There are considerable data showing that both Th17 and Th1 cells are increased within involved psoriatic skin (Uyemura et al., 1993
; Szabo et al., 1998
; Austin et al., 1999
; Chan et al., 2006
; Wolk et al., 2006
; Annunziato et al., 2007
; Wilson et al., 2007
; Zaba et al., 2007
; Kryczek et al., 2008
; Lowes et al., 2008
; Harper et al., 2009
). Here, we show clear evidence of significant elevations in circulating Th17 cells in psoriasis, using a number of criteria to define these cells. More specifically, we show increases in circulating CCR6+, IL-17A+, IL-22+, IL-17A + IFN-γ−, CCR6+ IL-17A+, CCR6+ IL-22+, CCR6+ TNF-α+, IL-17A + IL-22 + IFN-γ−, and IL-17A + IL-22−IFN-γ− CD4+ T cells in untreated psoriatics compared with healthy individuals (P
< 0.001 for all subsets) (– and ). Circulating Th1 cells, defined as IFN-γ+, IL-17A−IFN-γ+, and IL-17A−IL-22−IFN-γ + CD4+ cells, were also elevated (P
< 0.05 for the first two subsets and P
< 0.01 for the third subset) ( and ). It is noted that two groups recently reported normal numbers of circulating Th17 cells in psoriatics (Kryczek et al., 2008
; Lowes et al., 2008
). There are three possible reasons for this discrepancy: (1) these investigators used intracellular expression of IL-17A and IFN-γ alone to define Th17 cells (we used multiple criteria); (2) CD4+ T cells were not purified before their analyses as we have done; and (3) they studied smaller numbers of patients and controls compared with our current report.
It is unknown how circulating Th17 cells in psoriasis patients may contribute to skin disease and inflammation at sites other than skin. They may represent cells that are trafficking to and from skin, thus contributing significantly to cutaneous inflammation. However, the xenograft transplantation model of psoriasis, whereby human non-lesional skin from psoriatics is engrafted onto immunodeficient mice, is independent of circulating human T cells (Boyman et al., 2004
). Alternatively, and independent of skin disease severity, psoriatics may be genetically predisposed to make greater amounts of IL-23 and to create greater numbers of Th17 cells under the appropriate stimulation conditions, whether the cells are in the blood, skin, or elsewhere. This genetic tendency could be regulated, at least in part, by polymorphisms in IL-23p19, IL-12/23p40
, and IL-23R
(Nair et al., 2009
). Circulating Th17 cells could also have a role in systemic inflammation observed in psoriasis patients, including psoriatic arthritis and atherosclerosis (Gelfand et al., 2006
). Recent evidence suggests that Th17 cells and cytokines promote atherosclerosis (Cheng et al., 2008
; Eid et al., 2009
). Thus, we believe it would be important to study correlations between numbers and functional activity of circulating Th17 cells and the development of atherosclerosis and/or myocardial infarction in individuals with severe psoriasis.
Our data are strengthened by the utilization of 7-color flow cytometry to simultaneously examine both cell surface marker expression and intracellular cytokine expression in various circulating T cell subsets. As there is no universal cell surface marker to delineate Th17 cells from Th1 cells, we used several criteria to help distinguish these two subsets. CCR6 was expressed by 81% of circulating IL-17A+ cells and only 26% of circulating IFN-γ+ cells, making it the best cell surface marker we studied to delineate Th17 cells from Th1 cells (). By contrast, CD161 and IL-23R were expressed by fewer circulating Th17 cells and by a minority of circulating Th1 cells, which made them less useful markers in separating these two T cell subsets (). It is unclear why so few IL-17A+ cells expressed IL-23R, but this may have been due to downregulation of cell surface receptor expression following mitogenic stimulation of the cells. It is also possible that stimulation of cells by IL-23, and not by PMA/ionomycin, may be a more selective inducer of IL-17A specifically within the population of IL-23R + Th17 cells. Even though CCR6 was shown to be the best Th17 cell surface marker used here, sensitivity and specificity were too low for CCR6 to be used as a sole marker for Th17 cells. Combining expression of CCR6 with intracellular cytokine production may prove to be a useful tool in future analyses of Th17 and Th1 cells in human disease. Very recently, the skin-homing markers CCR4 and CCR10 have been identified on blood-derived Th22 cells (Duhen et al., 2009
; Trifari et al., 2009
). It will be interesting to further characterize Th22 and Th17 subsets in blood of psoriatics for their expression of cutaneous lymphocyte antigen, the prototypic skin-homing marker, and these two chemokine receptors.
