Induction of IL-17+ T cell trafficking and development by IFN-gamma: mechanism and pathological relevance in psoriasis

Abstract

Th1 and Th17 T cells are often colocalized in pathological environments, yet Th1-derived IFN-gamma inhibits Th17 cell development in vitro. We explored the physiologic basis of this paradox in humans. In this study, we demonstrate increased the number of CD4(+) and CD8(+) IL-17(+) T cells in skin lesions of psoriasis. Furthermore, we show that myeloid APCs potently support induction of IL-17(+) T cells, and that this activity is greatly increased in psoriasis. We tested stimuli that might account for this activity. Th1 cells and IFN-gamma are increased in psoriatic blood and lesional skin. We show that IFN-gamma programs myeloid APCs to induce human IL-17(+) T cells via IL-1 and IL-23. IFN-gamma also stimulates APC production of CCL20, supporting migration of IL-17(+) T cells, and synergizes with IL-17 in the production of human beta-defensin 2, an antimicrobial and chemotactic protein highly overexpressed by psoriatic keratinocytes. This study reveals a novel mechanistic interaction between Th1 and IL-17(+) T cells, challenges the view that Th1 cells suppress Th17 development through IFN-gamma, and suggests that Th1 and IL-17(+) T cells may collaboratively contribute to human autoimmune diseases.

FIGURE 1

IL-17⁺ T cells in psoriasis lesions. Skin biopsies and peripheral blood from healthy donors and patients with psoriasis were stained with specific antibodies as described in Materials and Methods. A-E, IL-17⁺ T cells were analyzed with FACS. A, IL-17⁺ T cells. Results were expressed as the mean percent of IL-17⁺ T cells in T cells, error bars indicate SEM, n = 8-10. B, Representative dot-plots are shown. C, Total number of IL-17⁺ T cells in the skin. Total number of IL-17⁺ T cells was calculated by multiplying the percentage of IL-17⁺ T cells by the absolute number of T cells per mm² of skin, as determined previously (26). Results are expressed as mean number ± SEM, n = 10. D, Coexpression of IL-17 and IFNγ on IL-17+ T cells. Single cell suspensions were made from skin tissues in healthy donors and patients with psoriasis. The expression of IFNγ and IL-17 was analyzed by intracellular cytokine staining gated on CD3+ T cells. E, Total number of IL-17⁺ T cell subsets in dermis and epidermis. Total number of IL-17⁺CD4⁺ and IL-17⁺CD8⁺ T cell subsets was calculated by multiplying the percentage of each IL-17⁺ T cell subset by the absolute number of T cell subsets per mm² skin (26) in the separated dermis and epidermis. Results are expressed as mean number ± SEM, n = 5. *P < 0.05 (A, C, E) compared to uninvolved and healthy donor skin.

FIGURE 2

IFNγ⁺ T cells in psoriasis lesions. A, T cells in psoriasis lesions. Psoriasis plaques were stained with anti-CD4 and anti-CD8. One of 5 representative individuals is shown. B, C, IFNγ⁺ T cells in psoriasis lesions. IFN-γ⁺ T cells in psoriatic lesions: T cells (CD45⁺CD3⁺) were analyzed by FACS in cell suspensions obtained from healthy skin and psoriatic plaque. B, IFN-γ⁺ T cells were analyzed by FACS and quantified in the skin. Results were expressed as the mean number per mm² skin ± SEM. (n = 3). C, Expression of IFN-γ in skin from healthy donors and patients with psoriasis. IFN-γ was quantified by real-time PCR. Results are expressed as the mean values of relative expression ± SEM. (n = 5, *P < 0.03 compared to uninvolved and healthy skin).

FIGURE 3

Psoriatic myeloid APCs stimulate memory IL-17⁺ T cell expansion. A, B, Myeloid APCs (CD45⁺CD14⁺) were analyzed by FACS in cell suspensions obtained from healthy skin and psoriatic plaque. A, One representative dot-plot of CD45⁺ leukocytes from the skin of a healthy donor and a patient with psoriasis is shown. Total number of CD3⁺ or CD14⁺ cells is indicated. B, Myeloid APCs were analyzed by FACS and quantified in the skin. Results were expressed as the mean number per mm² skin ± SEM (n = 3). C, D, Psoriatic myeloid APCs induce IL-17⁺ T cells. Normal peripheral blood T cells were stimulated for 5 days with myeloid APCs derived from skin and blood of healthy donors or patients with psoriasis in the presence of anti-CD3 and anti-CD28. C, IL-17 was detected in the culture supernatants. Results are expressed as mean ± SEM, n = 5, *P<0.05 compared to healthy donor. D, One representative dot-plot of IL-17⁺ T cells induced by myeloid APCs isolated from psoriatic skin or blood of one patient is shown.

FIGURE 4

IFN-γ programs myeloid APCs to stimulate IL-17⁺ T cells. Normal (A, B) or psoriatic (C, D) blood-derived ex vivo CD11c⁺ cells were conditioned for 72 hours with or without IFN-γ, and then cultured with normal T cells for 5 days in the presence of anti-CD3 and anti-CD28. A-C, IL-17⁺ T cells were detected by FACS. Results are expressed as the mean percentage of IL-17⁺ T cells in T cells ± SEM. D, IL-17 was measured in the supernatants by ELISA. Results are expressed as the mean value of IL-17 ± SEM. *P < 0.05 compared to control. (A, n = 11; B, one of 11; c, d, n = 2).

