Amelioration of epidermal hyperplasia by TNF inhibition is associated with reduced Th17 responses

Abstract

Biological agents have dramatically improved treatment options for patients with severe psoriasis. Etanercept (tumor necrosis factor [TNF] receptor-immunoglobulin fusion protein) is an effective treatment for many psoriasis patients, and blockade of TNF is considered to be its primary action. However, in this clinical trial, we show that etanercept has early inhibitory effects on a newly appreciated type of T cells: T helper type 17 (Th17) cells. Etanercept reduced the inflammatory dendritic cell products that drive Th17 cell proliferation (interleukin [IL] 23), as well as Th17 cell products and downstream effector molecules (IL-17, IL-22, CC chemokine ligand 20, and beta-defensin 4). In contrast, Th1 cellular products and effector molecules (interferon gamma, lymphotoxin alpha, and myxovirus resistance 1) were reduced late in disease resolution. This study suggests a role for Th17 in addition to Th1 cells in the pathogenesis of psoriasis. Th17 cells may be particularly important in driving epidermal activation in psoriatic plaques, whereas Th1 cells must also be eliminated for final disease resolution.

Figure 1.

Clinical and histological resolution of psoriasis with etanercept treatment. (A) Mean PASI scores, epidermal thickness, K16 mRNA expression, and Ki67 cell counts in histological responders (n = 16) during treatment with etanercept. Clinical response was measured at baseline and weeks 1, 2, 4, and 12; biopsies were evaluated in nonlesional skin (NL), lesional skin (LS), and in the lesional index plaque at weeks 1, 2, 4, and 12. Error bars represent the mean ± SEM. Baseline lesional values were compared with other time points. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (B) Histology and immunohistochemistry showing hematoxylin and eosin (H&E), K16, and Ki67 expression during treatment. Bar, 100 μm. (C) CD11c⁺ myeloid DCs, CD3⁺ T cells, and CD163⁺ macrophages per millimeter in nonlesional skin (NL), lesional skin (LS), and in the lesional index plaque at weeks 1, 2, 4, and 12. Horizontal bars represent the mean. Baseline lesional values were compared with other time points. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Ki67, CD11c, and CD3 baseline lesional cell counts have been previously reported (reference 50). (D) Immunohistochemistry showing CD11c, CD3, and CD163 expression during treatment. Bar, 100 μm.

Figure 2.

Th17 cell products and downstream mediators are rapidly down-modulated with etanercept treatment compared with Th1 and Th2 cell products. mRNA expression normalized to HARP for (A) Th17 cell products IL-17 and IL-22 and (B) Th1 cell products IFN-γ and LTA-1. Error bars represent the mean ± SEM. (C) Multivariate U-statistics correlating the change in Th17 or Th1 cell products with histological response (epidermal thickness, K16, and Ki67) over time. (D) Downstream effectors of Th17 cells, CCL20, and DEFB4. (E) MX-1, downstream effector of Th1 cells. (F) Th2 cell product IL-4. All mRNA was evaluated in nonlesional skin (NL), lesional skin (LS), and in the lesional index plaque at weeks 1, 2, 4, and 12. Error bars represent the mean ± SEM. Baseline lesional values were compared with other time points. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 3.

Inflammatory DC products are rapidly down-modulated with etanercept treatment. (A) mRNA expression normalized to HARP for the inflammatory DC cell products iNOS, IL-20, IL-23 p19, IL-23/IL-12 p40, and IL-12 p35 in nonlesional skin (NL), lesional skin (LS), and in the lesional index plaque at weeks 1, 2, 4, and 12. Baseline lesional values were compared with other time points. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (B–D) Double-label immunofluorescence of myeloid DCs (CD11c) and various mediators (IL-20, IL-12/23 p40, and TNF) demonstrating coexpression (yellow) in baseline lesional skin compared with weeks 2 and 12, showing a reduction in myeloid DCs and their products with etanercept treatment. (B) CD11c (green) and IL-20 (red); (C) CD11c (green) and IL-23/IL-12 p40 (red); and (D) CD11c (red) and TNF (green). The white lines identify the dermal epidermal junction. Autofluorescent keratinocytes appear in all panels. Bar, 100 μm.

Figure 4.

DCs down-regulate maturation and co-stimulatory molecules with etanercept treatment. (A) Immunohistochemistry for the mature DC markers CD83 and DC-LAMP in nonlesional skin (NL), lesional skin (LS), and in the lesional index plaque at weeks 1, 2, 4, and 12. Bar, 100 μm. (B) Quantification of CD83⁺ and DC-LAMP⁺ cells per millimeter (n = 16) during etanercept treatment. Horizontal bars represent the mean. Baseline lesional values were compared with other time points. *, P < 0.05; **, P < 0.01; ***, P < 0.001. CD83 and DC-LAMP baseline lesional cell counts have been previously reported (reference 50). (C) FACS analysis at baseline (week 0) and matched week 2 etanercept-treated lesional dermal single-cell suspensions. Acquired cells were gated on myeloid DCs (Lin⁻HLA-DR⁺CD11c⁺; dark gray). MFI is indicated in the top right corner of each histogram; isotypes are shown in light gray.

Figure 5.

In vitro MoDCs generated in the presence of etanercept are less mature and less immunostimulatory, and express macrophage antigen CD163. (A) FACS analysis of MoDCs generated without or with etanercept. Acquired cells were gated on myeloid DCs (Lin⁻HLA-DR⁺CD11c⁺; dark gray). MFI is indicated in top right corner of each histogram; isotypes are shown in light gray. (B) MLR comparing MoDCs matured with and without etanercept (T cells + iDC). T cells alone and T cells + CD3/28 beads serve as negative and positive controls, respectively. The percentage of proliferation is indicated in the bottom left corner of each FACS plot. (C) Comparison of CD163 mRNA expression (gene array) in MoDCs generated without (blue) or with (red) etanercept. Error bars represent the mean ± SEM. *, P < 0.05. (D) Increased surface expression of CD163 on MoDCs generated with etanercept was confirmed by flow cytometry. CFDA, carboxyfluorescein diacetate; iDC, inflammatory DC.

Figure 6.

Proposed role of Th17 and Th1 cells in psoriasis pathogenesis. TNF stimulates CD11c⁺ inflammatory DCs to produce IL-23 and IL-20. DC activation and production of IL-23 supports Th17 cell survival and proliferation and induces the production of IL-17 and IL-22. DC and Th17 cell products activate keratinocytes, promoting the release of innate inflammatory molecules such as DEFB4, S100A7, and IL-8. Concurrently, Th1 cells producing IFN-γ activate keratinocytes to up-regulate MHC class II molecules (HLA-DR) and integrins (ICAM), and release cytokines including membrane Ig (MIG) and IFN-inducible protein 10 (IP-10). Th1 and Th17 cells may suppress each other's development, but IFN-γ can also act synergistically with IL-17 to increase ICAM expression and IL-8 release from keratinocytes. In psoriasis, etanercept may proximally inhibit this IL-23–IL-17 pathway to normalize keratinocyte proliferation and leukocyte infiltration.