Little information is known about the function of Th17/Th1 cells and Th22 cells. Th17/Th1 cells were identified in the affected skin of psoriatics (Lowes et al., 2008
; Nograles et al., 2009
). Zaba et al. (2009)
recently reported that dermal dendritic cells within psoriasis lesions induce a population of activated T cells that simultaneously produce IL-17A and IFN-γ (i.e., Th17/Th1 cells), which was not observed when using dermal dendritic cells isolated from normal skin. We show here that Th17/Th1 cells, defined as either IL-17A + IFN-γ + or IL-17A + IL-22−IFN-γ + cells, are a rare population of circulating cells, representing only 0.05–0.11% of CD4+ cells in healthy blood and 0.13–0.21% of CD4+ cells in psoriatic blood (P
< 0.01 for both subsets) ( and ). Our data suggest that the tendency to develop Th17/Th1 cells may be a general immunologic feature seen in psoriasis patients, and not necessarily a phenomenon restricted to inflamed skin. Another relatively obscure subset of T cells, Th22 cells (defined as IL-17A−IL-22 + IFN-γ−), was more commonly detected when compared with Th17/Th1 cells: 1.29% of CD4+ cells in psoriatic versus 0.86% of CD4+ cells in healthy individuals (P
< 0.05) (). The clinical relevance of circulating Th22 cells remains to be determined, although Liu et al. (2009)
recently suggested that these cells are preferentially stimulated by Candida albicans
, and thus may have a role in defense against fungal infections.
We also measured plasma cytokine levels, which have been notoriously difficult in prior studies of psoriasis, as pro-inflammatory cytokines are usually active and elevated only at sites of tissue inflammation and not within circulation. We showed that plasma IL-22 concentration is higher in psoriatics when compared with healthy individuals and that levels are highly reflective of skin disease activity (Supplementary Figure S2
), which is consistent with two previous reports (Wolk et al., 2006
; Caproni et al., 2009
). We also detected increases in plasma TNF-α (Supplementary Figure S2
), similar to several prior reports (Mussi et al., 1997
; Arican et al., 2005
). In contrast to IL-22 and TNF-α, IL-17A and IFN-γ concentrations are not elevated in plasma of psoriatics (Supplementary Figure S2a
). Thus, despite some conflicting data on serum or plasma cytokine data, there appears to be consensus that circulating levels of IL-22 and TNF-α are increased in psoriatics, and that cytokine concentrations, especially for IL-22, correlate with extent of skin disease activity.
NF-κB is important in regulating expression of a wide variety of cytokines. Relevant to psoriasis, NF-κB expression is high in affected skin and normalizes following effective anti-psoriatic therapy with etanercept (Lizzul et al., 2005
). NF-κB regulates IL-17A production by inducing phosphorylation of IκB-α (Cho et al., 2006
). Here, we showed that specific inhibition of NF-κB by parthenolide led to the complete blockade of IL-17A, IL-22, IFN-γ, and TNF-α production by circulating CD4+ cells in vitro
(). Importantly, IL-17A production by Th17 cells is critically dependent on STAT3 activation through the PI3K pathway (Cho et al., 2006
). Furthermore, individuals with genetic mutations in STAT3
develop Job’s syndrome, also known as hyper-IgE syndrome, and are incapable of making Th17 cells (Ma et al., 2008
; Milner et al., 2008
). Accordingly, we showed that IL-17A, IL-22, IFN-γ, and TNF-α production by CD4+ cells could be completely blocked in vitro
by stattic, a specific inhibitor of STAT3 (). Although systemic inhibition may be impractical on the basis of feasibility and safety issues, skin-specific inhibition of either NF-κB or STAT3 may be a future therapeutic strategy for patients with localized psoriasis.
Infliximab is a chimeric anti-TNF-α mAb that is highly efficacious in the treatment of individuals with moderate-to-severe plaque psoriasis (Reich et al., 2005
). Here, we serially evaluated numbers of circulating Th17 and Th1 cells in five patients receiving induction therapy with infliximab. Infliximab led to decreases in clinical disease scores and circulating levels of both Th17 and Th1 cells, including Th17 and Th1 cells that produced TNF-α (). Drops in both clinical and immunologic parameters, however, were relatively minor and not statistically significant, although there was a trend toward greater decreases in Th17 cell numbers when compared to Th1 cell numbers. We believe dramatic changes were not observed because the study included too few patients and because of the potential residual clinical and immunologic effects induced by etanercept therapy, which these five patients were receiving 2–4 weeks before their first infliximab infusion. Thus, greater decreases in circulating Th17 and Th1 cells may have been observed if the patients showed more severe skin disease and/ or were treatment naive at the beginning of the study. It will be interesting to monitor circulating levels of Th17 and Th1 cells in patients receiving ustekinumab or ABT-874, two new highly effective human mAb therapies for moderate-to-severe psoriasis that target p40 (Kimball et al., 2008
; Leonardi et al., 2008
; Papp et al., 2008
), the subunit shared by IL-23 and IL-12. Drops in circulating levels of both Th17 and Th1 cells are expected, although it is unclear whether this will lead to decreases in systemic immune function.