FIGURE 5

Myeloid APCs stimulate IL-17⁺ T cell expansion through IL-1 and IL-23. A, IL-1 and IL-23 enhance the induction of IL-17⁺ T cells by CD11c⁺ cells. Normal peripheral blood T cells were stimulated for 5 days with normal CD11c⁺ myeloid APCs with or without IL-1 and IL-23 in the presence of anti-CD3 and anti-CD28. IL-17⁺ T cells were determined by FACS. Results are expressed as mean ± SEM, n = 7, * P < 0.03 compared to control. B, IFN-γ stimulates IL-1 and IL-23 expression by CD11c⁺ myeloid APCs. CD11c⁺ myeloid APCs were conditioned with or without IFN-γ. IL-1, IL-12 and IL-23 transcripts were quantified by QRT-PCR. Results are expressed as the mean of relative expression ± SEM, n = 7, *P < 0.01 compared to control. C, Blockade of IL-1 and IL-23 abrogates CD11c⁺ cell-mediated IL-17⁺ T cell induction. Blood-derived CD11c⁺ cells were transfected with IL-23-specific siRNA or control siRNA, then conditioned with IFN-γ. T cells were stimulated for 5 days with these myeloid APCs with or without neutralizing IL-1R antibodies. IL-17⁺ T cells were detected by FACS. Results are expressed as the mean of IL-17⁺ T cells in T cells ± SEM, n = 5, *P < 0.05 compared to control. D, E, Expression of IL-23, IL-12 and IL-1 were quantified by real-time PCR in skin from healthy donors and patients with psoriasis. Results are expressed as the mean of relative expression ± SEM, n = 5, *P < 0.03 compared to uninvolved and healthy skin.

FIGURE 6

CCR6⁺IL-17⁺ T cells and CCL20 in the psoriatic environment. A, B, Psoriatic IL-17⁺ T cells highly express CCR6. Expression of CCR6 was determined by FACS on IL-17⁺ and IL-17^- T cells in psoriatic skin. A, Results are expressed as the mean of CCR6⁺ T cells in IL-17⁺ T cells or IL-17^- T cells ± SEM. (n = 6, *P < 0.05 compared to IL-17^- T cells). B, One representative dot-plot of 6 is shown. Gated on CD4⁺ and CD8⁺ T cells. C, IL-17⁺ T cells migrate in response to CCL20. Migration assay was performed as described in Methods. The migrated T cells were subjected to intracellular staining for IL-17. The percent of IL-17⁺ T cells in the upper and lower chambers are shown. D, IFN-γ induces CCL-20 production. Blood CD11c⁺ cells were stimulated for 3 days with or without IFN-γ. CCL20 was detected by ELISA in the supernatants. Results are expressed as the mean value ± SEM (n = 7, *P < 0.01 compared to control). E, High expression of CCL20 in psoriatic skin. CCL20 transcript was quantified by real-time PCR. Results are expressed as the mean values of relative expression ± SEM. (n = 5, *P < 0.03 compared to uninvolved and healthy skin). F, High expression of CD103 on psoriatic IL-17⁺ T cells. CD103 expression was determined by FACS on IL-17⁺ and IL-17^- T cell subsets. Results are expressed as the mean of CD103⁺ T cells in each T cell subset ± SEM. (n = 5, *P < 0.05 compared to IL-17^- T cells). Right panel: One representative dot-plot of psoriatic CD103⁺IL-17⁺ T cells. Gated on CD4⁺ and CD8⁺ T cells.

FIGURE 7

Function of IL-17⁺ T cells in psoriasis. A, High levels of HBD-2 transcript in psoriatic plaque. Expression of HBD-2 was quantified by real-time PCR in the skin from healthy donors and patients with psoriasis. Results are expressed as the mean values of relative expression ± SEM. (n = 5, *P < 0.03 compared to uninvolved and healthy skin). B, IL-17 induces HBD-2 production by NHK. NHKs were cultured for 48 hours with variable concentrations of IL-17 with or without anti-IL-17 receptor. HBD-2 in the supernatants was detected by ELISA. One representative of 3 is shown. C, D, IL-17⁺ T cells stimulate NHK HBD-2 production through IL-17. T cells were stimulated with psoriatic myeloid APCs for 5 days to generate IL-17⁺ T cells. C, NHKs were cultured for 48 hours in the presence of 20% concentration of these T cell supernatants in the absence (control) or presence of anti-IL-17 receptor. HBD-2 in the NHK supernatants was detected by ELISA. (n = 10, *P < 0.01 compared to control). D, Strong correlation was observed between the amount of T cell derived IL-17 in the supernatants and NHK HBD-2 production (n = 10, R² = 0.8, P < 0.01). E, Synergistic effect of IFN-γ on IL-17 mediated HBD-2 production. NHK cells were cultured with IL-17 (10 ng/ml) in the presence of variable concentrations of IFN-γ. HBD-2 was detected in the NHK cell supernatants by ELISA. Results are expressed as the mean values of ± SEM. (n = 3, P < 0.05). F, Proposed schematic model of interaction between Th1, IL-17⁺ and myeloid APCs. IFN-γ derived from Th1 cells induces CCL-20 production by local APCs, which in turn promotes IL-17⁺ T cell trafficking to the psoriatic environment. IFN-γ also programs APCs and expands memory IL-17⁺ T cells through APC-derived IL-1 and IL-23. Additionally, IFN-γ derived from Th1 acts synergistically with IL-17 on keratinocytes. Thus, Th1 and IL-17⁺ T cells collaboratively contribute to psoriasis pathogenesis.