Circulating Th17, Th22, and Th1 cells are increased in psoriasis

Abstract

Th17, Th22, and Th1 cells are detected in psoriatic skin lesions and implicated in psoriasis pathogenesis, but inflammatory T cell numbers in blood, as well as the relative importance of each cell type, is unclear. Using 7-color flow cytometry, circulating Th17, Th22, and Th1 cells were quantified in 21 untreated psoriatics and 17 healthy individuals. CCR6 was the best cell surface marker for IL-17A+ cells when compared with IL-23R or CD161. CCR6+, IL-17A+, IL-22+, CCR6+IL-17A+, CCR6+IL-22+, CCR6+tumor necrosis factor-alpha+, IL-17A+IFN-gamma-, IL-17A+IL-22+IFN-gamma-, and IL-17A+IL-22-IFN-gamma- cells were increased in psoriatics (all values P<0.001), indicating elevations in circulating Th17 cells, using multiple criteria to define these cells. Th22 (IL-17A-IL-22+IFN-gamma-, P<0.05) and Th1 (IL-17A-IFN-gamma+, P<0.05) cells were also increased in psoriatics, but to a lesser extent. Inhibition of either NF-kappaB or STAT3 in vitro blocked cytokine production by both Th17 and Th1 cells. Circulating levels of Th17 and Th1 cells decreased in a subset of five psoriasis patients serially evaluated following induction therapy with infliximab. In summary, elevated numbers of circulating inflammatory T cells may contribute to cutaneous inflammation and systemic inflammatory disease that occurs in individuals with psoriasis.

Figure 1. Circulating CCR6+, IL-23R+, and CD161 + cells are increased in psoriasis

(a) The percentage of CD4+cells expressing CCR6, IL-23R, and CD161 among unstimulated cells from 17 healthy individuals and 21 untreated psoriatics (horizontal bars, mean; each circle, single donor). (b) Representative dot plot analyses of CCR6, IL-23R, and CD161 expression in circulating unstimulated CD4+ cells from the same individual. (c)The percentages of each subset divided by the total number of circulating CCR6, IL-23R, and CD161-positive cells in unstimulated CD4+cells of healthy individuals (white bars) and psoriatics (black bars). Data expressed as mean ± SD; *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 2. Circulating Th17, Th1, and Th17/Th1 cells are increased in psoriasis

(a)In single-color flow analyses, circulating IL-17A+, IL-22+, IFN-g+, and tumor necrosis factor (TNF)-alpha + cells are increased in psoriasis patients. (b)Representative two-color dot plot analyses of IL-17A and IFN-γ expression in stimulated CD4+ cells from a healthy volunteer and a psoriasis patient. (c)The percentages of circulating Th17 (IL-17A + IFN-γ−), Th1 (IL-17A−IFN-γ+), and Th17/Th1 (IL-17A + IFN-γ+) cells among stimulated CD4+ cells from healthy individuals and psoriatics. For a and c, each circle, single donor; horizontal bars, mean; and *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3. Most Th17 cells do not simultaneously express both IL-17A and IL-22

(a)Representative two-color dot plot analyses of IL-17A, IL-22, and IFN-γ expression in stimulated CD4+ cells from a single patient with psoriasis. (b)The percentages of circulating CD4+ cells that coexpress IL-17A, IL-22, and IFN-γ in healthy individuals (white bars) and psoriatics (black bars). Data expressed as mean ± SD; *P<0.05, **P<0.01, and ***P<0.001.

Figure 4. On the basis of IL-17A production, CCR6 is a more reliable cell surface marker for Th17 cells when compared with IL-23R or CD161

IL-17A + cells, IL-22 + cells, and IFN-γ + cells from psoriatics were gated and the percentage of cells coexpressing CCR6, IL-23R, CD161, or tumor necrosis factor (TNF)-α was determined (black bars). Similarly, IL-17A− cells, IL-22− cells, and IFN-γ− cells from psoriatics were gated and the percentage of cells coexpressing CCR6, IL-23R, CD161, or TNF-α was determined (white bars). Data expressed as mean ± SD; NS, not significant; *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 5. CCR6 + IL-17A+, CCR6 + IL-22+, and CCR6 + TNF-α+ cells are increased in psoriatics compared with healthy individuals

Representative two-color dot plot analyses of CCR6 and IL-17A/IL-22/tumor necrosis factor (TNF)-α expression in stimulated CD4+ cells from a single healthy volunteer (a) and a psoriasis patient (b).(c)The percentages of circulating IL-17A +cells that coexpress CCR6, IL-23R, and CD161 among stimulated CD4+ cells from healthy individuals and psoriatics. (d)The percentages of circulating IL-22 +cells that coexpress CCR6, IL-23R, and CD161 among stimulated CD4+ cells from healthy individuals and psoriatics. (e)The percentages of circulating TNF-α+ cells that coexpress CCR6, IL-23R, and CD161 among stimulated CD4+ cells from healthy individuals and psoriatics. Each circle, single donor; horizontal bars, mean; *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 6. NF-κB and STAT3 inhibition in vitro blocks cytokine production by both Th17 and Th1 cells in a dose-dependent manner

(a) CD4+ cells were cultured overnight with or without parthenolide or stattic at the indicated concentrations, and then activated with phorbol myristate acetate (PMA)/ionomycin for 6 hours. Representative two-color dot plot analyses of IFN-γ and tumor necrosis factor (TNF)-α expression from a single healthy volunteer are shown. (b)The percentage of circulating CD4+ cells producing IL-17A, IL-22, IFN-γ, and TNF-α in healthy individuals (solid lines) and psoriatics (dotted lines) with or without 2.5 µm of parthenolide or 1 µm of stattic. Each line, single donor.