduced in psoriatic Mo-MDSCs, suggesting a deficient Mo-MDSC control mechanism over T-cell proliferation in psoriatic patients. Whether this level of control is primarily in skin, draining lymph node, or in blood monocyte aggregates (
      • Golden J.B.
      • Groft S.G.
      • Squeri M.V.
      • Debanne S.M.
      • Ward N.L.
      • McCormick T.S.
      • et al.
      Chronic psoriatic skin inflammation leads to increased monocyte adhesion and aggregation.
      ) is currently unclear, but we were able to detect approximately equal numbers of dermal Mo-MDSCs in psoriatic and healthy skin using a combination of CD14 and HLA-DR as markers for tissue infiltrating Mo-MDSCs.
      In addition to a defect in direct suppression of T-cell proliferation, psoriatic Mo-MDSCs may indirectly influence Treg dysfunction, that is, both psoriatic and healthy control Mo-MDSCs induced Tregs, but importantly, these psoriatic Mo-MDSC iTregs exhibited a decreased capacity to suppress bead-activated CD8 T cells. We further examined the expression of glucocorticoid-induced tumor necrosis factor receptor-family related gene and cytotoxic T lymphocyte-associated protein-4, two of the key effector proteins used by Tregs, on the induced Tregs. We found that although glucocorticoid-induced tumor necrosis factor receptor-family related gene was highly expressed in both psoriatic and control induced Tregs, cytotoxic T lymphocyte-associated protein-4 was decreased in iTregs from psoriatic origin.
      Thus, we demonstrate an in vitro model for examining the ability of Mo-MDSCs to induce Tregs and show that the functionality of iTreg induced by psoriatic Mo-MDSCs is deficient in suppressive capacity. Despite an increase in circulating Mo-MDSCs in psoriasis, which would be expected to exert regulatory control over T-cell activation and expansion (
      • Albeituni S.H.
      • Ding C.
      • Yan J.
      Hampering immune suppressors: therapeutic targeting of myeloid-derived suppressor cells in cancer.
      ,
      • Dugast A.S.
      • Haudebourg T.
      • Coulon F.
      • Heslan M.
      • Haspot F.
      • Poirier N.
      • et al.
      Myeloid-derived suppressor cells accumulate in kidney allograft tolerance and specifically suppress effector T cell expansion.
      ,
      • Gros A.
      • Turcotte S.
      • Wunderlich J.R.
      • Ahmadzadeh M.
      • Dudley M.E.
      • Rosenberg S.A.
      Myeloid cells obtained from the blood but not from the tumor can suppress T-cell proliferation in patients with melanoma.
      ), psoriasis patient Mo-MDSCs are inferior in direct suppressive capacity as well as ability to induce iTregs that are capable of suppressing proliferation of expanding CD8 T cells (
      • Hoechst B.
      • Ormandy L.A.
      • Ballmaier M.
      • Lehner F.
      • Kruger C.
      • Manns M.P.
      • et al.
      A new population of myeloid-derived suppressor cells in hepatocellular carcinoma patients induces CD4(+)CD25(+)Foxp3(+) T cells.
      ). The combination of inducing a Treg phenotype while creating a dysfunctional Treg is consistent with observations that psoriasis patients, despite having normal levels of Tregs, exhibit dysfunctional suppression of activated T cells (
      • Goodman W.A.
      • Levine A.D.
      • Massari J.V.
      • Sugiyama H.
      • McCormick T.S.
      • Cooper K.D.
      IL-6 signaling in psoriasis prevents immune suppression by regulatory T cells.
      ,
      • Sugiyama H.
      • Gyulai R.
      • Toichi E.
      • Garaczi E.
      • Shimada S.
      • Stevens S.R.
      • et al.
      Dysfunctional blood and target tissue CD4+CD25high regulatory T cells in psoriasis: mechanism underlying unrestrained pathogenic effector T cell proliferation.
      ).

      Materials and Methods

      Cell isolation and sorting

      PBMCs were isolated from freshly obtained blood by Histopaque density gradient centrifugation (Sigma-Aldrich, St. Louis, MO) as described previously (
      • Soler D.C.
      • Sugiyama H.
      • Young A.B.
      • Massari J.V.
      • McCormick T.S.
      • Cooper K.D.
      Psoriasis patients exhibit impairment of the high potency CCR5(+) T regulatory cell subset.
      ). CD14+ monocytes were positively selected from PBMCs using magnetic CD14 microbeads (Miltenyi Biotech, San Diego, CA) with a magnet according to the manufacturer’s instructions. CD14+ cells were then flow sorted into Mo-MDSCs using a Becton Dickinson FACS Aria flow cytometer (Becton Dickinson, Franklin Lakes, NJ). The purity of the Mo-MDSC cells after sorting was >93%. All studies of human subjects were approved by the Institutional Review Board of University Hospitals Case Medical Center (Cleveland, OH). Peripheral blood samples were obtained from volunteer healthy controls or psoriasis patients after informed written patient consent was obtained according to the principles of the Declaration of Helsinki.

      Antibodies and flow cytometry

      To determine the frequency and phenotype of Mo-MDSC cells in PBMCs, multicolor fluorescence-activated cell sorting was done using the following antibodies: anti-CD14–APC (Invitrogen, Carlsbad, CA) and HLA-DR-FITC (BD Biosciences, San Jose, CA). Regulatory T cells were sorted using CD4-APC (Life Technologies, Grand Island, NY), CD25-PE, and Foxp3-647 (BD Biosciences). The following conjugated surface markers were used: PD-1–PE, PD-L1–PE., CCR4-PE, CCR10-PE, GITR-FITC (R&D Systems, Minneapolis, MN), and CTLA–PE (BD Biosciences). Analysis of FACS data was done using Winlist software, version 7.0 (Verity, Topsham, ME). Isotype-matched antibodies were used with all the samples as controls. Specifically, the population defined as HLA-DRlow/– was based on isotype staining with gating to include, at a minimum, 95% of the CD14+ population.

      Suppression assay

      Mo-MDSC cells were purified and sorted as described earlier. Autologous CD8 T cells were isolated from PBMCs using anti-CD8 microbeads and a magnetic column (Miltenyi Biotech). Psoriatic or healthy control Mo-MDSCs were seeded 2:1 in a 96-well round-bottom plate with CD8 cells previously labeled with 5 μM of e670 (eBiosciences, San Diego, CA). CD8 T-cell proliferation was induced by anti-CD2/CD3/CD28 stimulation beads (Miltenyi Biotech), and the suppressive capacity of Mo-MDSCs was measured using a BD C6 flow cytometer after 5 days of co-culture. Controls included a positive T-cell proliferation control (CD8 T cells alone), an induction negative control (CD8 T cells with medium only), and labeled but unstimulated CD8 cells. The CD8 T-cell proliferative capacity was titrated by decreasing the ratio of anti-CD2/CD3/CD28 bead to effector CD8 cell to reach approximately 60% proliferation (normalized to 100% proliferation) to allow for detection of either increased or decreased proliferation following cell-cell stimulation. Criss-cross experiments were performed in the same manner as described earlier with the modification of psoriatic Mo-MDSCs onto healthy control CD8 T cells stimulated with anti-CD2/CD3/CD28 bead and vice versa (control Mo-MDSCs onto psoriatic proliferating CD8 T cells).

      iTreg induction assay

      For analysis of in vitro-generated CD4+ CD25+ Foxp3 iTregs, Mo-MDSCs were sorted as described, and CD4 T cells were purified using a negative isolation kit (Miltenyi Biotech). Additionally enriched CD4 T cells were stained with CD4-APC and CD25-PE and sorted for CD4+ bottom 20% of CD25 cells. The CD4+ CD25 T cells were stimulated with anti-CD2/CD3/CD28 beads (Miltenyi Biotech) following the manufacturer’s instructions with a modified bead to cell ratio (1 bead per 8 cells) in the presence or absence of either psoriatic or healthy control Mo-MDSCs for 3 days in round-bottom 96-well plates. T cells were analyzed after 3 days and gated on a CD4+ CD25+ Foxp3 population. Alternatively, to functionally test in vitro Mo-MDSC–induced Tregs after 3 days as described earlier, Mo-MDSCs were removed magnetically with anti-CD14 microbeads (Miltenyi Biotech), and the negatively isolated iTregs were co-cultured 1:4 with e670-stained bead-activated CD8 T cells for 5 additional days. Suppression was measured using a BD C6 flow cytometer as described earlier.

      Oxidative stress measurements

      ROS were measured by flow cytometry using 2′,7′-dichlorofluorescein diacetate, a fluorogenic dye that measures hydroxyl, peroxyl, and other ROS species within isolated Mo-MDSCs. Psoriatic or healthy control PBMCs were incubated at room temperature in the presence of 300 pM 2′,7′-dichlorofluorescein diacetate for 10 minutes, washed with phosphate buffered saline (PBS), then labeled with anti-CD14–APC, anti–HLA-DR–FITC to allow for electronic selection of Mo-MDSCs. After incubation on ice for 20 minutes, cells were washed with PBS and analyzed using a BD C6 flow cytometer. After diffusion into Mo-MDSCs, 2′,7′-dichlorofluorescein diacetate fluorescence was reduced through deacetylation to a nonfluorescent compound, which was then oxidized by ROS into fluorescent 2′,7′-dichlorofluorescin and measured by flow cytometry.

      IL-10 enzyme-linked immunosorbent assay

      IL-10 from normal and psoriatic MDSCs was quantified using the human IL-10 enzyme-linked immunosorbent assay kit (R&D). MDSC and CD8 were mixed at a 2:1 ratio, and 200 μl of supernatant was collected from day 5 co-cultures according to the manufacturer’s recommendations. Absorbance 450 nm with wavelength correction to 540 nm was measured on a Perkin Elmer (Waltham, MA) Victor X3 spectrophotometer.

      Immunohistochemistry

      Tissue sections 7 μm thick were cut and fixed as described previously with some modifications (
      • Toichi E.
      • Torres G.
      • McCormick T.S.
      • Chang T.
      • Mascelli M.A.
      • Kauffman C.L.
      • et al.
      An anti-IL-12p40 antibody down-regulates type 1 cytokines, chemokines, and IL-12/IL-23 in psoriasis.
      ). Briefly, slides were dried for 20 minutes at room temperature, then fixed in cold acetone for 20 minutes. Rehydration was performed using DAKO (Carpinteria, CA) buffer for 1 minute. Slides were blocked with 10% goat and mouse serum for 30 minutes at room temperature. Mouse anti-human CD14 (Abcam clone 2Q1233, Cambridge, MA) was incubated overnight at 4 °C. Slides were then rinsed 5 minutes three times with PBS and stained for 60 minutes at room temperature using Alexa Fluor 647-conjugated goat anti-mouse IgG (Life Technologies, Grand Island, NY). Slides were then further rinsed for 5 minutes three times with PBS and stained with FITC-conjugated mouse-anti-human HLA-DR (BD Biosciences clone G46-6 San Jose, CA) for 2h at room temperature. Slides were then rinsed again 3 times 5 min each with PBS and mounted using Vectashield with DAPI (Vector Labs, Burlingame, CA). Images were acquired using an UltraVIEW VoX spinning disk confocal system (PerkinElmer, Waltham, MA) mounted on a Leica DMI6000B confocal microscope (Leica Microsystems, Bannockburn, IL) equipped with a HCX PL APO 20X/1.4 objective. All confocal images were analyzed using Volocity software (PerkinElmer).

      Statistical analysis

      The statistical significance between values was determined by Student t test or nonparametric Mann-Whitney test and equality of medians when samples were not distributed normally. All data are expressed as mean ± standard error of the mean. Probability values of P ≤ 0.05 were considered significant.

      Conflict of Interest

      The authors state no conflict of interest.

      Supplementary Material

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